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Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids.
The two primary methods used for the ionization of protein in mass spectrometry are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). These ionization techniques are used in conjunction with mass analyzers such as tandem mass spectrometry. In general, the proteins are analyzed either in a "top-down" approach in which proteins are analyzed intact, or a "bottom-up" approach in which protein are first digested into fragments. An intermediate "middle-down" approach in which larger peptide fragments are analyzed may also sometimes be used.
== History ==
The application of mass spectrometry to study proteins became popularized in the 1980s after the development of MALDI and ESI. These ionization techniques have played a significant role in the characterization of proteins. (MALDI) Matrix-assisted laser desorption ionization was coined in the late 1980s by Franz Hillenkamp and Michael Karas. Hillenkamp, Karas and their fellow researchers were able to ionize the amino acid alanine by mixing it with the amino acid tryptophan and irradiated with a pulse 266 nm laser. Though important, the breakthrough did not come until 1987. In 1987, Koichi Tanaka used the "ultra fine metal plus liquid matrix method" and ionized biomolecules the size of 34,472 Da protein carboxypeptidase-A.
In 1968, Malcolm Dole reported the first use of electrospray ionization with mass spectrometry. Around the same time MALDI became popularized, John Bennett Fenn was cited for the development of electrospray ionization. Koichi Tanaka received the 2002 Nobel Prize in Chemistry alongside John Fenn, and Kurt Wüthrich "for the development of methods for identification and structure analyses of biological macromolecules." These ionization methods have greatly facilitated the study of proteins by mass spectrometry. Consequently, protein mass spectrometry now plays a leading role in protein characterization.
== Methods and approaches ==
=== Techniques ===
Mass spectrometry of proteins requires that the proteins in solution or solid state be turned into an ionized form in the gas phase before they are injected and accelerated in an electric or magnetic field for analysis. The two primary methods for ionization of proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In electrospray, the ions are created from proteins in solution, and it allows fragile molecules to be ionized intact, sometimes preserving non-covalent interactions. In MALDI, the proteins are embedded within a matrix normally in a solid form, and ions are created by pulses of laser light. Electrospray produces more multiply-charged ions than MALDI, allowing for measurement of high mass protein and better fragmentation for identification, while MALDI is fast and less likely to be affected by contaminants, buffers and additives.
Whole-protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT-ICR). These two types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Electrospray ionization of a protein often results in generation of multiple charged species of 800 < m/z < 2000 and the resultant spectrum can be deconvoluted to determine the protein's average mass to within 50 ppm or better using TOF or ion-trap instruments.
Mass analysis of proteolytic peptides is a popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis are the MALDI-TOF instruments as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec). Multiple stage quadrupole-time-of-flight and the quadrupole ion trap also find use in this application.
Tandem mass spectrometry (MS/MS) is used to measure fragmentation spectra and identify proteins at high speed and accuracy. Collision-induced dissociation is used in mainstream applications to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of this simplicity in fragmentation, it is possible to use the observed fragment masses to match with a database of predicted masses for one of many given peptide sequences. Tandem MS of whole protein ions has been investigated recently using electron capture dissociation and has demonstrated extensive sequence information in principle but is not in common practice.
=== Approaches ===
In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as "top-down" strategy of protein analysis as it involves starting with the whole mass and then pulling it apart. The top-down approach however is mostly limited to low-throughput single-protein studies due to issues involved in handling whole proteins, their heterogeneity and the complexity of their analyses.
In the second approach, referred to as the "bottom-up" MS, proteins are enzymatically digested into smaller peptides using a protease such as trypsin. Subsequently, these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Hence, this approach uses identification at the peptide level to infer the existence of proteins pieced back together with de novo repeat detection. The smaller and more uniform fragments are easier to analyze than intact proteins and can be also determined with high accuracy, this "bottom-up" approach is therefore the preferred method of studies in proteomics. A further approach that is beginning to be useful is the intermediate "middle-down" approach in which proteolytic peptides larger than the typical tryptic peptides are analyzed.
=== Protein and peptide fractionation ===
Proteins of interest are usually part of a complex mixture of multiple proteins and molecules, which co-exist in the biological medium. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of material, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to "drown" or suppress signals from less abundant ones. Second, mass spectrum from a complex mixture is very difficult to interpret due to the overwhelming number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large number of peptide products.
In light of these problems, the methods of one- and two-dimensional gel electrophoresis and high performance liquid chromatography are widely used for separation of proteins. The first method fractionates whole proteins via two-dimensional gel electrophoresis. The first-dimension of 2D gel is isoelectric focusing (IEF). In this dimension, the protein is separated by its isoelectric point (pI) and the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This dimension separates the protein according to its molecular weight. Once this step is completed in-gel digestion occurs. In some situations, it may be necessary to combine both of these techniques. Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, usually the method of in-gel digestion is applied, where the protein spot of interest is excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometry for de novo sequencing. Small changes in mass and charge can be detected with 2D-PAGE. The disadvantages with this technique are its small dynamic range compared to other methods, some proteins are still difficult to separate due to their acidity, basicity, hydrophobicity, and size (too large or too small).
The second method, high performance liquid chromatography is used to fractionate peptides after enzymatic digestion. Characterization of protein mixtures using HPLC/MS is also called shotgun proteomics and MuDPIT (Multi-Dimensional Protein Identification Technology). A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography. The eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.
== Applications ==
=== Protein identification ===
There are two main ways MS is used to identify proteins. Peptide mass fingerprinting uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample. Purification steps therefore limit the throughput of the peptide mass fingerprinting approach. Alternatively, peptides can be fragmented with MS/MS to more definitively identify them.
MS is also the preferred method for the identification of post-translational modifications in proteins versus other approaches such as antibody-based methods.
=== De novo (peptide) sequencing ===
De novo peptide sequencing for mass spectrometry is typically performed without prior knowledge of the amino acid sequence. It is the process of assigning amino acids from peptide fragment masses of a protein. De novo sequencing has proven successful for confirming and expanding upon results from database searches.
As de novo sequencing is based on mass and some amino acids have identical masses (e.g. leucine and isoleucine), accurate manual sequencing can be difficult. Therefore, it may be necessary to utilize a sequence homology search application to work in tandem between a database search and de novo sequencing to address this inherent limitation.
Database searching has the advantage of quickly identifying sequences, provided they have already been documented in a database. Other inherent limitations of database searching include sequence modifications/mutations (some database searches do not adequately account for alterations to the 'documented' sequence, thus can miss valuable information), the unknown (if a sequence is not documented, it will not be found), false positives, and incomplete and corrupted data.
An annotated peptide spectral library can also be used as a reference for protein/peptide identification. It offers the unique strength of reduced search space and increased specificity. The limitations include spectra not included in the library will not be identified, spectra collected from different types of mass spectrometers can have quite distinct features, and reference spectra in the library may contain noise peaks, which may lead to false positive identifications. A number of different algorithmic approaches have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS), peptide de novo sequencing and sequence tag-based searching.
=== Antigen presentation ===
Antigen presentation is the first step in educating the immune system to recognize new pathogens. To this end, antigen presenting cells expose protein fragments via MHC molecules to the immune system. Not all protein fragments bind, however, to the MHC molecules of a certain individual. Using mass spectrometry, the true spectrum of molecules presented to the immune system can be determined.
=== Protein quantitation ===
Multiple methods allow for the quantitation of proteins by mass spectrometry, and recent advances have enabled quantifying thousands of proteins in single cells. Protein quantification by mass spectrometry benefits from efficient sampling (counting) of many ions per protein compared to other methods. Quantifications can be performed by label-free methods and by multiplexed methods, which use isotopic mass tags as labels. Multiplexed methods can improve both quantitative accuracy and throughput.
Typically, stable (e.g. non-radioactive) heavier isotopes of carbon (13C) or nitrogen (15N) are incorporated into one sample while the other one is labeled with corresponding light isotopes (e.g. 12C and 14N). The two samples are mixed before the analysis. Peptides derived from the different samples can be distinguished due to their mass difference. The ratio of their peak intensities corresponds to the relative abundance ratio of the peptides (and proteins). The first generation of methods for isotope labeling included SILAC (stable isotope labeling by amino acids in cell culture), trypsin-catalyzed 18O labeling, ICAT (isotope coded affinity tagging), and iTRAQ (isobaric tags for relative and absolute quantitation). The more recent generation of multiplexing methods include tandem mass tags (TMT) for DDA data and mTRAQ for multiplexed DIA (plexDIA).
"Semi-quantitative" mass spectrometry can be performed without labeling of samples. Typically, this is done with MALDI analysis (in linear mode). The peak intensity, or the peak area, from individual molecules (typically proteins) is here correlated to the amount of protein in the sample. However, the individual signal depends on the primary structure of the protein, on the complexity of the sample, and on the settings of the instrument. Other types of "label-free" quantitative mass spectrometry, uses the spectral counts (or peptide counts) of digested proteins as a means for determining relative protein amounts.
=== Protein structure determination ===
Characteristics indicative of the 3-dimensional structure of proteins can be probed with mass spectrometry in various ways. Comparing charge state distributions can give information about the structure of a protein. A wide variety of high charge states indicates disorder of the protein, whereas more compact, folded proteins result in lower charge states. By using chemical crosslinking to couple parts of the protein that are close in space, but far apart in sequence, information about the overall structure can be inferred. By following the exchange of amide protons with deuterium from the solvent, it is possible to probe the solvent accessibility of various parts of the protein. Hydrogen-deuterium exchange mass spectrometry has been used to study proteins and their conformations for over 20 years. This type of protein structural analysis can be suitable for proteins that are challenging for other structural methods. Another interesting avenue in protein structural studies is laser-induced covalent labeling. In this technique, solvent-exposed sites of the protein are modified by hydroxyl radicals. Its combination with rapid mixing has been used in protein folding studies.
=== Proteogenomics ===
In what is now commonly referred to as proteogenomics, peptides identified with mass spectrometry are used for improving gene annotations (for example, gene start sites) and protein annotations. Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events, especially when comparing multiple species.
== References == | Wikipedia/Protein_mass_spectrometry |
In chemistry, a salt bridge is a combination of two non-covalent interactions: hydrogen bonding and ionic bonding (Figure 1). Ion pairing is one of the most important noncovalent forces in chemistry, in biological systems, in different materials and in many applications such as ion pair chromatography. It is a most commonly observed contribution to the stability to the entropically unfavorable folded conformation of proteins. Although non-covalent interactions are known to be relatively weak interactions, small stabilizing interactions can add up to make an important contribution to the overall stability of a conformer. Not only are salt bridges found in proteins, but they can also be found in supramolecular chemistry. The thermodynamics of each are explored through experimental procedures to access the free energy contribution of the salt bridge to the overall free energy of the state.
== Salt bridges in chemical bonding ==
In water, formation of salt bridges or ion pairs is mostly driven by entropy, usually accompanied by unfavorable ΔH contributions on account of desolvation of the interacting ions upon association. Hydrogen bonds contribute to the stability of ion pairs with e.g. protonated ammonium ions, and with anions is formed by deprotonation as in the case of carboxylate, phosphate etc; then the association constants depend on the pH. Entropic driving forces for ion pairing (in absence of significant H-bonding contributions) are also found in methanol as solvent. In nonpolar solvents contact ion pairs with very high association constants are formed; in the gas phase the association energies of e.g. alkali halides reach up to 200 kJ/mol. The Bjerrum or the Fuoss equation describe ion pair association as function of the ion charges zA and zB and the dielectric constant ε of the medium; a corresponding plot of the stability ΔG vs. zAzB shows for over 200 ion pairs the expected linear correlation for a large variety of ions.
Inorganic as well as organic ions display at moderate ionic strength I similar salt bridge association ΔG values around 5 to 6 kJ/mol for a 1:1 combination of anion and cation, almost independent of the nature (size, polarizability etc) of the ions. The ΔG values are additive and approximately a linear function of the charges, the interaction of e.g. a doubly charged phosphate anion with a single charged ammonium cation accounts for about 2x5 = 10 kJ/mol. The ΔG values depend on the ionic strength I of the solution, as described by the Debye–Hückel equation, at zero ionic strength one observes ΔG = 8 kJ/mol. The stabilities of the alkali-ion pairs as function of the anion charge z by can be described by a more detailed equation.
== Salt bridges found in proteins ==
The salt bridge most often arises from the anionic carboxylate (RCOO−) of either aspartic acid or glutamic acid and the cationic ammonium (RNH3+) from lysine or the guanidinium (RNHC(NH2)2+) of arginine (Figure 2). Although these are the most common, other residues with ionizable side chains such as histidine, tyrosine, and serine can also participate, depending on outside factors perturbing their pKa's. The distance between the residues participating in the salt bridge is also cited as being important. The N-O distance required is less than 4 Å (400 pm). Amino acids greater than this distance apart do not qualify as forming a salt bridge. Due to the numerous ionizable side chains of amino acids found throughout a protein, the pH at which a protein is placed is crucial to its stability.
== Salt bridges found in protein - ligand complexes ==
Salt bridges also can form between a protein and small molecule ligands. Over 1100 unique protein-ligand complexes from the Protein Databank were found to form salt bridges with their protein targets, indicating that salt bridges are frequent in drug-protein interaction. These contain structures from different enzyme classes, including hydrolase, transferases, kinases, reductase, oxidoreductase, lyases, and G protein-coupled receptors (GPCRs).
== Methods for quantifying salt bridge stability in proteins ==
The contribution of a salt bridge to the overall stability to the folded state of a protein can be assessed through thermodynamic data gathered from mutagenesis studies and nuclear magnetic resonance techniques. Using a mutated pseudo-wild-type protein specifically mutated to prevent precipitation at high pH, the salt bridge’s contribution to the overall free energy of the folded protein state can be determined by performing a point-mutation, altering and, consequently, breaking the salt bridge. For example, a salt bridge was identified to exist in the T4 lysozyme between aspartic acid (Asp) at residue 70 and a histidine (His) at residue 31 (Figure 3). Site-directed mutagenesis with asparagine (Asn) (Figure 4) was done obtaining three new mutants: Asp70Asn His31 (Mutant 1), Asp70 His31Asn (Mutant 2), and Asp70Asn His31Asn (Double Mutant).
Once the mutants have been established, two methods can be employed to calculate the free energy associated with a salt bridge. One method involves the observation of the melting temperature of the wild-type protein versus that of the three mutants. The denaturation can be monitored through a change in circular dichroism. A reduction in melting temperature indicates a reduction in stability. This is quantified through a method described by Becktel and Schellman where the free energy difference between the two is calculated through ΔTΔS. There are some issues with this calculation and can only be used with very accurate data. In the T4 lysozyme example, ΔS of the pseudo-wild-type had previously been reported at pH 5.5 so the midpoint temperature difference of 11 °C at this pH multiplied by the reported ΔS of 360 cal/(mol·K) (1.5 kJ/(mol·K)) yields a free energy change of about −4 kcal/mol (−17 kJ/mol). This value corresponds to the amount of free energy contributed to the stability of the protein by the salt bridge.
The second method utilizes nuclear magnetic resonance spectroscopy to calculate the free energy of the salt bridge. A titration is performed, while recording the chemical shift corresponding to the protons of the carbon adjacent to the carboxylate or ammonium group. The midpoint of the titration curve corresponds to the pKa, or the pH where the ratio of protonated: deprotonated molecules is 1:1. Continuing with the T4 lysozyme example, a titration curve is obtained through observation of a shift in the C2 proton of histidine 31 (Figure 5). Figure 5 shows the shift in the titration curve between the wild-type and the mutant in which Asp70 is Asn. The salt bridge formed is between the deprotonated Asp70 and protonated His31. This interaction causes the shift seen in His31’s pKa. In the unfolded wild-type protein, where the salt bridge is absent, His31 is reported to have a pKa of 6.8 in H2O buffers of moderate ionic strength. Figure 5 shows a pKa of the wild-type of 9.05. This difference in pKa is supported by the His31’s interaction with Asp70. To maintain the salt bridge, His31 will attempt to keep its proton as long as possible. When the salt bridge is disrupted, like in the mutant D70N, the pKa shifts back to a value of 6.9, much closer to that of His31 in the unfolded state.
The difference in pKa can be quantified to reflect the salt bridge’s contribution to free energy. Using Gibbs free energy: ΔG = −RT ln(Keq), where R is the universal gas constant, T is the temperature in kelvins, and Keq is the equilibrium constant of a reaction in equilibrium. The deprotonation of His31 is an acid equilibrium reaction with a special Keq known as the acid dissociation constant, Ka: His31-H+ ⇌ His31 + H+. The pKa is then related to Ka by the following: pKa = −log(Ka). Calculation of the free energy difference of the mutant and wild-type can now be done using the free energy equation, the definition of pKa, the observed pKa values, and the relationship between natural logarithms and logarithms. In the T4 lysozyme example, this approach yielded a calculated contribution of about 3 kcal/mol to the overall free energy. A similar approach can be taken with the other participant in the salt bridge, such as Asp70 in the T4 lysozyme example, by monitoring its shift in pKa after mutation of His31.
A word of caution when choosing the appropriate experiment involves the location of the salt bridge within the protein. The environment plays a large role in the interaction. At high ionic strengths, the salt bridge can be completely masked since an electrostatic interaction is involved. The His31-Asp70 salt bridge in T4 lysozyme was buried within the protein. Entropy plays a larger role in surface salt bridges where residues that normally have the ability to move are constricted by their electrostatic interaction and hydrogen bonding. This has been shown to decrease entropy enough to nearly erase the contribution of the interaction. Surface salt bridges can be studied similarly to that of buried salt bridges, employing double mutant cycles and NMR titrations. Although cases exist where buried salt bridges contribute to stability, like anything else, exceptions do exist and buried salt bridges can display a destabilizing effect. Also, surface salt bridges, under certain conditions, can display a stabilizing effect. The stabilizing or destabilizing effect must be assessed on a case by case basis and few blanket statements are able to be made.
== Supramolecular chemistry ==
Supramolecular chemistry is a field concerned with non-covalent interactions between macromolecules. Salt bridges have been used by chemists within this field in both diverse and creative ways, including sensing of anions, the synthesis of molecular capsules and double helical polymers.
=== Anion complexation ===
Major contributions of supramolecular chemistry have been devoted to recognition and sensing of anions. Ion pairing is the most important driving force for anion complexation, but selectivity e.g. within the halide series has been achieved, mostly by hydrogen bonds contributions.
=== Molecular capsules ===
Molecular capsules are chemical scaffolds designed to capture and hold a guest molecule (see molecular encapsulation). Szumna and coworkers developed a novel molecular capsule with a chiral interior. This capsule is made of two halves, like a plastic easter egg (Figure 6). Salt bridge interactions between the two halves cause them to self-assemble in solution (Figure 7). They are stable even when heated to 60 °C.
=== Double helical polymers ===
Yashima and coworkers have used salt bridges to construct several polymers that adopt a double helix conformation much like DNA. In one example, they incorporated platinum to create a double helical metallopolymer. Starting from their monomer and platinum(II) biphenyl (Figure 8), their metallopolymer self assembles through a series of ligand exchange reactions. The two halves of the monomer are anchored together through the salt bridge between the deprotonated carboxylate and the protonated nitrogens.
== References == | Wikipedia/Salt_bridge_(protein) |
Proteins are essential nutrients for the human body. They are one of the building blocks of body tissue and can also serve as a fuel source. As a fuel, proteins provide as much energy density as carbohydrates: 17 kJ (4 kcal) per gram; in contrast, lipids provide 37 kJ (9 kcal) per gram. The most important aspect and defining characteristic of protein from a nutritional standpoint is its amino acid composition.
Proteins are polymer chains made of amino acids linked together by peptide bonds. During human digestion, proteins are broken down in the stomach to smaller polypeptide chains via hydrochloric acid and protease actions. This is crucial for the absorption of the essential amino acids that cannot be biosynthesized by the body.
There are nine essential amino acids which humans must obtain from their diet in order to prevent protein-energy malnutrition and resulting death. They are phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine. There has been debate as to whether there are 8 or 9 essential amino acids. The consensus seems to lean towards 9 since histidine is not synthesized in adults. There are five amino acids which humans are able to synthesize in the body. These five are alanine, aspartic acid, asparagine, glutamic acid and serine. There are six conditionally essential amino acids whose synthesis can be limited under special pathophysiological conditions, such as prematurity in the infant or individuals in severe catabolic distress. These six are arginine, cysteine, glycine, glutamine, proline and tyrosine. Dietary sources of protein include grains, legumes, nuts, seeds, meats, dairy products, fish, and eggs.
== Functions ==
After water, proteins account for more mass in an organism than any other type of molecule. Protein is present in every cell, and it is a structural component of every body tissue and organ, including hair, skin, blood, and bone. Protein is especially abundant in muscle. Cellular messengers (hormones) and transport molecules are constructed from proteins, including enzymes and antibodies, as are cell membrane components, such as glycoproteins, G proteins, and ion channels. The types of amino acids and their sequence determine the unique 3-dimensional structure and function of a protein.
Amino acids obtained through protein catabolism also enable the biosynthesis of non-protein molecules that are essential for life, such as nucleotides, certain neurotransmitters, and heme.
== Sources ==
Protein occurs in a wide range of food. On a worldwide basis, plant protein foods contribute over 60% of the per capita supply of protein. In North America, animal-derived foods contribute about 70% of protein sources. Insects are a source of protein in many parts of the world. In parts of Africa, up to 50% of dietary protein derives from insects. It is estimated that more than 2 billion people eat insects daily.
Meat, dairy, eggs, soybeans, fish, whole grains, and cereals are sources of protein. Examples of food staples and cereal sources of protein, each with a concentration greater than 7%, are (in no particular order) buckwheat, oats, rye, millet, maize (corn), rice, wheat, sorghum, amaranth, and quinoa. Game meat is an affordable protein source in some countries.
Plant sources of proteins include legumes, nuts, seeds, grains, and some vegetables and fruits. Plant foods with protein concentrations greater than 7% include (but are not limited to) soybeans, lentils, kidney beans, white beans, mung beans, chickpeas, cowpeas, lima beans, pigeon peas, lupines, wing beans, almonds, Brazil nuts, cashews, pecans, walnuts, cotton seeds, pumpkin seeds, hemp seeds, sesame seeds, and sunflower seeds.
Photovoltaic-driven microbial protein production uses electricity from solar panels and carbon dioxide from the air to create fuel for microbes, which are grown in bioreactor vats and then processed into dry protein powders. The process makes highly efficient use of land, water and fertiliser.
People eating a balanced diet do not need protein supplements.
The table below presents food groups as protein sources.
Colour key:
Protein source with highest density of respective amino acid.
Protein source with lowest density of respective amino acid.
Protein powders – such as casein, whey, egg, rice, soy and cricket flour– are processed and manufactured sources of protein.
== Testing in foods ==
=== Nitrogen-based crude protein ===
The classic assays for protein concentration in food are the Kjeldahl method and the Dumas method. These tests determine the total nitrogen in a sample. The only major component of most food which contains nitrogen is protein (fat, carbohydrate and dietary fiber do not contain nitrogen). If the amount of nitrogen is multiplied by a factor depending on the kinds of protein expected in the food the total protein can be determined. This value is known as the "crude protein" content. The use of correct conversion factors is heavily debated, specifically with the introduction of more plant-derived protein products. However, on food labels the protein is calculated as the amount of nitrogen multiplied by 6.25, because the average nitrogen content of proteins is about 16%. The Kjeldahl test is typically used, because it is the method the AOAC International has adopted and is therefore used by many food standards agencies around the world, though the Dumas method is also approved by some standards organizations.
Nitrogen-based protein measurement cannot distinguish between true protein and non-protein nitrogen (NPN). NPN occurs in significant amounts in milk, edible insects, and fish. In addition, accidental contamination and intentional adulteration of protein meals with NPN sources that inflate crude protein content measurements have been known to occur in the food industry for decades. To ensure food quality, purchasers of protein meals routinely conduct quality control tests designed to detect the most common non-protein nitrogen contaminants, such as urea and ammonium nitrate.
The limitations of the Kjeldahl method were at the heart of the Chinese protein export contamination in 2007 and the 2008 China milk scandal in which the industrial chemical melamine was added to the milk or glutens to increase the measured "protein".
=== True protein ===
In at least one segment of the food industry, the dairy industry, some countries (at least the U.S., Australia, France and Hungary) have adopted "true protein" measurement, as opposed to crude protein measurement, as the standard for payment and testing: "True protein is a measure of only the proteins in milk, whereas crude protein is a measure of all sources of nitrogen and includes nonprotein nitrogen, such as urea, which has no food value to humans. ... Current milk-testing equipment measures peptide bonds, a direct measure of true protein." Measuring peptide bonds in grains has also been put into practice in several countries including Canada, the UK, Australia, Russia and Argentina where near-infrared reflectance (NIR) technology, a type of infrared spectroscopy is used.
The more traditional approach to true protein analysis is amino acid analysis. Data from such analysis has additional nutritional meaning, as humans and other animals have specific requirements for essential amino acids. The Food and Agriculture Organization of the United Nations (FAO) recommends that only amino acid analysis be used to determine protein in, inter alia, foods used as the sole source of nourishment, such as infant formula, but also provides: "When data on amino acids analyses are not available, determination of protein based on total N content by Kjeldahl (AOAC, 2000) or similar method ... is considered acceptable." Using standard methods for amino acid analysis, the true protein content can be reported as the sum of the anhydrous masses of all 18 amino acids analyzed. AA analysis can be performed using standard methods including ISO 13903 (2005) and AOAC 988.15.
In the context of dairy products, NPN can also be calculated by precipitating away all protein and measuring the nitrogen content in the remaining fraction.
=== Ruminant metabolizable protein ===
The testing method for protein in beef cattle feed has grown into a science over the post-war years. The standard text in the United States, Nutrient Requirements of Beef Cattle, has been through eight editions over at least seventy years. The 1996 sixth edition substituted for the fifth edition's crude protein the concept of "metabolizeable protein", which was defined around the year 2000 as "the true protein absorbed by the intestine, supplied by microbial protein and undegraded intake protein". (This refers specifically to ruminant nutrition, where microbes living in the rumen can convert NPNs to proteins. Such conversion does not happen in non-ruminants such as humans.)
=== Protein quality ===
The most important aspect and defining characteristic of protein from a nutritional standpoint is its amino acid composition. There are multiple systems which rate proteins by their usefulness to an organism based on their relative percentage of amino acids and, in some systems, the digestibility of the protein source. They include biological value, net protein utilization, and PDCAAS (Protein Digestibility Corrected Amino Acids Score) which was developed by the FDA as a modification of the Protein efficiency ratio (PER) method. The PDCAAS rating was adopted by the US Food and Drug Administration (FDA) and the Food and Agricultural Organization of the United Nations/World Health Organization (FAO/WHO) in 1993 as "the preferred 'best'" method to determine protein quality. These organizations have suggested that other methods for evaluating the quality of protein are inferior.
In 2013 FAO proposed changing to Digestible Indispensable Amino Acid Score.
== Digestion ==
Most proteins are decomposed to single amino acids by digestion in the gastro-intestinal tract.
Digestion typically begins in the stomach when pepsinogen is converted to pepsin by the action of hydrochloric acid, and continued by trypsin and chymotrypsin in the small intestine.
Before the absorption in the small intestine, most proteins are already reduced to single amino acid or peptides of several amino acids. Most peptides longer than four amino acids are not absorbed. Absorption into the intestinal absorptive cells is not the end. There, most of the peptides are broken into single amino acids.
Absorption of the amino acids and their derivatives into which dietary protein is degraded is done by the gastrointestinal tract. The absorption rates of individual amino acids are highly dependent on the protein source; for example, the digestibilities of many amino acids in humans, the difference between soy and milk proteins and between individual milk proteins, beta-lactoglobulin and casein. For milk proteins, about 50% of the ingested protein is absorbed between the stomach and the jejunum and 90% is absorbed by the time the digested food reaches the ileum. Biological value (BV) is a measure of the proportion of absorbed protein from a food which becomes incorporated into the proteins of the organism's body.
=== Newborn ===
Newborns of mammals are exceptional in protein digestion and assimilation in that they can absorb intact proteins at the small intestine. This enables passive immunity, i.e., transfer of immunoglobulins from the mother to the newborn, via milk.
== Dietary requirements ==
Considerable debate has taken place regarding issues surrounding protein intake requirements. The amount of protein required in a person's diet is determined in large part by overall energy intake, the body's need for nitrogen and essential amino acids, body weight and composition, rate of growth in the individual, physical activity level, the individual's energy and carbohydrate intake, and the presence of illness or injury. Physical activity and exertion as well as enhanced muscular mass increase the need for protein. Requirements are also greater during childhood for growth and development, during pregnancy, or when breastfeeding in order to nourish a baby or when the body needs to recover from malnutrition or trauma or after an operation.
== Dietary recommendations ==
According to US & Canadian Dietary Reference Intake guidelines, women ages 19–70 need to consume 46 grams of protein per day while men ages 19–70 need to consume 56 grams of protein per day to minimize risk of deficiencies. These Recommended Dietary Allowances (RDAs) were calculated based on 0.8 grams protein per kilogram body weight and average body weights of 57 kg (126 pounds) and 70 kg (154 pounds), respectively. However, this recommendation is based on structural requirements but disregards use of protein for energy metabolism. This requirement is for a normal sedentary person. In the United States, average protein consumption is higher than the RDA. According to results of the National Health and Nutrition Examination Survey (NHANES 2013–2014), average protein consumption for women ages 20 and older was 69.8 grams and for men 98.3 grams/day. According to research from Harvard University, the National Academy of Medicine suggests that adults should consume at least 0.8 grams of protein per kilogram of body weight daily, which is roughly equivalent to a little more than 7 grams for every 20 pounds of body weight. This recommendation is widely accepted by health professionals as a guideline for maintaining muscle mass, supporting metabolic functions, and promoting overall health.
=== Active people ===
Several studies have concluded that active people and athletes may require elevated protein intake (compared to 0.8 g/kg) due to increase in muscle mass and sweat losses, as well as need for body repair and energy source. Indeed, it has been shown that protein contribute around 3% of the total energy expenditure during exercise. Suggested amounts vary from 1.2 to 1.4 g/kg for those doing endurance exercise to as much as 1.6-1.8 g/kg for strength exercise and up to 2.0 g/kg/day for older people, while a proposed maximum daily protein intake would be approximately 25% of energy requirements i.e. approximately 2 to 2.5 g/kg. However, many questions still remain to be resolved.
In addition, some have suggested that athletes using restricted-calorie diets for weight loss should further increase their protein consumption, possibly to 1.8–2.0 g/kg, in order to avoid loss of lean muscle mass.
=== Aerobic exercise protein needs ===
Endurance athletes differ from strength-building athletes in that endurance athletes do not build as much muscle mass from training as strength-building athletes do. Research suggests that individuals performing endurance activity require more protein intake than sedentary individuals so that muscles broken down during endurance workouts can be repaired. Although the protein requirement for athletes still remains controversial (for instance see Lamont, Nutrition Research Reviews, pages 142 - 149, 2012), research does show that endurance athletes can benefit from increasing protein intake because the type of exercise endurance athletes participate in still alters the protein metabolism pathway. The overall protein requirement increases because of amino acid oxidation in endurance-trained athletes. Endurance athletes who exercise over a long period (2–5 hours per training session) use protein as a source of 5–10% of their total energy expended. Therefore, a slight increase in protein intake may be beneficial to endurance athletes by replacing the protein lost in energy expenditure and protein lost in repairing muscles. One review concluded that endurance athletes may increase daily protein intake to a maximum of 1.2–1.4 g per kg body weight.
=== Anaerobic exercise protein needs ===
Research also indicates that individuals performing strength training activity require more protein than sedentary individuals. Strength-training athletes may increase their daily protein intake to a maximum of 1.4–1.8 g per kg body weight to enhance muscle protein synthesis, or to make up for the loss of amino acid oxidation during exercise. Many athletes maintain a high-protein diet as part of their training. In fact, some athletes who specialize in anaerobic sports (e.g., weightlifting) believe a very high level of protein intake is necessary, and so consume high protein meals and also protein supplements.
=== Special populations ===
==== Protein allergies ====
A food allergy is an abnormal immune response to proteins in food. The signs and symptoms may range from mild to severe. They may include itchiness, swelling of the tongue, vomiting, diarrhea, hives, trouble breathing, or low blood pressure. These symptoms typically occurs within minutes to one hour after exposure. When the symptoms are severe, it is known as anaphylaxis. The following eight foods are responsible for about 90% of allergic reactions: cow's milk, eggs, wheat, shellfish, fish, peanuts, tree nuts and soy.
==== Chronic kidney disease ====
While there is no conclusive evidence that a high protein diet can cause chronic kidney disease, there is a consensus that people with this disease should decrease consumption of protein. According to one 2009 review updated in 2018, people with chronic kidney disease who reduce protein consumption have less likelihood of progressing to end stage kidney disease. Moreover, people with this disease while using a low protein diet (0.6 g/kg/d - 0.8 g/kg/d) may develop metabolic compensations that preserve kidney function, although in some people, malnutrition may occur.
==== Phenylketonuria ====
Individuals with phenylketonuria (PKU) must keep their intake of phenylalanine – an essential amino acid – extremely low to prevent a mental disability and other metabolic complications. Phenylalanine is a component of the artificial sweetener aspartame, so people with PKU need to avoid low calorie beverages and foods with this ingredient.
== Excess consumption ==
The U.S. and Canadian Dietary Reference Intake review for protein concluded that there was not sufficient evidence to establish a Tolerable upper intake level, i.e., an upper limit for how much protein can be safely consumed.
When amino acids are in excess of needs, the liver takes up the amino acids and deaminates them, a process converting the nitrogen from the amino acids into ammonia, further processed in the liver into urea via the urea cycle. Excretion of urea occurs via the kidneys. Other parts of the amino acid molecules can be converted into glucose and used for fuel. When food protein intake is periodically high or low, the body tries to keep protein levels at an equilibrium by using the "labile protein reserve" to compensate for daily variations in protein intake. However, unlike body fat as a reserve for future caloric needs, there is no protein storage for future needs.
Excessive protein intake may increase calcium excretion in urine, occurring to compensate for the pH imbalance from oxidation of sulfur amino acids. This may lead to a higher risk of kidney stone formation from calcium in the renal circulatory system. One meta-analysis reported no adverse effects of higher protein intakes on bone density. Another meta-analysis reported a small decrease in systolic and diastolic blood pressure with diets higher in protein, with no differences between animal and plant protein.
High protein diets have been shown to lead to an additional 1.21 kg of weight loss over a period of 3 months versus a baseline protein diet in a meta-analysis. Benefits of decreased body mass index as well as HDL cholesterol were more strongly observed in studies with only a slight increase in protein intake rather where high protein intake was classified as 45% of total energy intake. Detrimental effects to cardiovascular activity were not observed in short-term diets of 6 months or less. There is little consensus on the potentially detrimental effects to healthy individuals of a long-term high protein diet, leading to caution advisories about using high protein intake as a form of weight loss.
The 2015–2020 Dietary Guidelines for Americans (DGA) recommends that men and teenage boys increase their consumption of fruits, vegetables and other under-consumed foods, and that a means of accomplishing this would be to reduce overall intake of protein foods. The 2015–2020 DGA report does not set a recommended limit for the intake of red and processed meat. While the report acknowledges research showing that lower intake of red and processed meat is correlated with reduced risk of cardiovascular diseases in adults, it also notes the value of nutrients provided from these meats. The recommendation is not to limit intake of meats or protein, but rather to monitor and keep within daily limits the sodium (< 2300 mg), saturated fats (less than 10% of total calories per day), and added sugars (less than 10% of total calories per day) that may be increased as a result of consumption of certain meats and proteins. While the 2015 DGA report does advise for a reduced level of consumption of red and processed meats, the 2015–2020 DGA key recommendations recommend that a variety of protein foods be consumed, including both vegetarian and non-vegetarian sources of protein.
== Protein deficiency ==
Protein deficiency and malnutrition (PEM) can lead to a variety of ailments, including Intellectual disability and kwashiorkor. Symptoms of kwashiorkor include apathy, diarrhea, inactivity, failure to grow, flaky skin, fatty liver, and edema of the belly and legs. This edema is explained by the action of lipoxygenase on arachidonic acid to form leukotrienes and the normal functioning of proteins in fluid balance and lipoprotein transport.
PEM is fairly common worldwide in both children and adults and accounts for 6 million deaths annually. In the industrialized world, PEM is predominantly seen in hospitals, is associated with disease, or is often found in the elderly.
== See also ==
Azotorrhea
Biological value
Bodybuilding supplement
Leaf protein concentrate
Low-protein diet
Ninja diet
Protein bar
Protein toxicity
Single-cell protein
List of proteins in the human body
== References == | Wikipedia/Protein_(nutrient) |
Protein–protein interaction prediction is a field combining bioinformatics and structural biology in an attempt to identify and catalog physical interactions between pairs or groups of proteins. Understanding protein–protein interactions is important for the investigation of intracellular signaling pathways, modelling of protein complex structures and for gaining insights into various biochemical processes.
Experimentally, physical interactions between pairs of proteins can be inferred from a variety of techniques, including yeast two-hybrid systems, protein-fragment complementation assays (PCA), affinity purification/mass spectrometry, protein microarrays, fluorescence resonance energy transfer (FRET), and Microscale Thermophoresis (MST). Efforts to experimentally determine the interactome of numerous species are ongoing. Experimentally determined interactions usually provide the basis for computational methods to predict interactions, e.g. using homologous protein sequences across species. However, there are also methods that predict interactions de novo, without prior knowledge of existing interactions.
== Methods ==
Proteins that interact are more likely to co-evolve, therefore, it is possible to make inferences about interactions between pairs of proteins based on their phylogenetic distances. It has also been observed in some cases that pairs of interacting proteins have fused orthologues in other organisms. In addition, a number of bound protein complexes have been structurally solved and can be used to identify the residues that mediate the interaction so that similar motifs can be located in other organisms.
=== Phylogenetic profiling ===
The phylogenetic profile method is based on the hypothesis that if two or more proteins are concurrently present or absent across several genomes, then they are likely functionally related. Figure A illustrates a hypothetical situation in which proteins A and B are identified as functionally linked due to their identical phylogenetic profiles across 5 different genomes. The Joint Genome Institute provides an Integrated Microbial Genomes and Microbiomes database (JGI IMG) that has a phylogenetic profiling tool for single genes and gene cassettes.
=== Prediction of co-evolved protein pairs based on similar phylogenetic trees ===
It was observed that the phylogenetic trees of ligands and receptors were often more similar than due to random chance. This is likely because they faced similar selection pressures and co-evolved. This method uses the phylogenetic trees of protein pairs to determine if interactions exist. To do this, homologs of the proteins of interest are found (using a sequence search tool such as BLAST) and multiple-sequence alignments are done (with alignment tools such as Clustal) to build distance matrices for each of the proteins of interest. The distance matrices should then be used to build phylogenetic trees. However, comparisons between phylogenetic trees are difficult, and current methods circumvent this by simply comparing distance matrices. The distance matrices of the proteins are used to calculate a correlation coefficient, in which a larger value corresponds to co-evolution. The benefit of comparing distance matrices instead of phylogenetic trees is that the results do not depend on the method of tree building that was used. The downside is that difference matrices are not perfect representations of phylogenetic trees, and inaccuracies may result from using such a shortcut. Another factor worthy of note is that there are background similarities between the phylogenetic trees of any protein, even ones that do not interact. If left unaccounted for, this could lead to a high false-positive rate. For this reason, certain methods construct a background tree using 16S rRNA sequences which they use as the canonical tree of life. The distance matrix constructed from this tree of life is then subtracted from the distance matrices of the proteins of interest. However, because RNA distance matrices and DNA distance matrices have different scale, presumably because RNA and DNA have different mutation rates, the RNA matrix needs to be rescaled before it can be subtracted from the DNA matrices. By using molecular clock proteins, the scaling coefficient for protein distance/RNA distance can be calculated. This coefficient is used to rescale the RNA matrix.
=== Rosetta stone (gene fusion) method ===
The Rosetta Stone or Domain Fusion method is based on the hypothesis that interacting proteins are sometimes fused into a single protein. For instance, two or more separate proteins in a genome may be identified as fused into one single protein in another genome. The separate proteins are likely to interact and thus are likely functionally related. An example of this is the Human Succinyl coA Transferase enzyme, which is found as one protein in humans but as two separate proteins, Acetate coA Transferase alpha and Acetate coA Transferase beta, in Escherichia coli. In order to identify these sequences, a sequence similarity algorithm such as the one used by BLAST is necessary. For example, if we had the amino acid sequences of proteins A and B and the amino acid sequences of all proteins in a certain genome, we could check each protein in that genome for non-overlapping regions of sequence similarity to both proteins A and B. Figure B depicts the BLAST sequence alignment of Succinyl coA Transferase with its two separate homologs in E. coli. The two subunits have non-overlapping regions of sequence similarity with the human protein, indicated by the pink regions, with the alpha subunit similar to the first half of the protein and the beta similar to the second half. One limit of this method is that not all proteins that interact can be found fused in another genome, and therefore cannot be identified by this method. On the other hand, the fusion of two proteins does not necessitate that they physically interact. For instance, the SH2 and SH3 domains in the src protein are known to interact. However, many proteins possess homologs of these domains and they do not all interact.
=== Conserved gene neighborhood ===
The conserved neighborhood method is based on the hypothesis that if genes encoding two proteins are neighbors on a chromosome in many genomes, then they are likely functionally related. The method is based on an observation by Bork et al. of gene pair conservation across nine bacterial and archaeal genomes. The method is most effective in prokaryotes with operons as the organization of genes in an operon is generally related to function. For instance, the trpA and trpB genes in Escherichia coli encode the two subunits of the tryptophan synthase enzyme known to interact to catalyze a single reaction. The adjacency of these two genes was shown to be conserved across nine different bacterial and archaeal genomes.
=== Classification methods ===
Classification methods use data to train a program (classifier) to distinguish positive examples of interacting protein/domain pairs with negative examples of non-interacting pairs. Popular classifiers used are Random Forest Decision (RFD) and Support Vector Machines. RFD produces results based on the domain composition of interacting and non-interacting protein pairs. When given a protein pair to classify, RFD first creates a representation of the protein pair in a vector. The vector contains all the domain types used to train RFD, and for each domain type the vector also contains a value of 0, 1, or 2. If the protein pair does not contain a certain domain, then the value for that domain is 0. If one of the proteins of the pair contains the domain, then the value is 1. If both proteins contain the domain, then the value is 2. Using training data, RFD constructs a decision forest, consisting of many decision trees. Each decision tree evaluates several domains, and based on the presence or absence of interactions in these domains, makes a decision as to if the protein pair interacts. The vector representation of the protein pair is evaluated by each tree to determine if they are an interacting pair or a non-interacting pair. The forest tallies up all the input from the trees to come up with a final decision. The strength of this method is that it does not assume that domains interact independent of each other. This makes it so that multiple domains in proteins can be used in the prediction. This is a big step up from previous methods which could only predict based on a single domain pair. The limitation of this method is that it relies on the training dataset to produce results. Thus, usage of different training datasets could influence the results. A caveat of most methods is the lacks negative data, e.g non-interactions for proteins which can be overcome using topology-driven negative sampling.
=== Inference of interactions from homologous structures ===
This group of methods makes use of known protein complex structures to predict and structurally model interactions between query protein sequences. The prediction process generally starts by employing a sequence based method (e.g. Interolog) to search for protein complex structures that are homologous to the query sequences. These known complex structures are then used as templates to structurally model the interaction between query sequences. This method has the advantage of not only inferring protein interactions but also suggests models of how proteins interact structurally, which can provide some insights into the atomic level mechanism of that interaction. On the other hand, the ability for these methods to make a prediction is constrained by a limited number of known protein complex structures.
=== Association methods ===
Association methods look for characteristic sequences or motifs that can help distinguish between interacting and non-interacting pairs. A classifier is trained by looking for sequence-signature pairs where one protein contains one sequence-signature, and its interacting partner contains another sequence-signature. They look specifically for sequence-signatures that are found together more often than by chance. This uses a log-odds score which is computed as log2(Pij/PiPj), where Pij is the observed frequency of domains i and j occurring in one protein pair; Pi and Pj are the background frequencies of domains i and j in the data. Predicted domain interactions are those with positive log-odds scores and also having several occurrences within the database. The downside with this method is that it looks at each pair of interacting domains separately, and it assumes that they interact independently of each other.
=== Identification of structural patterns ===
This method builds a library of known protein–protein interfaces from the PDB, where the interfaces are defined as pairs of polypeptide fragments that are below a threshold slightly larger than the Van der Waals radius of the atoms involved. The sequences in the library are then clustered based on structural alignment and redundant sequences are eliminated. The residues that have a high (generally >50%) level of frequency for a given position are considered hotspots. This library is then used to identify potential interactions between pairs of targets, providing that they have a known structure (i.e. present in the PDB).
=== Bayesian network modelling ===
Bayesian methods integrate data from a wide variety of sources, including both experimental results and prior computational predictions, and use these features to assess the likelihood that a particular potential protein interaction is a true positive result. These methods are useful because experimental procedures, particularly the yeast two-hybrid experiments, are extremely noisy and produce many false positives, while the previously mentioned computational methods can only provide circumstantial evidence that a particular pair of proteins might interact.
=== Domain-pair exclusion analysis ===
The domain-pair exclusion analysis detects specific domain interactions that are hard to detect using Bayesian methods. Bayesian methods are good at detecting nonspecific promiscuous interactions and not very good at detecting rare specific interactions. The domain-pair exclusion analysis method calculates an E-score which measures if two domains interact. It is calculated as log(probability that the two proteins interact given that the domains interact/probability that the two proteins interact given that the domains don’t interact). The probabilities required in the formula are calculated using an Expectation Maximization procedure, which is a method for estimating parameters in statistical models. High E-scores indicate that the two domains are likely to interact, while low scores indicate that other domains form the protein pair are more likely to be responsible for the interaction. The drawback with this method is that it does not take into account false positives and false negatives in the experimental data.
=== Supervised learning problem ===
The problem of PPI prediction can be framed as a supervised learning problem. In this paradigm the known protein interactions supervise the estimation of a function that can predict whether an interaction exists or not between two proteins given data about the proteins (e.g., expression levels of each gene in different experimental conditions, location information, phylogenetic profile, etc.).
== Relationship to docking methods ==
The field of protein–protein interaction prediction is closely related to the field of protein–protein docking, which attempts to use geometric and steric considerations to fit two proteins of known structure into a bound complex. This is a useful mode of inquiry in cases where both proteins in the pair have known structures and are known (or at least strongly suspected) to interact, but since so many proteins do not have experimentally determined structures, sequence-based interaction prediction methods are especially useful in conjunction with experimental studies of an organism's interactome.
== See also ==
Interactome
Protein–protein interaction
Protein function prediction
Protein structure prediction
Protein structure prediction software
Gene prediction
Macromolecular docking
Protein–DNA interaction site predictor
Two-hybrid screening
FastContact
== References ==
== External links ==
Overview of protein interaction databases | Wikipedia/Protein–protein_interaction_prediction |
In molecular biology, fibrous proteins or scleroproteins are one of the three main classifications of protein structure (alongside globular and membrane proteins). Fibrous proteins are made up of elongated or fibrous polypeptide chains which form filamentous and sheet-like structures. This kind of protein can be distinguished from globular protein by its low solubility in water. In contrast, globular proteins are spherical and generally soluble in water, performing dynamic functions like enzymatic activity or transport. Such proteins serve protective and structural roles by forming connective tissue, tendons, bone matrices, and muscle fiber.
Fibrous proteins consist of many families including keratin, collagen, elastin, fibrin or spidroin. Collagen is the most abundant of these proteins which exists in vertebrate connective tissue including tendon, cartilage, and bone.
== Biomolecular structure ==
A fibrous protein forms long protein filaments, which are shaped like rods or wires. Fibrous proteins are structural or storage proteins that are typically inert and water-insoluble. A fibrous protein occurs as an aggregate due to hydrophobic side chains that protrude from the molecule.
A fibrous protein's peptide sequence often has limited residues with repeats; these can form unusual secondary structures, such as a collagen helix. The structures often feature cross-links between chains (e.g., cys-cys disulfide bonds between keratin chains).
Fibrous proteins tend not to denature as easily as globular proteins.
Miroshnikov et al. (1998) are among the researchers who have attempted to synthesize fibrous proteins.
== References ==
== External links ==
Scleroproteins at the U.S. National Library of Medicine Medical Subject Headings (MeSH) | Wikipedia/Fibrous_protein |
Protein function prediction methods are techniques that bioinformatics researchers use to assign biological or biochemical roles to proteins. These proteins are usually ones that are poorly studied or predicted based on genomic sequence data. These predictions are often driven by data-intensive computational procedures. Information may come from nucleic acid sequence homology, gene expression profiles, protein domain structures, text mining of publications, phylogenetic profiles, phenotypic profiles, and protein-protein interaction. Protein function is a broad term: the roles of proteins range from catalysis of biochemical reactions to transport to signal transduction, and a single protein may play a role in multiple processes or cellular pathways.
Generally, function can be thought of as, "anything that happens to or through a protein". The Gene Ontology Consortium provides a useful classification of functions, based on a dictionary of well-defined terms divided into three main categories of molecular function, biological process and cellular component. Researchers can query this database with a protein name or accession number to retrieve associated Gene Ontology (GO) terms or annotations based on computational or experimental evidence.
While techniques such as microarray analysis, RNA interference, and the yeast two-hybrid system can be used to experimentally demonstrate the function of a protein, advances in sequencing technologies have made the rate at which proteins can be experimentally characterized much slower than the rate at which new sequences become available. Thus, the annotation of new sequences is mostly by prediction through computational methods, as these types of annotation can often be done quickly and for many genes or proteins at once. The first such methods inferred function based on homologous proteins with known functions (homology-based function prediction). The development of context-based and structure based methods have expanded what information can be predicted, and a combination of methods can now be used to get a picture of complete cellular pathways based on sequence data. The importance and prevalence of computational prediction of gene function is underlined by an analysis of 'evidence codes' used by the GO database: as of 2010, 98% of annotations were listed under the code IEA (inferred from electronic annotation) while only 0.6% were based on experimental evidence.
== Homology-based methods ==
Proteins of similar sequence are usually homologous and thus have a similar function. Hence proteins in a newly sequenced genome are routinely annotated using the sequences of similar proteins in related genomes.
However, closely related proteins do not always share the same function. For example, the yeast Gal1 and Gal3 proteins are paralogs (73% identity and 92% similarity) that have evolved very different functions with Gal1 being a galactokinase and Gal3 being a transcriptional inducer.
There is no hard sequence-similarity threshold for "safe" function prediction; many proteins of barely detectable sequence similarity have the same function while others (such as Gal1 and Gal3) are highly similar but have evolved different functions. As a rule of thumb, sequences that are more than 30-40% identical are usually considered as having the same or a very similar function.
For enzymes, predictions of specific functions are especially difficult, as they only need a few key residues in their active site, hence very different sequences can have very similar activities. By contrast, even with sequence identity of 70% or greater, 10% of any pair of enzymes have different substrates; and differences in the actual enzymatic reactions are not uncommon near 50% sequence identity.
=== Sequence motif-based methods ===
The development of protein domain databases such as Pfam (Protein Families Database) allow us to find known domains within a query sequence, providing evidence for likely functions. The dcGO website contains annotations to both the individual domains and supra-domains (i.e., combinations of two or more successive domains), thus via dcGO Predictor allowing for the function predictions in a more realistic manner. Within protein domains, shorter signatures known as 'motifs' are associated with particular functions, and motif databases such as PROSITE ('database of protein domains, families and functional sites') can be searched using a query sequence. Motifs can, for example, be used to predict subcellular localization of a protein (where in the cell the protein is sent after synthesis). Short signal peptides direct certain proteins to a particular location such as the mitochondria, and various tools exist for the prediction of these signals in a protein sequence. For example, SignalP, which has been updated several times as methods are improved.
Thus, aspects of a protein's function can be predicted without comparison to other full-length homologous protein sequences.
== Structure-based methods ==
Because 3D protein structure is generally more well conserved than protein sequence, structural similarity is a good indicator of similar function in two or more proteins. Many programs have been developed to screen a known protein structure against the Protein Data Bank and report similar structures (for example, FATCAT (Flexible structure AlignmenT by Chaining AFPs (Aligned Fragment Pairs) with Twists), CE (combinatorial extension)) and DeepAlign (protein structure alignment beyond spatial proximity). Similarly, the main protein databases, such as UniProt, have built-in tools to search any given protein sequences against structure databases, and link to related proteins of known structure.
=== Protein structure prediction ===
To deal with the situation that many protein sequences have no solved structures, some function prediction servers such as RaptorX are also developed that can first predict the 3D model of a sequence and then use structure-based method to predict functions based upon the predicted 3D model. In many cases instead of the whole protein structure, the 3D structure of a particular motif representing an active site or binding site can be targeted. The Structurally Aligned Local Sites of Activity (SALSA) method, developed by Mary Jo Ondrechen and students, utilizes computed chemical properties of the individual amino acids to identify local biochemically active sites. Databases such as Catalytic Site Atlas have been developed that can be searched using novel protein sequences to predict specific functional sites.
=== Computational solvent mapping ===
One of the challenges involved in protein function prediction is discovery of the active site. This is complicated by certain active sites not being formed – essentially existing – until the protein undergoes conformational changes brought on by the binding of small molecules. Most protein structures have been determined by X-ray crystallography which requires a purified protein crystal. As a result, existing structural models are generally of a purified protein and as such lack the conformational changes that are created when the protein interacts with small molecules.
Computational solvent mapping utilizes probes (small organic molecules) that are computationally 'moved' over the surface of the protein searching for sites where they tend to cluster. Multiple different probes are generally applied with the goal being to obtain a large number of different protein-probe conformations. The generated clusters are then ranked based on the cluster's average free energy. After computationally mapping multiple probes, the site of the protein where relatively large numbers of clusters form typically corresponds to an active site on the protein.
This technique is a computational adaptation of 'wet lab' work from 1996. It was discovered that ascertaining the structure of a protein while it is suspended in different solvents and then superimposing those structures on one another produces data where the organic solvent molecules (that the proteins were suspended in) typically cluster at the protein's active site. This work was carried out as a response to realizing that water molecules are visible in the electron density maps produced by X-ray crystallography. The water molecules are interacting with the protein and tend to cluster at the protein's polar regions. This led to the idea of immersing the purified protein crystal in other solvents (e.g. ethanol, isopropanol, etc.) to determine where these molecules cluster on the protein. The solvents can be chosen based on what they approximate, that is, what molecule this protein may interact with (e.g. ethanol can probe for interactions with the amino acid serine, isopropanol a probe for threonine, etc.). It is vital that the protein crystal maintains its tertiary structure in each solvent. This process is repeated for multiple solvents and then this data can be used to try to determine potential active sites on the protein. Ten years later this technique was developed into an algorithm by Clodfelter et al.
== Genome context-based methods ==
Many of the newer methods for protein function prediction are not based on comparison of sequence or structure as above, but on some type of correlation between novel genes/proteins and those that already have annotations. Several methods have been developed to predict gene function on the local genomic or phylogenomic context and structure of genes:
Phylogenetic profiling is based on the observation that two or more proteins with the same pattern of presence or absence in many different genomes most likely have a functional link. Whereas homology-based methods can often be used to identify molecular functions of a protein, context-based approaches can be used to predict cellular function, or the biological process in which a protein acts. For example, proteins involved in the same metabolic pathway are likely to be present in a genome together or are absent altogether, suggesting that these genes work together in a functional context.
Operons are clusters of genes that are transcribed together. Based on co-transcription data but also based on the fact that the order of genes in operons is often conserved across many bacteria, indicates that they act together.
Gene fusion occurs when two or more genes encode two or more proteins in one organism and have, through evolution, combined to become a single gene in another organism (or vice versa for gene fission). This concept has been used, for example, to search all E. coli protein sequences for homology in other genomes and find over 6000 pairs of sequences with shared homology to single proteins in another genome, indicating potential interaction between each of the pairs. Because the two sequences in each protein pair are non-homologous, these interactions could not be predicted using homology-based methods.
== Gene expression and location-based methods ==
In prokaryotes, clusters of genes that are physically close together in the genome often conserve together through evolution, and tend to encode proteins that interact or are part of the same operon. Thus, chromosomal proximity also called the gene neighbour method can be used to predict functional similarity between proteins, at least in prokaryotes. Chromosomal proximity has also been seen to apply for some pathways in selected eukaryotic genomes, including Homo sapiens, and with further development gene neighbor methods may be valuable for studying protein interactions in eukaryotes.
Genes involved in similar functions are also often co-transcribed, so that an unannotated protein can often be predicted to have a related function to proteins with which it co-expresses. The guilt by association algorithms developed based on this approach can be used to analyze large amounts of sequence data and identify genes with expression patterns similar to those of known genes. Often, a guilt by association study compares a group of candidate genes (unknown function) to a target group (for example, a group of genes known to be associated with a particular disease), and rank the candidate genes by their likelihood of belonging to the target group based on the data. Based on recent studies, however, it has been suggested that some problems exist with this type of analysis. For example, because many proteins are multifunctional, the genes encoding them may belong to several target groups. It is argued that such genes are more likely to be identified in guilt by association studies, and thus predictions are not specific.
With the accumulation of RNA-seq data that are capable of estimating expression profiles for alternatively spliced isoforms, machine learning algorithms have also been developed for predicting and differentiating functions at the isoform level. This represents an emerging research area in function prediction, which integrates large-scale, heterogeneous genomic data to infer functions at the isoform level.
== Network-based methods ==
Guilt by association type algorithms may be used to produce a functional association network for a given target group of genes or proteins. These networks serve as a representation of the evidence for shared/similar function within a group of genes, where nodes represent genes/proteins and are linked to each other by edges representing evidence of shared function.
=== Integrated networks ===
Several networks based on different data sources can be combined into a composite network, which can then be used by a prediction algorithm to annotate candidate genes or proteins. For example, the developers of the bioPIXIE system used a wide variety of Saccharomyces cerevisiae (yeast) genomic data to produce a composite functional network for that species. This resource allows the visualization of known networks representing biological processes, as well as the prediction of novel components of those networks. Many algorithms have been developed to predict function based on the integration of several data sources (e.g. genomic, proteomic, protein interaction, etc.), and testing on previously annotated genes indicates a high level of accuracy. Disadvantages of some function prediction algorithms have included a lack of accessibility, and the time required for analysis. Faster, more accurate algorithms such as GeneMANIA (multiple association network integration algorithm) have however been developed in recent years and are publicly available on the web, indicating the future direction of function prediction.
== Tools and databases for protein function prediction ==
STRING: web tool that integrates various data sources for function prediction.
VisANT: Visual analysis of networks and integrative visual data-mining.
Mantis: A consensus-driven function prediction tool that dynamically integrates multiple reference databases.
== See also ==
Gene prediction
Protein–protein interaction prediction
Protein structure prediction
Structural genomics
Functional genomics
== References ==
== External links ==
The dcGO database
Protein Data Bank
Catalytic Site Atlas
RaptorX Server for model-assisted protein function prediction
Blast2GO, high-throughput tool for protein function prediction and functional annotation (webpage). | Wikipedia/Protein_function_prediction |
The Kjeldahl method or Kjeldahl digestion (Danish pronunciation: [ˈkʰelˌtɛˀl]) in analytical chemistry is a method for the quantitative determination of a sample's organic nitrogen plus ammonia/ammonium (NH3/NH4+). Without modification, other forms of inorganic nitrogen, for instance nitrate, are not included in this measurement. Using an empirical relation between Kjeldahl nitrogen and protein, it is an important method for indirectly quantifying protein content of a sample. This method was developed by the Danish chemist Johan Kjeldahl in 1883.
== Method ==
The method consists of heating a sample to 360–410 °C with concentrated sulfuric acid (H2SO4), which decomposes, or digests, the organic sample by oxidation to liberate the reduced nitrogen as stable ammonium sulfate: (NH4)2SO4.
Hot concentrated sulfuric acid oxidizes carbon (as bituminous coal) and sulfur (see sulfuric acid's reactions with carbon):
C + 2 H2SO4 → CO2 + 2 SO2 + 2 H2O
S + 2 H2SO4 → 3 SO2 + 2 H2O
Most of organic carbon and sulfur are decomposed and eliminated as gaseous CO2 and SO2.
In contrast to organic carbon and sulfur, the digested organic nitrogen remains preserved in the concentrated sulfuric acid as stable ammonium cation (NH+4). Ammonium does not further oxidize to gaseous N2, or a higher oxidized form of nitrogen, such as, e.g., N2O, NO, NO−2, NO2, or NO−3. If it was the case, the Kjeldahl method would not work.
Catalysts like selenium, Hg2SO4 or CuSO4 are often added to accelerate the digestion. Na2SO4 or K2SO4 is also added to increase the boiling point of H2SO4. Digestion is complete when the liquor clarifies with the release of fumes.
After complete digestion of the sample, to recover ammonia (NH3) from the ammonium sulfate, sodium hydroxide (NaOH) is first added to the residual sulfuric acid to neutralize it and to convert the soluble ammonium ion into volatile ammonia:
NH+4 + OH− → NH3 + H2O
Then, ammonia is recovered by distillation using the system below (right side of the figure).
The end of the condenser is dipped into a known volume of standard acid (i.e. acid of known concentration). A weak acid like boric acid (H3BO3) in excess of ammonia is often used. Standardized HCl, H2SO4 or some other strong acid can be used instead, but this is less commonplace. The sample solution is then distilled with a small excess of sodium hydroxide (NaOH). NaOH can also be added with a dropping funnel. NaOH converts dissolved ammonium (NH4+) to gaseous ammonia (NH3), which boils off the sample solution. Ammonia bubbles through the standard acid solution and reacts back to ammonium salts with the weak or strong acid.
Ammonium ion concentration in the acid solution, and thus the amount of nitrogen in the sample, is measured via titration. If boric acid (or some other weak acid) was used, direct acid–base titration is done with a strong acid of known concentration. HCl or H2SO4 can be used. Indirect back titration is used instead if strong acids were used to make the standard acid solution: a strong base of known concentration (like NaOH) is used to neutralize the solution. In this case, the amount of ammonia is calculated as the difference between the amount of HCl and NaOH. In the case of direct titration, it is not necessary to know the exact amount of weak acid (e.g. boric acid) because it does not interfere with the titration (it does have to be in excess of ammonia to trap it efficiently). Thus, one standard solution (e.g., HCl) is needed in the direct titration, while two are needed (e.g., HCl and NaOH) in the back-titration. One of the suitable indicators for these titration reactions is Tashiro's indicator.
In practice, this analysis is largely automated; specific catalysts accelerate the decomposition. Originally, the catalyst of choice was mercuric oxide. However, while it was very effective, health concerns resulted in its replacement with cupric sulfate. Cupric sulfate was less efficient than mercuric oxide and yielded lower protein results. It was soon supplemented with titanium dioxide, the approved catalyst in all protein analysis methods in the Official Methods and Recommended Practices of AOAC International.
== Applications ==
The Kjeldahl method's universality, precision and reproducibility have made it the internationally recognized method for estimating the protein content in foods. It is the standard method against which all other methods are judged. It is also used to assay soils, waste waters, fertilizers and other materials. However, it does not allow for determining the true protein content, as it measures non-protein nitrogen in addition to the nitrogen in proteins. This is evidenced by the 2007 pet food incident and the 2008 Chinese milk powder scandal, when melamine, a nitrogen-rich chemical, was added to raw materials to fake high protein contents. Also, different correction factors are needed for different proteins to account for different amino acid sequences. Additional disadvantages, such as the need to use concentrated sulfuric acid at high temperature and the relatively long testing time (an hour or more), compare unfavorably with the Dumas method for measuring crude protein content.
=== Total Kjeldahl nitrogen ===
Total Kjeldahl nitrogen or TKN is the sum of nitrogen bound in organic substances, nitrogen in ammonia (NH3-N) and in ammonium (NH4+-N) in the chemical analysis of soil, water, or waste water (e.g. sewage treatment plant effluent).
Today, TKN is a required parameter for regulatory reporting at many treatment plants and for monitoring plant operations.
=== Conversion factors ===
TKN is often used as a surrogate for protein in food samples. The conversion from TKN to protein depends on the type of protein present in the sample and what fraction of the protein is composed of nitrogenous amino acids, like arginine and histidine. However, the range of conversion factors is relatively narrow. Example conversion factors, known as N factors, for foods range from 6.38 for dairy and 6.25 for meat, eggs, maize (corn) and sorghum to 5.83 for most grains; 5.95 for rice, 5.70 for wheat flour, and 5.46 for peanuts. In practice, 6.25 is used for almost all food and feed regardless of applicability. The factor 6.25 is specifically required by US Nutrition Label regulations in the absence of another published factor.
=== Sensitivity ===
The Kjeldahl method is poorly sensitive in the original version. Other detection methods have been used to quantify NH4+ after mineralisation and distillation, achieving improved sensitivity: in-line generator of hydride coupled to a plasma atomic emission spectrometer (ICP-AES-HG, 10–25 mg/L), potentiometric titration (> 0.1 mg of nitrogen), zone capillary electrophoresis (1.5 μg/mL of nitrogen), and ion chromatography (0.5 μg/mL).
=== Limitations ===
Kjeldahl method does not apply to compounds containing nitrogen in nitro and azo groups and nitrogen present in rings (e.g. pyridine, quinoline, isoquinoline) as nitrogen of these compounds does not convert to ammonium sulfate under the conditions of this method.
== See also ==
Dumas method, another nitrogen analysis method
Devarda's alloy, a powerful reducing agent for nitrate analysis
Bicinchoninic acid assay, a colorimetric assay for protein-nitrogen
Combustion analysis another carbon, hydrogen and nitrogen analysis method
== References ==
== Bibliography ==
Wastewater Engineering: Treatment and Reuse, Metcalf & Eddy, McGraw-Hill Higher Education; 4th edition, 1 May 2002, ISBN 978-0071241403
== External links ==
Solutions for automation of the Kjeldahl method
Kjeldahl Proficiency guide | Wikipedia/Kjeldahl_method |
Kaede is a photoactivatable fluorescent protein naturally originated from a stony coral, Trachyphyllia geoffroyi. Its name means "maple" in Japanese. With the irradiation of ultraviolet light (350–400 nm), Kaede undergoes irreversible photoconversion from green fluorescence to red fluorescence.
Kaede is a homotetrameric protein with the size of 116 kDa. The tetrameric structure was deduced as its primary structure is only 28 kDa. This tetramerization possibly makes Kaede have a low tendency to form aggregates when fused to other proteins.
== Discovery ==
The property of photoconverted fluorescence Kaede protein was serendipitously discovered and first reported by Ando et al. in Proceedings of the United States National Academy of Sciences. An aliquot of Kaede protein was discovered to emit red fluorescence after being left on the bench and exposed to sunlight. Subsequent verification revealed that Kaede, which is originally green fluorescent, after exposure to UV light is photoconverted, becoming red fluorescent. It was then named Kaede.
== Properties ==
The property of photoconversion in Kaede is contributed by the tripeptide, His62-Tyr63-Gly64, that acts as a green chromophore that can be converted to red. Once Kaede is synthesized, a chromophore, 4-(p-hydroxybenzylidene)-5-imidazolinone, derived from the tripeptide mediates green fluorescence in Kaede. When exposed to UV, Kaede protein undergoes unconventional cleavage between the amide nitrogen and the α carbon (Cα) at His62 via a formal β-elimination reaction. Followed by the formation of a double bond between His62-Cα and –Cβ, the π-conjugation is extended to the imidazole ring of His62. A new chromophore, 2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolinone, is formed with the red-emitting property.
The cleavage of the tripeptide was analysed by SDS-PAGE analysis. Unconverted green Kaede shows one band at 28 kDa, whereas two bands at 18 kDa and 10 kDa are observed for converted red Kaede, indicating that the cleavage is crucial for the photoconversion.
A shifting of the absorption and emission spectrum in Kaede is caused by the cleavage of the tripeptide. Before the photoconversion, Kaede displays a major absorption wavelength maximum at 508 nm, accompanied with a slight shoulder at 475 nm. When it is excited at 480 nm, green fluorescence is emitted with a peak of 518 nm.
When Kaede is irradiated with UV or violet light, the major absorption peak shifts to 572 nm. When excited at 540 nm, Kaede showed an emission maximum at 582 nm with a shoulder at 627 nm and the 518-nm peak. Red fluorescence is emitted after this photoconversion.
The photoconversion in Kaede is irreversible. Exposure in dark or illumination at 570 nm cannot restore its original green fluorescence. A reduced fluorescence is observed in red, photoconverted Kaede when it is intensively exposed to 405 nm light, followed by partial recover after several minutes.
== Applications ==
As all other fluorescent proteins, Kaede can be the regional optical markers for gene expression and protein labeling for the study of cell behaviors.
One of the most useful applications is the visualization of neurons. Delineation of an individual neuron is difficult due to the long and thin processes which entangle with other neurons. Even when cultured neurons are labeled with fluorescent proteins, they are still difficult to identify individually because of the dense package.
In the past, such visualization could be done conventionally by filling neurons with Lucifer yellow or sulforhodamine, which is a laborious technique.[1] After the discovery of Kaede protein, it was found to be useful in delineating individual neurons. The neurons are transfected by Kaede protein cDNA, and are UV irradiated. The red, photoconverted Kaede protein has free diffusibility in the cell except for the nucleus, and spreads over the entire cell including dendrites and axon. This technique help disentangle the complex networks established in a dense culture. Besides, by labeling neurons with different colors by UV irradiating with different duration times, contact sites between the red and green neurons of interest are allowed to be visualized.
The ability of visualization of individual cells is also a powerful tool to identify the precise morphology and migratory behaviors of individual cells within living cortical slices. By Kaede protein, a particular pair of daughter cells in neighboring Kaede-positive cells in the ventricular zone of mouse brain slices can be followed. The cell-cell borders of daughter cells are visualized and the position and distance between two or more cells can be described.
As the change in the fluorescent colour is induced by UV light, marking of cells and subcellular structures is efficient even when only a partial photoconversion is induced.
== Advantages as an optical marker ==
Due to the special property of photo-switchable fluorescence, Kaede protein possesses several advantages as an optical cell marker.
After the photoconversion, the photoconverted Kaede protein emits bright and stable red fluorescence. This fluorescence can last for months without anaerobic conditions. As this red state of Kaede is bright and stable compared to the green state, and because the unconverted green Kaede emits very low intensity of red fluorescence, the red signals provides contrast.
Besides, before the photoconversion, Kaede emits bright green fluorescence which enables the visualization of the localization of the non-photoacivated protein. This is superior to other fluorescent proteins such as PA-GFP and KFP1, which only show low fluorescence before photoactivation.
In addition, as both green and red fluorescence of Kaede are excited by blue light at 480 nm for observation, this light will not induce photoconversion. Therefore, illumination lights for observation and photoconversion can be separated completely.
== Limitations ==
In spite of the usefulness in cell tracking and cell visualization of Kaede, there are some limitations.
Although Kaede will shift to red upon the exposure of UV or violet light and display a 2,000-fold increase in red-to-green fluorescence ratio, using both the red and green fluorescence bands can cause problems in multilabel experiments. The tetramerization of Kaede may disturb the localization and trafficking of fusion proteins. This limits the usefulness of Kaede as a fusion protein tag.
== Ecological significance ==
The photoconversion property of Kaede does not only contribute to the application on protein labeling and cell tracking, it is also responsible for the vast variation in the colour of stony corals, Trachyphyllia geoffroyi. Under sunlight, due to the photoconversion of Kaede, the tentacles and disks will turn red. As green fluorescent Kaede is synthesized continuously, these corals appear green again as more unconverted Kaede is created. By the different proportion of photoconverted and unconverted Kaede, great diversity of colour is found in corals.
== References ==
Tomura, M.; Yoshida, N.; Tanaka, J.; Karasawa, S.; Miwa, Y.; Miyawaki, A.; Kanagawa, O. (2008). "Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice". Proceedings of the National Academy of Sciences. 105 (31): 10871–10876. Bibcode:2008PNAS..10510871T. doi:10.1073/pnas.0802278105. PMC 2504797. PMID 18663225.
Dittrich, P. S.; Schäfer, S. P.; Schwille, P. (2005). "Characterization of the Photoconversion on Reaction of the Fluorescent Protein Kaede on the Single-Molecule Level". Biophysical Journal. 89 (5): 3446–3455. Bibcode:2005BpJ....89.3446D. doi:10.1529/biophysj.105.061713. PMC 1366840. PMID 16055537. | Wikipedia/Kaede_(protein) |
In molecular biology, SUMO (Small Ubiquitin-like Modifier) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. This process is called SUMOylation (pronounced soo-muh-lā-shun and sometimes written sumoylation). SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. In human proteins, there are over 53,000 SUMO binding sites, making it a substantial component of fundamental biology.
SUMO proteins are similar to ubiquitin and are considered members of the ubiquitin-like protein family. SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.
SUMO family members often have dissimilar names; the SUMO homologue in yeast, for example, is called SMT3 (suppressor of mif two 3). Several pseudogenes have been reported for SUMO genes in the human genome.
== Function ==
SUMO modification of proteins has many functions. Among the most frequent and best studied are protein stability, nuclear-cytosolic transport, and transcriptional regulation. Typically, only a small fraction of a given protein is SUMOylated and this modification is rapidly reversed by the action of deSUMOylating enzymes. SUMOylation of target proteins has been shown to cause a number of different outcomes including altered localization and binding partners. The SUMO-1 modification of RanGAP1 (the first identified SUMO substrate) leads to its trafficking from cytosol to nuclear pore complex. The SUMO modification of ninein leads to its movement from the centrosome to the nucleus. In many cases, SUMO modification of transcriptional regulators correlates with inhibition of transcription. One can refer to the GeneRIFs of the SUMO proteins, e.g. human SUMO-1, to find out more.
There are 4 confirmed SUMO isoforms in humans; SUMO-1, SUMO-2, SUMO-3 and SUMO-4. At the amino acid level, SUMO1 is about 50% identical to SUMO2. SUMO-2/3 show a high degree of similarity to each other and are distinct from SUMO-1. SUMO-4 shows similarity to SUMO-2/3 but differs in having a Proline instead of Glutamine at position 90. As a result, SUMO-4 isn't processed and conjugated under normal conditions, but is used for modification of proteins under stress-conditions like starvation. During mitosis, SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to the mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. One of the major SUMO conjugation products associated with mitotic chromosomes arose from SUMO-2/3 conjugation of topoisomerase II, which is modified exclusively by SUMO-2/3 during mitosis. SUMO-2/3 modifications seem to be involved specifically in the stress response. SUMO-1 and SUMO-2/3 can form mixed chains, however, because SUMO-1 does not contain the internal SUMO consensus sites found in SUMO-2/3, it is thought to terminate these poly-SUMO chains.
Serine 2 of SUMO-1 is phosphorylated, raising the concept of a 'modified modifier'.
=== DNA damage response ===
Cellular DNA is regularly exposed to DNA damaging agents. A DNA damage response (DDR) that is well regulated and intricate is usually employed to deal with the potential deleterious effects of the damage. When DNA damage occurs, SUMO protein has been shown to act as a molecular glue to facilitate the assembly of large protein complexes in repair foci. Also, SUMOylation can alter a protein's biochemical activities and interactions. SUMOylation plays a role in the major DNA repair pathways of base excision repair, nucleotide excision repair, non-homologous end joining and homologous recombinational repair. SUMOylation also facilitates error prone translation synthesis.
== Structure ==
SUMO proteins are small; most are around 100 amino acids in length and 12 kDa in mass. The exact length and mass varies between SUMO family members and depends on which organism the protein comes from. Although SUMO has very little sequence identity with ubiquitin (less than 20%) at the amino acid level, it has a nearly identical structural fold. SUMO protein has a unique N-terminal extension of 10-25 amino acids which other ubiquitin-like proteins do not have. This N-terminal is found related to the formation of SUMO chains.
The structure of human SUMO1 is depicted on the right. It shows SUMO1 as a globular protein with both ends of the amino acid chain (shown in red and blue) sticking out of the protein's centre. The spherical core consists of an alpha helix and a beta sheet. The diagrams shown are based on an NMR analysis of the protein in solution.
== Prediction of SUMO attachment ==
Most SUMO-modified proteins contain the tetrapeptide consensus motif Ψ-K-x-D/E where Ψ is a hydrophobic residue, K is the lysine conjugated to SUMO, x is any amino acid (aa), D or E is an acidic residue. Substrate specificity appears to be derived directly from Ubc9 and the respective substrate motif. Currently available prediction programs are:
SUMOplot - online free access software developed to predict the probability for the SUMO consensus sequence (SUMO-CS) to be engaged in SUMO attachment. The SUMOplot score system is based on two criteria: 1) direct amino acid match to the SUMO-CS observed and shown to bind Ubc9, and 2) substitution of the consensus amino acid residues with amino acid residues exhibiting similar hydrophobicity. SUMOplot has been used in the past to predict Ubc9 dependent sites.
seeSUMO - uses random forests and support vector machines trained on the data collected from the literature
SUMOsp - uses PSSM to score potential SUMOylation peptide sites. It can predict sites followed the ψKXE motif and unusual SUMOylation sites contained other non-canonical motifs.
JASSA - online free access predictor of SUMOylation sites (classical and inverted consensus) and SIMs (SUMO interacting motif). JASSA uses a scoring system based on a Position Frequency Matrix derived from the alignment of experimental SUMOylation sites or SIMs. Novel features were implemented towards a better evaluation of the prediction, including identification of database hits matching the query sequence and representation of candidate sites within the secondary structural elements and/or the 3D fold of the protein of interest, retrievable from deposited PDB files.
SumoPred-PLM or SUMOylation site Prediction using Protein Language Model - An AI deep learning utility to predict based on known biological rules around SUMO2 and SUMO3 binding in human proteins incorporating knowledge from a separate pretrained PLM tool developed previously in 2021 by Elnaggar et al. known as ProtT5-XL-UniRef50. Such collaboration between multidisciplinary AI tools is becoming common practice.
== SUMO attachment (SUMOylation) ==
SUMO attachment to its target is similar to that of ubiquitin (as it is for the other ubiquitin-like proteins such as NEDD 8). The SUMO precursor has some extra amino acids that need to be removed, therefore a C-terminal peptide is cleaved from the SUMO precursor by a protease (in human these are the SENP proteases or Ulp1 in yeast) to reveal a di-glycine motif. The obtained SUMO then becomes bound to an E1 enzyme (SUMO Activating Enzyme (SAE)) which is a heterodimer (subunits SAE1 and SAE2). It is then passed to an E2, which is a conjugating enzyme (Ubc9). Finally, one of a small number of E3 ligating proteins attaches it to the protein.
In budding yeast, there are four SUMO E3 proteins, Cst9, Mms21, Siz1 and Siz2. While in ubiquitination an E3 is essential to add ubiquitin to its target, evidence suggests that the E2 is sufficient in SUMOylation as long as the consensus sequence is present. It is thought that the E3 ligase promotes the efficiency of SUMOylation and in some cases has been shown to direct SUMO conjugation onto non-consensus motifs. E3 enzymes can be largely classed into PIAS proteins, such as Mms21 (a member of the Smc5/6 complex) and Pias-gamma and HECT proteins. On Chromosome 17 of the human genome, SUMO2 is near SUMO1+E1/E2 and SUMO2+E1/E2, among various others. Some E3's, such as RanBP2, however, are neither.
Recent evidence has shown that PIAS-gamma is required for the SUMOylation of the transcription factor yy1 but it is independent of the zinc-RING finger (identified as the functional domain of the E3 ligases). SUMOylation is reversible and is removed from targets by specific SUMO proteases. In budding yeast, the Ulp1 SUMO protease is found bound at the nuclear pore, whereas Ulp2 is nucleoplasmic. The distinct subnuclear localisation of deSUMOylating enzymes is conserved in higher eukaryotes.
== DeSUMOylation ==
SUMO can be removed from its substrate, which is called deSUMOylation. Specific proteases mediate this procedure (SENP in human or Ulp1 and Ulp2 in yeast).
In yeast, SMT3 encodes the SUMO protein, and SUMO E3 ligase attaches SUMO to target proteins. In cell cycle regulation, the base case is that SUMO ligation is constantly taking place, leading to polySUMOylation of eligible target proteins. This is countered by the SUMO protease Ulp2 which cleaves polySUMO groups, leaving the protein in a monoSUMOylated state. As shown in the Biorender figure, there is a feedback mechanism in which ULP2 maintains the monoSUMOylated state by passively and diligently cleaving SUMO such that the polySUMOyated state is never stabilized enough to be acted upon by downstream actors. This deSUMOylation is critical to prevent precocious advancement of the cell cycle as discussed in several studies.
The deSUMOylation may be arrested by the inhibitory phosphorylation of the Ulp2 SUMO protease by the Polo-like kinase Cdc5. By inhibiting the deSUMOylation of Ulp2, polySUMOylation is then promoted as the new stable state of target proteins, which are often but not always bound to other proteins in order to regulate major changes within the cell. Cdc5 is countered by the Rts1-PP2A phosphatase, which maintains the active state of the Ulp2 SUMO protease by removing the phosphate group added by Cdc5 kinase.
The consequence of disrupting the counteracting deSUMOylation is the following: First, the targeted protein becomes polySUMOylated. Second, SUMO Targeted Ubiquitin Ligase, or STUbL, (SLX5 or SLX8 in the case of yeast) may then bind the polySUMOylated target and attach Ubiquitin groups (often polyUbiquitinating the already polySUMOylated protein). Third, segregases such as Cdc48 may then dissociate the SUMOylated and ubiquitinated target from its bound protein. Fourth, while the unbound protein it had been bound to is now free to do what it could not do while bound, the dissociated protein may then be degraded by the canonical Ubiquitin-Proteasome pathway.
As studied with budding yeast, in the case of Tof2-Cdc14, Cdc14 release from the nucleolus allows the Mitotic Exit Network to commence, but it is regulated by the binding of Tof2, a protein subject to SUMOylation. Likewise, the Cohesin protein which binds sister chromatids in metaphase is able to be targeted by SUMOylation to allow the Cdc48 segregase to separate Cohesin and allow sister chromatid separation in early anaphase.
In research as is often the case, scientists test drugs known to have significant effects on living systems; one such example is Rapamycin (known in pharmaceuticals as Sirolimus), the well-known inhibitor of mechanistic Target of Rapamycin, or mTOR. With respect to SUMOylation, Rapamycin may be thought of as having a "Sledge Hammer" effect, in which the drug promotes cellular autophagy, part of which includes broad-spectrum promotion of nonspecific SUMOylation for many proteins. This may be beneficial in some circumstances as it supports the breakdown of accumulated waste products.
The importance of these studies in models such as yeast lies in their potential to inform scientists in the research and development of precise biomedical interventions that can translate to the improvement of human health in an array of clinical aspects.
== Role in Human Pathology ==
SUMO protein is implicated in the etiology of many biomedical disease states not limited to: cancer, atherosclerosis, cardiovascular disease, neurodegenerative disease, diabetes, liver disease, intestinal disorders, and even infectious disease.
In the case of the well-studied cancer tumor suppressor known as p53, there is a regulatory ubiquitin ligase protein in humans called Mouse Double Minute 2 protein, or MDM2, which acts to remove p53 from the cell. MDM2 regulates itself through self-ubiquitination by way of a RING finger domain, targeting itself for proteasomal destruction. When it is SUMOylated at the RING finger domain, MDM2 no longer limits its own function in the cell. When protected from itself, it likewise ubiquitinates p53, marking the protective p53 for destruction instead, whose absence is understood to promote cancer. Here again, the base case is SUMOylation, which is actively being undone by newly discovered SUMO protease SUSP4 and also by the SUMO protease interaction of SMT3IP1/SENP3 which is understood to deSUMOylate both MDM2 and p53. One of the ways p53 functions is as a DNA-binding tetramer; interestingly, SUMOylation of p53 delocalizes it from the nucleus, which prevents such activity. The critical nature of p53 cannot be overstated: in fact, if a human carries only one non-functioning copy of p53, it results in a deadly cancer prognosis known as Li-Fraumeni syndrome. Beyond p53, in cancer, many oncogenes and tumor suppressors have been discovered to be SUMOylated in order for the cancer to progress or not, with each SUMOylation event having one of a variety of effects. When IκB is SUMOylated, the SUMO post-translational modification outcompetes ubiquitination, protecting it from degradation, and by extension, the transcription factor NF-κB is bound in a complex with IκB, preventing the expression of genes that may otherwise cause cells with DNA damage to apoptose. In hypoxic conditions as arise in some cancers, HIF-1α, which is usually SUMOylated followed by subsequent ubiquitination and degradation through the von Hippel-Lindau tumor suppressor's ubiquitin ligase activity, is instead deSUMOylated thereby promoting survival of the tumorigenic cells. The fallout from deSUMOylation of HIF-1α includes promotion of MMPs which are understood to contribute to the progression of EMT, a hallmark of cancer.
In atherosclerosis, both p53 and ERK5 are SUMOylated by the stimulus of disturbed blood flow. The stimulus is transduced by the activation of a serine/threonine kinsase called p90RSK, which phosphorylates the human SUMO protease SENP2 at the throenine amino acid residue 368. That phosphorylation is sufficient for the delocalization of the SENP2 from the nucleus. The effects of this phosphorylation-dependent SENP2 inhibition by nuclear export include the SUMOylation of p53 which leads to endothelial cell apoptosis, and SUMOylation of ERK5 which leads to inflammation. Nuclear export of SENP2 additionally downregulates endothelial nitric oxide synthase, eNOS while it upregulates inflammatory adhesion molecules. As eNOS is required for healthy vascular physiology, pathological oxidative stress ensues in vascular endothelial cells. With the oxidative stress comes subsequent accumulation of cellular lipids; this results in the inflammatory foamy cell state that is typified by atherosclerosis as well as the similarly inflammatory myelin-laden macrophages known to produce chronic inflammation in SCI.
In cardiovascular disease, many proteins are subject to SUMOylation. To say SUMOylation itself is bad or good regarding this or any other class of disease is to overlook the role of the multiple proteins in question. One common denominator among many conditions is fibrosis; in myocardial fibrosis, PPARγ1 is understood to have a role in regulating expression of some key genes, and its transcriptional activity is generally inhibited by SUMOylation. Therefore, one possible therapeutic intervention in the case of cardiac hypertrophy may be countering the SUMOylation of PPARγ1.
In neurodegenerative disease, we often observe pathological accumulation of proteins. Inclusion bodies form when for example, the Huntington's disease protein, aptly named Huntingtin, accumulates and folds into a form which is impervious to the proteasome. In Huntington's disease, sufficient SUMOylation of the anomalous Huntingtin protein prior to such refolding could perhaps delay the progression of the disease state by enabling timely destruction of the protein while the polypeptide chains are still accessible to the protease subunits within the proteasome. Other accumulating proteins which threaten neurodegenerative disorders include α-synuclein (associated with Parkinson's) and Amyloid β (associated with Alzheimer's), and if acted upon early enough, disease could perhaps be better mitigated.
== Human SUMO proteins ==
SUMO1
SUMO2
SUMO3
SUMO4
NSMCE2
== Role in protein purification ==
Recombinant proteins expressed in E. coli may fail to fold properly, instead forming aggregates and precipitating as inclusion bodies. This insolubility may be due to the presence of codons read inefficiently by E. coli, differences in eukaryotic and prokaryotic ribosomes, or lack of appropriate molecular chaperones for proper protein folding. In order to purify such proteins it may be necessary to fuse the protein of interest with a solubility tag such as SUMO or MBP (maltose-binding protein) to increase the protein's solubility. SUMO can later be cleaved from the protein of interest using a SUMO-specific protease such as Ulp1 peptidase.
== See also ==
Ubiquitin
Prokaryotic ubiquitin-like protein
== References ==
=== Further reading ===
== External links ==
SUMO1 homology group from HomoloGene
human SUMO proteins on ExPASy: SUMO1 SUMO2 SUMO3 SUMO4
Programs for prediction SUMOylation:
SUMOplot Analysis Program — predicts and scores SUMOylation sites in your protein (by Abgent)
seeSUMO - prediction of SUMOylation sites
SUMOsp - prediction of SUMOylation sites
JASSA - Predicts and scores SUMOylation sites and SIM (SUMO interacting motif)
=== Research laboratories === | Wikipedia/SUMO_protein |
Protein acetylation (and deacetylation) are acetylation reactions that occur within living cells as drug metabolism, by enzymes in the liver and other organs (e. g., the brain). Pharmaceuticals frequently employ acetylation to enable such esters to cross the blood–brain barrier (and placenta), where they are deacetylated by enzymes (carboxylesterases) in a manner similar to acetylcholine. Examples of acetylated pharmaceuticals are diacetylmorphine (heroin), acetylsalicylic acid (aspirin), THC-O-acetate, and diacerein. Conversely, drugs such as isoniazid are acetylated within the liver during drug metabolism. A drug that depends on such metabolic transformations in order to act is termed a prodrug.
Acetylation is an important modification of proteins in cell biology; and proteomics studies have identified thousands of acetylated mammalian proteins. Acetylation occurs as a co-translational and post-translational modification of proteins, for example, histones, p53, and tubulins. Among these proteins, chromatin proteins and metabolic enzymes are highly represented, indicating that acetylation has a considerable impact on gene expression and metabolism. In bacteria, 90% of proteins involved in central metabolism of Salmonella enterica are acetylated.
== N-terminal acetylation ==
N-terminal acetylation is one of the most common co-translational covalent modifications of proteins in eukaryotes, and it is crucial for the regulation and function of different proteins. N-terminal acetylation plays an important role in the synthesis, stability and localization of proteins. About 85% of all human proteins and 68% in yeast are acetylated at their Nα-terminus. Several proteins from prokaryotes and archaea are also modified by N-terminal acetylation.
N-terminal Acetylation is catalyzed by a set of enzyme complexes, the N-terminal acetyltransferases (NATs). NATs transfer an acetyl group from acetyl-coenzyme A (Ac-CoA) to the α-amino group of the first amino acid residue of the protein. Different NATs are responsible for the acetylation of nascent protein N-terminal, and the acetylation was found to be irreversible so far.
=== N-terminal acetyltransferases ===
To date, seven different NATs have been found in humans - NatA, NatB, NatC, NatD, NatE, NatF and NatH. Each of these different enzyme complexes is specific for different amino acids or amino acid sequences which is shown in the following table.
Table 1. The Composition and Substrate specificity of NATs.
==== NatA ====
NatA is composed of two subunits, the catalytic subunit Naa10 and the auxiliary subunit Naa15. NatA subunits are more complex in higher eukaryotes than in lower eukaryotes. In addition to the genes NAA10 and NAA15, the mammal-specific genes NAA11 and NAA16, make functional gene products, which form different active NatA complexes. Four possible hNatA catalytic-auxiliary dimers are formed by these four proteins. However, Naa10/Naa15 is the most abundant NatA.
NatA acetylates Ser, Ala-, Gly-, Thr-, Val- and Cys N-termini after the initiator methionine is removed by methionine amino-peptidases. These amino acids are more frequently expressed in the N-terminal of proteins in eukaryotes, so NatA is the major NAT corresponding to the whole number of its potential substrates.
Several different interaction partners are involved in the N-terminal acetylation by NatA. Huntingtin interacting protein K (HYPK) interacts with hNatA on the ribosome to affect the N-terminal acetylation of a subset of NatA substrates. Subunits hNaa10 and hNaa15 will increase the tendency for aggregation of Huntingtin if HYPK is depleted. Hypoxia-inducible factor (HIF)-1α has also been found to interact with hNaa10 to inhibit hNaa10-mediated activation of β-catenin transcriptional activity.
==== NatB ====
NatB complexes are composed of the catalytic subunit Naa20p and the auxiliary subunit Naa25p, which are both found in yeast and humans. In yeast, all the NatB subunits are ribosome-associated; but in humans, NatB subunits are both found to be ribosome-associated and non-ribosomal form. NatB acetylates the N-terminal methionine of substrates starting with Met-Glu-, Met-Asp-, Met-Asn- or Met-Gln- N termini.
==== NatC ====
NatC complex consists of one catalytic subunit Naa30p and two auxiliary subunits Naa35p and Naa38p. All three subunits are found on the ribosome in yeast, but they are also found in non-ribosomal NAT forms like Nat2. NatC complex acetylates the N-terminal methionine of substrates Met-Leu-, Met-Ile-, Met-Trp- or Met-Phe N-termini.
==== NatD ====
NatD is only composed with the catalytic unit Naa40p and Naa40p and it is conceptually different form the other NATs. At first, only two substrates, H2A and H4 have been identified in yeast and humans. Secondly, the substrate specificity of Naa40p lies within the first 30-50 residues which are quite larger than the substrate specificity of other NATs. The acetylation of histones by NatD is partially associate with ribosomes and the amino acids substrates are the very N-terminal residues, which makes it different from lysine N-acetyltransferases (KATs).
==== NatE ====
NatE complex consists with subunit Naa50p and two NatA subunits, Naa10p and Naa15p. The N terminus of Naa50p substrates is different from those acetylated by the NatA activity of Naa10p. NAA50 in plants is essential to control plant growth, development, and stress responses and NAA50 function is highly conserved between humans and plants.
==== NatF ====
NatF is a NAT that is composed of the Naa60 enzyme. Initially, it was thought that NatF was only found in higher eukaryotes, since it was absent from yeast. However, it was later found that Naa60 is found throughout the eukaryotic domain, but was secondarily lost in the fungi lineage. Compared to yeast, NatF contributes to the higher abundance of N-terminal acetylation in humans. NatF complex acetylates the N-terminal methionine of substrates Met-Lys-, Met-Leu-, Met-Ile-, Met-Trp- and Met-Phe N termini which are partly overlapping with NatC and NatE. NatF has been shown to have an organellar localization and acetylates cytosolic N-termini of transmembrane proteins. The organellar localization of Naa60 is mediated by its unique C-terminus, which consists of two alpha helices that peripherally associate with the membrane and mediate interactions with PI(4)P.
==== NAA80/NatH ====
NAA80/NatH is an N-terminal acetyltransferase that specifically acetylates the N-terminus of actin.
=== N-terminal acetylation function ===
==== Protein stability ====
N-terminal acetylation of proteins can affect protein stability, but the results and mechanism were not very clear until now. It was believed that N-terminal acetylation protects proteins from being degraded as Nα-acetylation N-termini were supposed to block N-terminal ubiquitination and subsequent protein degradation. However, several studies have shown that the N-terminal acetylated protein have a similar degradation rate as proteins with a non-blocked N-terminus.
==== Protein localization ====
N-terminal acetylation has been shown that it can steer the localization of proteins. Arl3p is one of the ‘Arf-like’ (Arl) GTPases, which is crucial for the organization of membrane traffic. It requires its Nα-acetyl group for its targeting to the Golgi membrane by the interaction with Golgi membrane-residing protein Sys1p. If the Phe or Tyr is replaced by an Ala at the N-terminal of Arl3p, it can no longer localized to the Golgi membrane, indicating that Arl3p needs its natural N-terminal residues which could be acetylated for proper localization.
==== Metabolism and apoptosis ====
Protein N-terminal acetylation has also been proved to relate with cell cycle regulation and apoptosis with protein knockdown experiments. Knockdown of the NatA or the NatC complex leads to the induction of p53-dependent apoptosis, which may indicate that the anti-apoptotic proteins were less or no longer functional because of reduced protein N-terminal acetylation. But in contrast, the caspase-2, which is acetylated by NatA, can interact with the adaptor protein RIP associated Ich-1/Ced-3 homologous protein with a death domain (RAIDD). This could activate caspase-2 and induce cell apoptosis.
==== Protein synthesis ====
Ribosome proteins play an important role in the protein synthesis, which could also be N-terminal acetylated. The N-terminal acetylation of the ribosome proteins may have an effect on protein synthesis. A decrease of 27% and 23% in the protein synthesis rate was observed with NatA and NatB deletion strains. A reduction of translation fidelity was observed in the NatA deletion strain and a defect in ribosome was noticed in the NatB deletion strain.
=== Cancer ===
NATs have been suggested to act as both onco-proteins and tumor suppressors in human cancers, and NAT expression may be increased and decreased in cancer cells. Ectopic expression of hNaa10p increased cell proliferation and up regulation of gene involved in cell survival proliferation and metabolism. Overexpression of hNaa10p was in the urinary bladder cancer, breast cancer and cervical carcinoma. But a high level expression of hNaa10p could also suppress tumor growth and a reduced level of expressed hNaa10p is associated with a poor prognosis, large tumors and more lymph node metastases.
Table 2. Overview of the expression of NatA subunits in various cancer tissues
== Lysine acetylation and deacetylation ==
Proteins are typically acetylated on lysine residues and this reaction relies on acetyl-coenzyme A as the acetyl group donor.
In histone acetylation and deacetylation, histone proteins are acetylated and deacetylated on lysine residues in the N-terminal tail as part of gene regulation. Typically, these reactions are catalyzed by enzymes with histone acetyltransferase (HAT) or histone deacetylase (HDAC) activity, although HATs and HDACs can modify the acetylation status of non-histone proteins as well.
The regulation of transcription factors, effector proteins, molecular chaperones, and cytoskeletal proteins by acetylation and deacetylation is a significant post-translational regulatory mechanism These regulatory mechanisms are analogous to phosphorylation and dephosphorylation by the action of kinases and phosphatases. Not only can the acetylation state of a protein modify its activity but there has been recent suggestion that this post-translational modification may also crosstalk with phosphorylation, methylation, ubiquitination, sumoylation, and others for dynamic control of cellular signaling. The regulation of tubulin protein is an example of this in mouse neurons and astroglia. A tubulin acetyltransferase is located in the axoneme, and acetylates the α-tubulin subunit in an assembled microtubule. Once disassembled, this acetylation is removed by another specific deacetylase in the cell cytosol. Thus axonemal microtubules, which have a long half-life, carry a "signature acetylation," which is absent from cytosolic microtubules that have a shorter half-life.
In the field of epigenetics, histone acetylation (and deacetylation) have been shown to be important mechanisms in the regulation of gene transcription. Histones, however, are not the only proteins regulated by posttranslational acetylation. The following are examples of various other proteins with roles in regulating signal transduction, whose activities are also affected by acetylation and deacetylation.
=== p53 ===
The p53 protein is a tumor suppressor that plays an important role in the signal transactions in cells, especially in maintaining the stability of the genome by preventing mutation. Therefore, it is also known as “the guardian of the genome". It also regulates the cell cycle and arrests cell growth by activating a regulator of the cell cycle, p21. Under severe DNA damage, it also initiates programmed cell death.The function of p53 is negatively regulated by oncoprotein Mdm2. Studies suggested that Mdm2 will form a complex with p53 and prevent it from binding to specific p53-responsive genes.
==== Acetylation of p53 ====
The acetylation of p53 is indispensable for its activation. It has been reported that the acetylation level of p53 will increase significantly when the cell undergoes stress. Acetylation sites have been observed on the DNA binding domain (K164 and K120) and the C terminus. Acetylation sites demonstrate significant redundancy: if only one acetylation site is inactivated by mutation to arginine, the expression of p21 is still observed. However, if multiple acetylation sites are blocked, the expression of p21 and the suppression of cell growth caused by p53 is completely lost. In addition, the acetylation of p53 prevents its binding to the repressor Mdm2 on DNA. In addition, it is suggested that the p53 acetylation is crucial for its transcription-independent proapoptotic functions. An acetylation site of the C-terminus was investigated by molecular dynamics simulations and circular dichroism spectroscopy, and it was suggested that the acetylation changes the structural ensemble of the C-terminus.
==== Implications for cancer therapy ====
Since the major function of p53 is tumor suppressor, the idea that activation of p53 is an appealing strategy for cancer treatment. Nutlin-3 is a small molecule designed to target p53 and Mdm2 interaction that kept p53 from deactivation. Reports also shown that the cancer cell under the Nutilin-3a treatment, acetylation of lys 382 was observed in the c-terminal of p53.
=== Microtubule ===
The structure of microtubules is long, hollow cylinder dynamically assembled from α/β-tubulin dimers. They play an essential role in maintaining the structure of the cell as well as cell processes, for example, movement of organelles. In addition, microtubule is responsible of forming mitotic spindle in eukaryotic cells to transport chromosomes in cell division.
==== Acetylation of tubulin ====
The acetylated residue of α-tubulin is K40, which is catalyzed by α-tubulin acetyl-transferase (α-TAT) in human. The acetylation of K40 on α-tubulin is a hallmark of stable microtubules. The active site residues D157 and C120 of α-TAT1 are responsible for the catalysis because of the shape complementary to α-Tubulin. In addition, some unique structural features such as β4-β5 hairpin, C-terminal loop, and α1-α2 loop regions are important for specific α-Tubulin molecular recognition. The reverse reaction of the acetylation is catalyzed by histone deacetylase 6.
==== Implications for cancer therapy ====
Since microtubules play an important role in cell division, especially in the G2/M phase of the cell cycle, attempts have been made to impede microtubule function using small molecule inhibitors, which have been successfully used in clinics as cancer therapies. For example, the vinca alkaloids and taxanes selectively bind and inhibit microtubules, leading to cell cycle arrest. The identification of the crystal structure of acetylation of α-tubulin acetyl-transferase (α-TAT) also sheds a light on the discovery of small molecule that could modulate the stability or de-polymerization of tubulin. In other words, by targeting α-TAT, it is possible to prevent the tubulin from acetylation and result in the destabilization of tubulin, which is a similar mechanism for tubulin destabilizing agents.
=== STAT3 ===
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is phosphorylated by receptor associated kinases, for example, Janus-family tyrosine kinases, and translocate to nucleus. STAT3 regulates several genes in response to growth factors and cytokines and play an important role in cell growth. Therefore, STAT3 facilitates oncogenesis in a variety of cell growth related pathways. On the other hand, it also play a role in the tumor suppressor.
==== Acetylation of STAT3 ====
The acetylation of Lys685 of STAT3 is important for STAT3 homo-dimerization, which is essential for the DNA-binding and the transcriptional activation of oncogenes. The acetylation of STAT3 is catalyzed by histone acetyltransferase p300, and reversed by type 1 histone deacetylase. The lysine acetylation of STAT3 is also elevated in cancer cells.
==== Therapeutic implications for cancer therapy ====
Since the acetylation of STAT3 is important for its oncogenic activity and the fact that the level of acetylated STAT3 is high in cancer cells, it is implied that targeting acetylated STAT3 for chemoprevention and chemotherapy is a promising strategy. This strategy is supported by treating resveratrol, an inhibitor of acetylation of STAT3, in cancer cell line reverses aberrant CpG island methylation.
== See also ==
Compendium of protein lysine acetylation
Glycosylation
Lipidation
Proteolysis
== References == | Wikipedia/Protein_acetylation |
Protein methylation is a type of post-translational modification featuring the addition of methyl groups to proteins. It can occur on the nitrogen-containing side-chains of arginine and lysine, but also at the amino- and carboxy-termini of a number of different proteins. In biology, methyltransferases catalyze the methylation process, activated primarily by S-adenosylmethionine.
Protein methylation has been most studied in histones, where the transfer of methyl groups from S-adenosyl methionine is catalyzed by histone methyltransferases. Histones that are methylated on certain residues can act epigenetically to repress or activate gene expression.
== Methylation by substrate ==
Multiple sites of proteins can be methylated. For some types of methylation, such as N-terminal methylation and prenylcysteine methylation, additional processing is required, whereas other types of methylation such as arginine methylation and lysine methylation do not require pre-processing.
=== Arginine ===
Arginine can be methylated once (monomethylated arginine) or twice (dimethylated arginine). Methylation of arginine residues is catalyzed by three different classes of protein arginine methyltransferases (PRMTs): Type I PRMTs (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8) attach two methyl groups to a single terminal nitrogen atom, producing asymmetric dimethylarginine (N G,N G-dimethylarginine). In contrast, type II PRMTs (PRMT5 and PRMT9) catalyze the formation of symmetric dimethylarginine with one methyl group on each terminal nitrogen (symmetric N G,N' G-dimethylarginine). Type I and II PRMTs both generate N G-monomethylarginine intermediates; PRMT7, the only known type III PRMT, produces only monomethylated arginine.
Arginine-methylation usually occurs at glycine and arginine-rich regions referred to as "GAR motifs", which is likely due to the enhanced flexibility of these regions that enables insertion of arginine into the PRMT active site. Nevertheless, PRMTs with non-GAR consensus sequences exist. PRMTs are present in the nucleus as well as in the cytoplasm. In interactions of proteins with nucleic acids, arginine residues are important hydrogen bond donors for the phosphate backbone — many arginine-methylated proteins have been found to interact with DNA or RNA.
Enzymes that facilitate histone acetylation as well as histones themselves can be arginine methylated. Arginine methylation affects the interactions between proteins and has been implicated in a variety of cellular processes, including protein trafficking, signal transduction and transcriptional regulation. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.
=== Lysine ===
Lysine can be methylated once, twice, or three times by lysine methyltransferases (PKMTs). Most lysine methyltransferases contain an evolutionarily conserved SET domain, which possesses S-adenosylmethionine-dependent methyltransferase activity, but are structurally distinct from other S-adenosylmethionine binding proteins. Lysine methylation plays a central part in how histones interact with proteins. Lysine methylation can be reverted by lysine demethylases (PKDMs).
Different SET domain-containing proteins possess distinct substrate specificities. For example, SET1, SET7 and MLL methylate lysine 4 of histone H3, whereas Suv39h1, ESET and G9a specifically methylate lysine 9 of histone H3. Methylation at lysine 4 and lysine 9 are mutually exclusive and the epigenetic consequences of site-specific methylation are diametrically opposed: Methylation at lysine 4 correlates with an active state of transcription, whereas methylation at lysine 9 is associated with transcriptional repression and heterochromatin. Other lysine residues on histone H3 and histone H4 are also important sites of methylation by specific SET domain-containing enzymes. Although histones are the prime target of lysine methyltransferases, other cellular proteins carry N-methyllysine residues, including elongation factor 1A and the calcium sensing protein calmodulin.
=== N-terminal methylation ===
Many eukaryotic proteins are post-translationally modified on their N-terminus. A common form of N-terminal modification is N-terminal methylation (Nt-methylation) by N-terminal methyltransferases (NTMTs). Proteins containing the consensus motif H2N-X-Pro-Lys- (where X can be Ala, Pro or Ser) after removal of the initiator methionine (iMet) can be subject to N-terminal α-amino-methylation. Monomethylation may have slight effects on α-amino nitrogen nucleophilicity and basicity, whereas trimethylation (or dimethylation in case of proline) will result in abolition of nucleophilicity and a permanent positive charge on the N-terminal amino group. Although from a biochemical point of view demethylation of amines is possible, Nt-methylation is considered irreversible as no N-terminal demethylase has been described to date.
Histone variants CENP-A and CENP-B have been found to be Nt-methylated in vivo.
=== Prenylcysteine ===
Eukaryotic proteins with C-termini that end in a CAAX motif are often subjected to a series of posttranslational modifications. The CAAX-tail processing takes place in three steps: First, a prenyl lipid anchor is attached to the cysteine through a thioester linkage. Then endoproteolysis occurs to remove the last three amino acids of the protein to expose the prenylcysteine α-COOH group. Finally, the exposed prenylcysteine group is methylated. The importance of this modification can be seen in targeted disruption of the methyltransferase for mouse CAAX proteins, where loss of isoprenylcysteine carboxyl methyltransferase resulted in mid-gestation lethality.
The biological function of prenylcysteine methylation is to facilitate the targeting of CAAX proteins to membrane surfaces within cells. Prenylcysteine can be demethylated and this reverse reaction is catalyzed by isoprenylcysteine carboxyl methylesterases. CAAX box containing proteins that are prenylcysteine methylated include Ras, GTP-binding proteins, nuclear lamins and certain protein kinases. Many of these proteins participate in cell signaling, and they utilize prenylcysteine methylation to concentrate them on the cytosolic surface of the plasma membrane where they are functional.
Methylations on the C-terminus can increase a protein's chemical repertoire and are known to have a major effect on the functions of a protein.
=== Protein phosphatase 2 ===
In eukaryotic cells, phosphatases catalyze the removal of phosphate groups from tyrosine, serine and threonine phosphoproteins. The catalytic subunit of the major serine/threonine phosphatases, like Protein phosphatase 2 is covalently modified by the reversible methylation of its C-terminus to form a leucine carboxy methyl ester. Unlike CAAX motif methylation, no C-terminal processing is required to facilitate methylation. This C-terminal methylation event regulates the recruitment of regulatory proteins into complexes through the stimulation of protein–protein interactions, thus indirectly regulating the activity of the serine-threonine phosphatases complex. Methylation is catalyzed by a unique protein phosphatase methyltransferase. The methyl group is removed by a specific protein phosphatase methylesterase. These two opposed enzymes make serine-threonine phosphatases methylation a dynamic process in response to stimuli.
=== L-isoaspartyl ===
Damaged proteins accumulate isoaspartyl which causes protein instability, loss of biological activity and stimulation of autoimmune responses. The spontaneous age-dependent degradation of L-aspartyl residues results in the formation of a succinimidyl intermediate, a succinimide radical. This is spontaneously hydrolyzed either back to L-aspartyl or, in a more favorable reaction, to abnormal L-isoaspartyl. A methyltransferase dependent pathway exists for the conversion of L-isoaspartyl back to L-aspartyl. To prevent the accumulation of L-isoaspartyl, this residue is methylated by the protein L-isoaspartyl methyltransferase, which catalyzes the formation of a methyl ester, which in turn is converted back to a succinimidyl intermediate.
Loss and gain of function mutations have unmasked the biological importance of the L-isoaspartyl O-methyltransferase in age-related processes: Mice lacking the enzyme die young of fatal epilepsy, whereas flies engineered to over-express it have an increase in life span of over 30%.
== Physical effects ==
A common theme with methylated proteins, as with phosphorylated proteins, is the role this modification plays in the regulation of protein–protein interactions. The arginine methylation of proteins can either inhibit or promote protein–protein interactions depending on the type of methylation. The asymmetric dimethylation of arginine residues in close proximity to proline-rich motifs can inhibit the binding to SH3 domains. The opposite effect is seen with interactions between the survival of motor neurons protein and the snRNP proteins SmD1, SmD3 and SmB/B', where binding is promoted by symmetric dimethylation of arginine residues in the snRNP proteins.
A well-characterized example of a methylation dependent protein–protein interaction is related to the selective methylation of lysine 9, by SUV39H1 on the N-terminal tail of the histone H3. Di- and tri-methylation of this lysine residue facilitates the binding of heterochromatin protein 1 (HP1). Because HP1 and Suv39h1 interact, it is thought the binding of HP1 to histone H3 is maintained and even allowed that to spread along the chromatin. The HP1 protein harbors a chromodomain which is responsible for the methyl-dependent interaction between it and lysine 9 of histone H3. It is likely that additional chromodomain-containing proteins will bind the same site as HP1, and to other lysine methylated positions on histones H3 and Histone H4.
C-terminal protein methylation regulates the assembly of protein phosphatase. Methylation of the protein phosphatase 2A catalytic subunit enhances the binding of the regulatory B subunit and facilitates holoenzyme assembly.
== References == | Wikipedia/Protein_methylation |
Protein phosphorylation is a reversible post-translational modification of proteins in which an amino acid residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Phosphorylation alters the structural conformation of a protein, causing it to become activated, deactivated, or otherwise modifying its function. Approximately 13,000 human proteins have sites that are phosphorylated.
The reverse reaction of phosphorylation is called dephosphorylation, and is catalyzed by protein phosphatases. Protein kinases and phosphatases work independently and in a balance to regulate the function of proteins.
The amino acids most commonly phosphorylated are serine, threonine, tyrosine, and histidine. These phosphorylations play important and well-characterized roles in signaling pathways and metabolism. However, other amino acids can also be phosphorylated post-translationally, including arginine, lysine, aspartic acid, glutamic acid and cysteine, and these phosphorylated amino acids have been identified to be present in human cell extracts and fixed human cells using a combination of antibody-based analysis (for pHis) and mass spectrometry (for all other amino acids).
Protein phosphorylation was first reported in 1906 by Phoebus Levene at the Rockefeller Institute for Medical Research with the discovery of phosphorylated vitellin. However, it was nearly 50 years until the enzymatic phosphorylation of proteins by protein kinases was discovered.
== History ==
In 1906, Phoebus Levene at the Rockefeller Institute for Medical Research identified phosphate in the protein vitellin (phosvitin) and by 1933 had detected phosphoserine in casein, with Fritz Lipmann. However, it took another 20 years before Eugene P. Kennedy described the first "enzymatic phosphorylation of proteins". The first phosphorylase enzyme was discovered by Carl and Gerty Cori in the late 1930s. Carl and Gerty Cori found two forms of glycogen phosphorylase which they named A and B but did not correctly understand the mechanism of the B form to A form conversion. The interconversion of phosphorylase b to phosphorylase a was later described by Edmond Fischer and Edwin Krebs, as well as, Wosilait and Sutherland, involving a phosphorylation/dephosphorylation mechanism. It was found that an enzyme, named phosphorylase kinase and Mg-ATP were required to phosphorylate glycogen phosphorylase by assisting in the transfer of the γ-phosphoryl group of ATP to a serine residue on phosphorylase b. Protein phosphatase 1 is able to catalyze the dephosphorylation of phosphorylated enzymes by removing the phosphate group. Earl Sutherland explained in 1950, that the activity of phosphorylase was increased and thus glycogenolysis stimulated when liver slices were incubated with adrenalin and glucagon. Phosphorylation was considered a specific control mechanism for one metabolic pathway until the 1970s, when Lester Reed discovered that mitochondrial pyruvate dehydrogenase complex was inactivated by phosphorylation. Also in the 1970s, the term multisite phosphorylation was coined in response to the discovery of proteins that are phosphorylated on two or more residues by two or more kinases. In 1975, it was shown that cAMP-dependent proteins kinases phosphorylate serine residues on specific amino acid sequence motifs. Ray Erikson discovered that v-Src was a kinase and Tony Hunter found that v-Src phosphorylated tyrosine residues on proteins in the 1970s. In the early 1980, the amino-acid sequence of the first protein kinase was determined which helped geneticists understand the functions of regulatory genes. In the late 1980s and early 1990s, the first protein tyrosine phosphatase (PTP1B) was purified and the discovery, as well as, cloning of JAK kinases was accomplished which led to many in the scientific community to name the 1990s as the decade of protein kinase cascades. Edmond Fischer and Edwin Krebs were awarded the Nobel prize in 1992 "for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism".
== Abundance ==
Reversible phosphorylation of proteins is abundant in both prokaryotic and even more so in eukaryotic organisms. For instance, in bacteria 5–10% of all proteins are thought to be phosphorylated. By contrast, it is estimated that one third of all human proteins is phosphorylated at any point in time, with 230,000, 156,000, and 40,000 unique phosphorylation sites existing in human, mouse, and yeast, respectively. In yeast, about 120 kinases (out of ~6,000 proteins total) cause 8,814 known regulated phosphorylation events, generating about 3,600 phosphoproteins (about 60% of all yeast proteins). Hence, phosphorylation is a universal regulatory mechanism that affects a large portion of proteins. Even if a protein is not phosphorylated itself, its interactions with other proteins may be regulated by phosphorylation of these interacting proteins.
== Mechanisms and functions of phosphorylation ==
Phosphorylation introduces a charged and hydrophilic group in the side chain of amino acids, possibly changing a protein's structure by altering interactions with nearby amino acids. Some proteins such as p53 contain multiple phosphorylation sites, facilitating complex, multi-level regulation. Because of the ease with which proteins can be phosphorylated and dephosphorylated, this type of modification is a flexible mechanism for cells to respond to external signals and environmental conditions.
Kinases phosphorylate proteins and phosphatases dephosphorylate proteins. Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Reversible phosphorylation results in a conformational change in the structure in many enzymes and receptors, causing them to become activated or deactivated. Phosphorylation usually occurs on serine, threonine, tyrosine and histidine residues in eukaryotic proteins. Histidine phosphorylation of eukaryotic proteins appears to be much more frequent than tyrosine phosphorylation. In prokaryotic proteins phosphorylation occurs on the serine, threonine, tyrosine, histidine, arginine or lysine residues. The addition of a phosphate (PO43-) molecule to a non-polar R group of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic portion of a molecule. In this way protein dynamics can induce a conformational change in the structure of the protein via long-range allostery with other hydrophobic and hydrophilic residues in the protein.
One such example of the regulatory role that phosphorylation plays is the p53 tumor suppressor protein. The p53 protein is heavily regulated and contains more than 18 different phosphorylation sites. Activation of p53 can lead to cell cycle arrest, which can be reversed under some circumstances, or apoptotic cell death. This activity occurs only in situations wherein the cell is damaged or physiology is disturbed in normal healthy individuals.
Upon the deactivating signal, the protein becomes dephosphorylated again and stops working. This is the mechanism in many forms of signal transduction, for example the way in which incoming light is processed in the light-sensitive cells of the retina.
Regulatory roles of phosphorylation include:
Biological thermodynamics of energy-requiring reactions
Phosphorylation of Na+/K+-ATPase during the transport of sodium (Na+) and potassium (K+) ions across the cell membrane in osmoregulation to maintain homeostasis of the body's water content.
Mediates enzyme inhibition
Phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) as part of the insulin signaling pathway.
Phosphorylation of src (pronounced "sarc") tyrosine kinase by C-terminal Src kinase (Csk) induces a conformational change in the enzyme, resulting in a fold in the structure, which masks its kinase domain, and is thus shut "off".
=== Membrane transport ===
Phosphorylation of Na+/K+-ATPase during the transport of sodium (Na+) and potassium (K+) ions across the cell membrane in osmoregulation to maintain homeostasis of the body's water content.
ATP-binding cassette transporter
=== Protein degradation ===
Arginine phosphorylation by McsB kinase marks proteins for degradation by a Clp protease. The arginine phosphorylation system, which is widely distributed across Gram-positive bacteria, appears to be functionally analogous to the eukaryotic ubiquitin–proteasome system.
=== Enzyme regulation (activation and inhibition) ===
The first example of protein regulation by phosphorylation to be discovered was glycogen phosphorylase. Eddie Fisher and Ed Krebs described how phosphorylation of glycogen phosphorylase b converted it to the active glycogen phosphorylase a. It was soon discovered that glycogen synthase, another metabolic enzyme, is inactivated by phosphorylation.
Phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) as part of the insulin signaling pathway.
Phosphorylation of Src tyrosine kinase by C-terminal Src kinase inactivates Src by inducing a conformational change which masks its kinase domain.
Phosphorylation of the H2AX histones on serine 139, within two million bases (0.03% of the chromatin) surrounding a double-strand break in DNA, is needed for repair of the double-strand break. Phosphorylation of methylpurine DNA glycosylase at serine 172 is required for base excision repair of alkylated base damage.
=== Protein-protein interactions ===
Phosphorylation of the cytosolic components of NADPH oxidase, a large membrane-bound, multi-protein enzyme present in phagocytic cells, plays an important role in the regulation of protein-protein interactions in the enzyme.
Important in protein degradation.
In the late 1990s, it was recognized that phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. These target proteins become substrates for particular E3 ubiquitin ligases only when they are phosphorylated.
=== Signaling networks ===
Elucidating complex signaling pathway phosphorylation events can be difficult. In cellular signaling pathways, protein A phosphorylates protein B, and B phosphorylates C. However, in another signaling pathway, protein D phosphorylates A, or phosphorylates protein C. Global approaches such as phosphoproteomics, the study of phosphorylated proteins, which is a sub-branch of proteomics, combined with mass spectrometry-based proteomics, have been utilised to identify and quantify dynamic changes in phosphorylated proteins over time. These techniques are becoming increasingly important for the systematic analysis of complex phosphorylation networks. They have been successfully used to identify dynamic changes in the phosphorylation status of more than 6,000 sites after stimulation with epidermal growth factor. Another approach for understanding Phosphorylation Network, is by measuring the genetic interactions between multiple phosphorylating proteins and their targets. This reveals interesting recurring patterns of interactions – network motifs. Computational methods have been developed to model phosphorylation networks and predict their responses under different perturbations.
=== Phosphorylation of histones ===
Eukaryotic DNA is organized with histone proteins in specific complexes called chromatin. The chromatin structure functions and facilitates the packaging, organization and distribution of eukaryotic DNA. However, it has a negative impact on several fundamental biological processes such as transcription, replication and DNA repair by restricting the accessibility of certain enzymes and proteins. Post-translational modification of histones such as histone phosphorylation has been shown to modify the chromatin structure by changing protein:DNA or protein:protein interactions. Histone post-translational modifications modify the chromatin structure. The most commonly associated histone phosphorylation occurs during cellular responses to DNA damage, when phosphorylated histone H2A separates large chromatin domains around the site of DNA breakage. Researchers investigated whether modifications of histones directly impact RNA polymerase II directed transcription. Researchers choose proteins that are known to modify histones to test their effects on transcription, and found that the stress-induced kinase, MSK1, inhibits RNA synthesis. Inhibition of transcription by MSK1 was most sensitive when the template was in chromatin, since DNA templates not in chromatin were resistant to the effects of MSK1. It was shown that MSK1 phosphorylated histone H2A on serine 1, and mutation of serine 1 to alanine blocked the inhibition of transcription by MSK1. Thus results suggested that the acetylation of histones can stimulate transcription by suppressing an inhibitory phosphorylation by a kinase as MSK1.
== Kinases ==
Within a protein, phosphorylation can occur on several amino acids. Phosphorylation on serine is thought to be the most common, followed by threonine. Tyrosine phosphorylation is relatively rare but lies at the head of many protein phosphorylation signalling pathways (e.g. in tyrosine kinase-linked receptors) in most of the eukaryotes. Phosphorylation on amino acids, such as serine, threonine, and tyrosine results in the formation of a phosphoprotein, when the phosphate group of the phosphoprotein reacts with the -OH group of a Ser, Thr, or Tyr sidechain in an esterification reaction. However, since tyrosine phosphorylated proteins are relatively easy to purify using antibodies, tyrosine phosphorylation sites are relatively well understood. Histidine and aspartate phosphorylation occurs in prokaryotes as part of two-component signaling and in some cases in eukaryotes in some signal transduction pathways. The analysis of phosphorylated histidine using standard biochemical and mass spectrometric approaches is much more challenging than that of Ser, Thr or Tyr. and In prokaryotes, archaea, and some lower eukaryotes, histidine's nitrogen act as a nucleophile and binds to a phosphate group. Once histidine is phosphorylated the regulatory domain of the response regulator catalyzes the transfer of the phosphate to aspartate.
== Receptor tyrosine kinases ==
While tyrosine phosphorylation is found in relatively low abundance, it is well studied due to the ease of purification of phosphotyrosine using antibodies. Receptor tyrosine kinases are an important family of cell surface receptors involved in the transduction of extracellular signals such as hormones, growth factors, and cytokines. Binding of a ligand to a monomeric receptor tyrosine kinase stabilizes interactions between two monomers to form a dimer, after which the two bound receptors phosphorylate tyrosine residues in trans. Phosphorylation and activation of the receptor activates a signaling pathway through enzymatic activity and interactions with adaptor proteins. Signaling through the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is critical for the development of multiple organ systems including the skin, lung, heart, and brain. Excessive signaling through the EGFR pathway is found in many human cancers.
=== Cyclin-dependent kinases ===
Cyclin-dependent kinases (CDKs) are serine-threonine kinases which regulate progression through the eukaryotic cell cycle. CDKs are catalytically active only when bound to a regulatory cyclin. Animal cells contain at least nine distinct CDKs which bind to various cyclins with considerable specificity. CDK inhibitors (CKIs) block kinase activity in the cyclin-CDK complex to halt the cell cycle in G1 or in response to environmental signals or DNA damage. The activity of different CDKs activate cell signaling pathways and transcription factors that regulate key events in mitosis such as the G1/S phase transition. Earlier cyclin-CDK complexes provide the signal to activate subsequent cyclin-CDK complexes.
== Sites ==
There are thousands of distinct phosphorylation sites in a given cell since:
There are thousands of proteins in any particular cell.
An estimated 1/10 to 1/2 of proteins are phosphorylated in some cellular state.
30–65% of proteins in humans and ~50% of proteins in yeast may be phosphorylated.
An estimated 230,000, 156,000, and 40,000 phosphorylation sites exist in human, mouse, and yeast, respectively.
Phosphorylation often occurs on multiple distinct sites on a given protein.
Since phosphorylation of any site on a given protein can change the function or localization of that protein, understanding the "state" of a cell requires knowing the phosphorylation state of its proteins. For example, generally, if amino acid Serine-473 in the protein AKT is phosphorylated, AKT is functionally active as a kinase, and if it is not phosphorylated, AKT is an inactive kinase.
Phosphorylation sites are crucial for proteins and their transportation and functions. They are the covalent modification of proteins through reversible phosphorylation. This enables proteins to stay inbound within a cell since the negative phosphorylated site disallows their permeability through the cellular membrane. Protein dephosphorylation allows the cell to replenish phosphates through release of pyrophosphates which saves ATP use in the cell. An example of phosphorylating enzyme is found in E. coli bacteria. It possesses alkaline phosphatase in its periplasmic region of its membrane. The outermost membrane is permeable to phosphorylated molecules however the inner cytoplasmic membrane is impermeable due to large negative charges. In this way, the E. coli bacteria stores proteins and pyrophosphates in its periplasmic membrane until either are needed within the cell.
Recent advancement in phosphoproteomic identification has resulted in the discoveries of countless phosphorylation sites in proteins. This required an integrative medium for accessible data in which known phosphorylation sites of proteins are organized. A curated database of dbPAF was created, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. The database currently holds 294,370 non-redundant phosphorylation sites of 40,432 proteins. Other tools of phosphorylation prediction in proteins include NetPhos for eukaryotes, NetPhosBac for bacteria, and ViralPhos for viruses.
=== Serine and threonine ===
There are a large variety of serine residues, and the phosphorylation of each residue can lead to different metabolic consequences.
Protein kinase N1 is responsible for the phosphorylation of the TNF receptor-associated factor (TRAF1) on serine 139 under specific conditions. Murine TRAF1 is also phosphorylated by the same kinase, which leads to the silencing of IKK/NF-κB activity. The elimination of phosphorylation on serine 139 can be achieved by the replacement of TRAF1 with an Alanine residue, which consequently leads to the improved recruitment of TBK1.
At the serine 789 residue, FGFR1 is phosphorylated by RSK2 when the kinase is in its active form. The signaling capabilities of FGFR1 at the serine 777 site can be weakened by phosphorylation. Serine 1047 and serine 1048 have been linked to the decreased binding affinity of ubiquitin ligase c-Cbl to EFGR when they are phosphorylated.
When serine 349 is phosphorylated, the binding affinity between protein complex p62 and the protein Keap1 is strengthened, which is linked to stress response.
When serine 337 is phosphorylated by protein kinase A in vitro, the DNA binding efficiency of the p50 subunit of NF-κB is greatly increased.
Phosphorylation of serine and threonine residues is known to crosstalk with O-GlcNAc modification of serine and threonine residues.
=== Tyrosine ===
Tyrosine phosphorylation is a fast, reversible reaction, and one of the major regulatory mechanisms in signal transduction. Cell growth, differentiation, migration, and metabolic homeostasis are cellular processes maintained by tyrosine phosphorylation. The function of protein tyrosine kinases and protein-tyrosine phosphatase counterbalances the level of phosphotyrosine on any protein. The malfunctioning of specific chains of protein tyrosine kinases and protein tyrosine phosphatase has been linked to multiple human diseases such as obesity, insulin resistance, and type 2 diabetes mellitus. Phosphorylation on tyrosine occurs in eukaryotes, select bacterial species, and is present among prokaryotes. Phosphorylation on tyrosine maintains the cellular regulation in bacteria similar to its function in eukaryotes.
=== Arginine ===
Arginine phosphorylation in many Gram-positive bacteria marks proteins for degradation by a Clp protease.
=== Non-canonical phosphorylation on His, Asp, Cys, Glu, Arg and Lys in human cells ===
Widespread human protein phosphorylation occurs on multiple non-canonical amino acids, including motifs containing phosphorylated histidine (1 and 3 positions), aspartate, cysteine, glutamate, arginine, and lysine in HeLa cell extracts. Due to the chemical and thermal lability of these phosphorylated residues, special procedures and separation techniques are required for preservation alongside the heat stable 'classical' Ser, Thr and Tyr phosphorylation.
== Detection and characterization ==
Antibodies can be used as powerful tool to detect whether a protein is phosphorylated at a particular site. Antibodies bind to and detect phosphorylation-induced conformational changes in the protein. Such antibodies are called phospho-specific antibodies; hundreds of such antibodies are now available. They are becoming critical reagents both for basic research and for clinical diagnosis.
Post-translational modification (PTM) isoforms are easily detected on 2D gels. Indeed, phosphorylation replaces neutral hydroxyl groups on serines, threonines, or tyrosines with negatively charged phosphates with pKs near 1.2 and 6.5. Thus, below pH 5.5, phosphates add a single negative charge; near pH 6.5, they add 1.5 negative charges; above pH 7.5, they add 2 negative charges. The relative amount of each isoform can also easily and rapidly be determined from staining intensity on 2D gels.
In some very specific cases, the detection of the phosphorylation as a shift in the protein's electrophoretic mobility is possible on simple 1-dimensional SDS-PAGE gels, as it is described for instance for a transcriptional coactivator by Kovacs et al. Strong phosphorylation-related conformational changes (that persist in detergent-containing solutions) are thought to underlie this phenomenon. Most of the phosphorylation sites for which such a mobility shift has been described fall in the category of SP and TP sites (i.e. a proline residue follows the phosphorylated serine or threonine residue).
Large-scale mass spectrometry analyses have been used to determine sites of protein phosphorylation. Dozens of studies have been published, each identifying thousands of sites, many of which were previously undescribed. Mass spectrometry is ideally suited for such analyses using HCD or ETD fragmentation, as the addition of phosphorylation results in an increase in the mass of the protein and the phosphorylated residue. Advanced, highly accurate mass spectrometers are needed for these studies, limiting the technology to labs with high-end mass spectrometers. However, the analysis of phosphorylated peptides by mass spectrometry is still not as straightforward as for "regular", unmodified peptides. EThcD has been developed combining electron-transfer and higher-energy collision dissociation. Compared to the usual fragmentation methods, EThcD scheme provides more informative MS/MS spectra for unambiguous phosphosite localization.
A detailed characterization of the sites of phosphorylation is very difficult, and the quantitation of protein phosphorylation by mass spectrometry requires isotopic internal standard approaches. A relative quantitation can be obtained with a variety of differential isotope labeling technologies. There are also several quantitative protein phosphorylation methods, including fluorescence immunoassays, microscale thermophoresis, FRET, TRF, fluorescence polarization, fluorescence-quenching, mobility shift, bead-based detection, and cell-based formats.
== Evolution ==
Protein phosphorylation is common among all clades of life, including all animals, plants, fungi, bacteria, and archaea. The origins of protein phosphorylation mechanisms are ancestral and have diverged greatly between different species. In eukaryotes, it is estimated that between 30 – 65% of all proteins may be phosphorylated, with tens or even hundreds of thousands of distinct phosphorylation sites. Some phosphorylation sites appear to have evolved as conditional "off" switches, blocking the active site of an enzyme, such as in the prokaryotic metabolic enzyme isocitrate dehydrogenase. However, in the case of proteins that must be phosphorylated to be active, it is less clear how they could have emerged from non-phosphorylated ancestors. It has been shown that a subset of serine phosphosites are often replaced by acidic residues such as aspartate and glutamate between different species. These anionic residues can interact with cationic residues such as lysine and arginine to form salt bridges, stable non-covalent interactions that alter a protein's structure. These phosphosites often participate in salt bridges, suggesting that some phosphorylation sites evolved as conditional "on" switches for salt bridges, allowing these proteins to adopt an active conformation only in response to a specific signal.
There are around 600 known eukaryotic protein kinases, making them one of the largest eukaryotic gene families. Most phosphorylation is carried out by a single superfamily of protein kinases that share a conserved kinase domain. Protein phosphorylation is highly conserved in pathways central to cell survival, such as cell cycle progression relying on cyclin-dependent kinases (CDKs), but individual phosphorylation sites are often flexible. Targets of CDK phosphorylation often have phosphosites in disordered segments, which are found in non-identical locations even in close species. Conversely, targets of CDK phosphorylation in structurally defined regions are more highly conserved. While CDK activity is critical for cell growth and survival in all eukaryotes, only very few phosphosites show strong conservation of their precise positions. Positioning is likely to be highly important for phosphates that allosterically regulate protein structure, but much more flexible for phosphates that interact with phosphopeptide-binding domains to recruit regulatory proteins.
=== Comparisons between eukaryotes and prokaryotes ===
Protein phosphorylation is a reversible post-translational modification of proteins. In eukaryotes, protein phosphorylation functions in cell signaling, gene expression, and differentiation. It is also involved in DNA replication during the cell cycle, and the mechanisms that cope with stress-induced replication blocks. Compared to eukaryotes, prokaryotes use Hanks-type kinases and phosphatases for signal transduction. Whether or not the phosphorylation of proteins in bacteria can also regulate processes like DNA repair or replication still remains unclear.
Compared to the protein phosphorylation of prokaryotes, studies of protein phosphorylation in eukaryotes from yeast to human cells have been rather extensive. It is known that eukaryotes rely on the phosphorylation of the hydroxyl group on the side chains of serine, threonine, and tyrosine for cell signaling. These are the main regulatory post-translational modifications in eukaryotic cells but the protein phosphorylation of prokaryotes are less intensely studied. While serine, threonine, and tyrosine are phosphorylated in eukaryotes, histidine and aspartate is phosphorylated in prokaryotes and eukaryotes. In bacteria, histidine phosphorylation occurs in the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs), which are involved in the process of internalization as well as the phosphorylation of sugars.
Protein phosphorylation by protein kinase was first shown in E. coli and Salmonella typhimurium and has since been demonstrated in many other bacterial cells. It was found that bacteria use histidine and aspartate phosphorylation as a model for bacterial signaling transduction. Serine, threonine and tyrosine phosphorylation are also present in bacteria. Bacteria carry kinases and phosphatases similar to that of their eukaryotic equivalent and have also developed unique kinases and phosphatases not found in eukaryotes.
== Pathology ==
Abnormal protein phosphorylation has been implicated in a number of diseases, including cancer, Alzheimer's disease, Parkinson's disease, and other degenerative disorders.
Tau protein belongs to a group of microtubule associated proteins (MAPs) which help stabilize microtubules in cells, including neurons. Association and stabilizing activity of tau protein depends on its phosphorylated state. In Alzheimer's disease, due to misfoldings and abnormal conformational changes in tau protein structure, it is rendered ineffective at binding to microtubules and unable to keep the neural cytoskeletal structure organized during neural processes. Abnormal tau inhibits and disrupts microtubule organization and disengages normal tau from microtubules into cytosolic phase. The misfoldings lead to the abnormal aggregation into fibrillary tangles inside the neurons. The tau protein needs to be phosphorylated to function, but hyperphosphorylation of tau protein is one of the major influences on its incapacity to associate. Phosphatases PP1, PP2A, PP2B, and PP2C dephosphorylate tau protein in vitro, and their activities are reduced in areas of the brain in Alzheimer patients. Tau phosphoprotein is three to fourfold hyperphosphorylated in an Alzheimer patient compared to an aged non-afflicted individual. Alzheimer disease tau seems to remove MAP1 and MAP2 (two other major associated proteins) from microtubules and this deleterious effect is reversed when dephosphorylation is performed, evidencing hyperphosphorylation as the sole cause of the crippling activity.
=== Parkinson's disease ===
α-Synuclein is a protein that is associated with Parkinson's disease. In humans, this protein is encoded by the SNCA gene. α-Synuclein is involved in recycling synaptic vesicles that carry neurotransmitters and naturally occurs in an unfolded form. Elevated levels of α-Synuclein are found in patients with Parkinson's disease. There is a correlation between the concentration of unphosphorylated α-Synuclein present in the patient and the severity of Parkinson's disease. Specifically, phosphorylation of Ser129 in α-Synuclein has an impact on severity. Healthy patients have higher levels of unphosphorylated α-Synuclein than patients with Parkinson's disease. The measurement of change in the ratio of concentrations of phosphorylated α-Synuclein to unphosphorylated α-Synuclein within a patient could be a marker of the disease progression. Antibodies that target α-Synuclein at phosphorylated Ser129 are used to study the molecular aspects of synucleinopathies.
Phosphorylation of Ser129 is associated with the aggregation of the protein and further damage to the nervous system. The aggregation of phosphorylated α-Synuclein can be enhanced if a presynaptic scaffold protein, Sept4, is present in insufficient quantities. Direct interaction of α-Synuclein with Sept4 inhibits the phosphorylation of Ser129. However, phosphorylation of Ser129 can be observed without synuclein aggregation in conditions of overexpression.
== References == | Wikipedia/Protein_phosphorylation |
The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets.
The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form an internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than molecular oxygen.
In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. It has been used in modified forms to make biosensors, and many animals have been created that express GFP, which demonstrates a proof of concept that a gene can be expressed throughout a given organism, in selected organs, or in cells of interest. GFP can be introduced into animals or other species through transgenic techniques, and maintained in their genome and that of their offspring. GFP has been expressed in many species, including bacteria, yeasts, fungi, fish and mammals, including in human cells. Scientists Roger Y. Tsien, Osamu Shimomura, and Martin Chalfie were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein.
Most commercially available genes for GFP and similar fluorescent proteins are around 730 base-pairs long. The natural protein has 238 amino acids. Its molecular mass is 27 kD. Therefore, fusing the GFP gene to the gene of a protein of interest can significantly increase the protein's size and molecular mass, and can impair the protein's natural function or change its location or trajectory of transport within the cell.
== Background ==
=== Wild-type GFP (wtGFP) ===
In the 1960s and 1970s, GFP, along with the separate luminescent protein aequorin (an enzyme that catalyzes the breakdown of luciferin, releasing light), was first purified from the jellyfish Aequorea victoria and its properties studied by Osamu Shimomura. In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca2+ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green. However, its utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene. The funding for this project had run out, so Prasher sent cDNA samples to several labs. The lab of Martin Chalfie expressed the coding sequence of wtGFP, with the first few amino acids deleted, in heterologous cells of E. coli and C. elegans, publishing the results in Science in 1994. Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later. Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this near-wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, pH sensitivity, chloride sensitivity, poor fluorescence quantum yield, poor photostability and poor folding at 37 °C (99 °F).
The first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in Science in 1996. One month later, the Phillips group independently reported the wild-type GFP structure in Nature Biotechnology. These crystal structures provided vital background on chromophore formation and neighboring residue interactions. Researchers have modified these residues by directed and random mutagenesis to produce the wide variety of GFP derivatives in use today. Further research into GFP has shown that it is resistant to detergents, proteases, guanidinium chloride (GdmCl) treatments, and drastic temperature changes.
=== GFP derivatives ===
Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered. The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm. This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher. A 37 °C folding efficiency (F64L) point mutant to this scaffold, yielding enhanced GFP (EGFP), was discovered in 1995 by the laboratories of Thastrup and Falkow. EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M−1cm−1. The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M−1cm−1.
Superfolder GFP (sfGFP), a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.
Many other mutations have been made, including color mutants; in particular, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). BFP derivatives (except mKalama1) contain the Y66H substitution. They exhibit a broad absorption band in the ultraviolet centered close to 380 nanometers and an emission maximum at 448 nanometers. A green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) and Cu(II) has been developed. BFPms1 have several important mutations including and the BFP chromophore (Y66H),Y145F for higher quantum yield, H148G for creating a hole into the beta-barrel and several other mutations that increase solubility. Zn(II) binding increases fluorescence intensity, while Cu(II) binding quenches fluorescence and shifts the absorbance maximum from 379 to 444 nm. Therefore, they can be used as a Zn biosensor.
More color variants are possible via chromophore binding. The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an indole rather than phenol component. Several additional compensatory mutations in the surrounding barrel are required to restore brightness to this modified chromophore due to the increased bulk of the indole group. In ECFP and Cerulean, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching.
Additional site-directed random mutagenesis in combination with fluorescence lifetime based screening has further stabilized the seventh β-strand resulting in a bright variant, mTurquoise2, with a quantum yield (QY) of 0.93. The red-shifted wavelength of the YFP derivatives is accomplished by the T203Y mutation and is due to π-electron stacking interactions between the substituted tyrosine residue and the chromophore. These two classes of spectral variants are often employed for Förster resonance energy transfer (FRET) experiments. Genetically encoded FRET reporters sensitive to cell signaling molecules, such as calcium or glutamate, protein phosphorylation state, protein complementation, receptor dimerization, and other processes provide highly specific optical readouts of cell activity in real time.
Semirational mutagenesis of a number of residues led to pH-sensitive mutants known as pHluorins, and later super-ecliptic pHluorins. By exploiting the rapid change in pH upon synaptic vesicle fusion, pHluorins tagged to synaptobrevin have been used to visualize synaptic activity in neurons.
Redox sensitive GFP (roGFP) was engineered by introduction of cysteines into the beta barrel structure. The redox state of the cysteines determines the fluorescent properties of roGFP.
=== Nomenclature ===
The nomenclature of modified GFPs is often confusing due to overlapping mapping of several GFP versions onto a single name. For example, mGFP often refers to a GFP with an N-terminal palmitoylation that causes the GFP to bind to cell membranes. However, the same term is also used to refer to monomeric GFP, which is often achieved by the dimer interface breaking A206K mutation. Wild-type GFP has a weak dimerization tendency at concentrations above 5 mg/mL. mGFP also stands for "modified GFP," which has been optimized through amino acid exchange for stable expression in plant cells.
== In nature ==
The purpose of both the (primary) bioluminescence (from aequorin's action on luciferin) and the (secondary) fluorescence of GFP in jellyfish is unknown. GFP is co-expressed with aequorin in small granules around the rim of the jellyfish bell. The secondary excitation peak (480 nm) of GFP does absorb some of the blue emission of aequorin, giving the bioluminescence a more green hue. The serine 65 residue of the GFP chromophore is responsible for the dual-peaked excitation spectra of wild-type GFP. It is conserved in all three GFP isoforms originally cloned by Prasher. Nearly all mutations of this residue consolidate the excitation spectra to a single peak at either 395 nm or 480 nm. The precise mechanism of this sensitivity is complex, but, it seems, involves donation of a hydrogen from serine 65 to glutamate 222, which influences chromophore ionization. Since a single mutation can dramatically enhance the 480 nm excitation peak, making GFP a much more efficient partner of aequorin, A. victoria appears to evolutionarily prefer the less-efficient, dual-peaked excitation spectrum. Roger Tsien has speculated that varying hydrostatic pressure with depth may affect serine 65's ability to donate a hydrogen to the chromophore and shift the ratio of the two excitation peaks. Thus, the jellyfish may change the color of its bioluminescence with depth. However, a collapse in the population of jellyfish in Friday Harbor, where GFP was originally discovered, has hampered further study of the role of GFP in the jellyfish's natural environment.
Most species of lancelet are known to produce GFP in various regions of their body. Unlike A. victoria, lancelets do not produce their own blue light, and the origin of their endogenous GFP is still unknown. Some speculate that it attracts plankton towards the mouth of the lancelet, serving as a passive hunting mechanism. It may also serve as a photoprotective agent in the larvae, preventing damage caused by high-intensity blue light by converting it into lower-intensity green light. However, these theories have not been tested.
GFP-like proteins have been found in multiple species of marine copepods, particularly from the Pontellidae and Aetideidae families. GFP isolated from Pontella mimocerami has shown high levels of brightness with a quantum yield of 0.92, making them nearly two-fold brighter than the commonly used EGFP isolated from A. victoria.
== Other fluorescent proteins ==
There are many GFP-like proteins that, despite being in the same protein family as GFP, are not directly derived from Aequorea victoria. These include dsRed, eqFP611, Dronpa, TagRFPs, KFP, EosFP/IrisFP, Dendra, and so on. Having been developed from proteins in different organisms, these proteins can sometimes display unanticipated approaches to chromophore formation. Some of these, such as KFP, are developed from naturally non- or weakly-fluorescent proteins to be greatly improved upon by mutagenesis. When GFP-like barrels of different spectral characteristics are used, the excitation spectrum of one chromophore can be used to power another chromophore (FRET), allowing for conversion between wavelengths of light.
FMN-binding fluorescent proteins (FbFPs) were developed in 2007 and are a class of small (11–16 kDa), oxygen-independent fluorescent proteins that are derived from blue-light receptors. They are intended especially for the use under anaerobic or hypoxic conditions, since the formation and binding of the flavin chromophore does not require molecular oxygen, as it is the case with the synthesis of the GFP chromophore.
Fluorescent proteins with other chromophores, such as UnaG with bilirubin, can display unique properties like red-shifted emission above 600 nm or photoconversion from a green-emitting state to a red-emitting state. They can have excitation and emission wavelengths far enough apart to achieve conversion between red and green light.
A new class of fluorescent protein was engineered from α-allophycocyanin, a phycobiliprotein found in the cyanobacterium Trichodesmium erythraeum, and was named small ultra red fluorescent protein (smURFP) in 2016. smURFP autocatalytically incorporates the chromophore biliverdin without the need for an external protein known as a lyase. Jellyfish- and coral-derived GFP-like proteins require oxygen and produce a stoichiometric amount of hydrogen peroxide upon chromophore formation. smURFP does not require oxygen or produce hydrogen peroxide. smURFP has a large extinction coefficient (180,000 M−1 cm−1) and has a modest quantum yield (0.20), which makes it comparable biophysical brightness to eGFP and ~2-fold brighter than most red or far-red fluorescent proteins derived from coral. smURFP spectral properties are similar to the organic dye Cy5.
Reviews on new classes of fluorescent proteins and applications can be found in the cited reviews.
== Structure ==
GFP has a beta barrel structure consisting of eleven β-strands with a pleated sheet arrangement, with an alpha helix containing the covalently bonded chromophore 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) running through the center. Five shorter alpha helices form caps on the ends of the structure. The beta barrel structure is a nearly perfect cylinder, 42Å long and 24Å in diameter (some studies have reported a diameter of 30Å), creating what is referred to as a "β-can" formation, which is unique to the GFP-like family. HBI, the spontaneously modified form of the tripeptide Ser65–Tyr66–Gly67, is nonfluorescent in the absence of the properly folded GFP scaffold and exists mainly in the un-ionized phenol form in wtGFP. Inward-facing sidechains of the barrel induce specific cyclization reactions in Ser65–Tyr66–Gly67 that induce ionization of HBI to the phenolate form and chromophore formation. This process of post-translational modification is referred to as maturation. The hydrogen-bonding network and electron-stacking interactions with these sidechains influence the color, intensity and photostability of GFP and its numerous derivatives. The tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water. In addition to the auto-cyclization of the Ser65-Tyr66-Gly67, a 1,2-dehydrogenation reaction occurs at the Tyr66 residue. Besides the three residues that form the chromophore, residues such as Gln94, Arg96, His148, Thr203, and Glu222 all act as stabilizers. The residues of Gln94, Arg96, and His148 are able to stabilize by delocalizing the chromophore charge. Arg96 is the most important stabilizing residue due to the fact that it prompts the necessary structural realignments that are necessary from the HBI ring to occur. Any mutation to the Arg96 residue would result in a decrease in the development rate of the chromophore because proper electrostatic and steric interactions would be lost. Tyr66 is the recipient of hydrogen bonds and does not ionize in order to produce favorable electrostatics.
Blue fluorescent protein (BFP) is the blue variant of green fluorescent protein (GFP). BFP has a very similar structure to GFP. In the BFP structure, two substitution mutations in the amino acid sequence change its fluorescence from green to blue. The first mutation occurs inside the chromophore of GFP at position 66 which changes a tyrosine to a histidine. The other mutation in BFP is on the tyrosine at position 145 which mutates to phenylalanine. The autocatalytic cyclization and oxidation of the serine, tyrosine, and glycine form the GFP chromophore. These three residues at positions 65-67 make up the green fluorescent chromophore. When the tyrosine in the chromophore is substituted by a histidine, it changes the folding structure of the protein and emission spectra. The T145F mutation is also added to increase the stability of the protein and well as intensify the fluorescence. These mutations are what change GFP to BFP.
=== Autocatalytic formation of the chromophore in wtGFP ===
Mechanistically, the process involves base-mediated cyclization followed by dehydration and oxidation. In the reaction of 7a to 8 involves the formation of an enamine from the imine, while in the reaction of 7b to 9 a proton is abstracted. The formed HBI fluorophore is highlighted in green.
The reactions are catalyzed by residues Glu222 and Arg96. An analogous mechanism is also possible with threonine in place of Ser65.
== Applications ==
=== Reporter assays ===
Green fluorescent protein may be used as a reporter gene.
For example, GFP can be used as a reporter for environmental toxicity levels. This protein has been shown to be an effective way to measure the toxicity levels of various chemicals including ethanol, p-formaldehyde, phenol, triclosan, and paraben. GFP is great as a reporter protein because it has no effect on the host when introduced to the host's cellular environment. Due to this ability, no external visualization stain, ATP, or cofactors are needed. With regards to pollutant levels, the fluorescence was measured in order to gauge the effect that the pollutants have on the host cell. The cellular density of the host cell was also measured. Results from the study conducted by Song, Kim, & Seo (2016) showed that there was a decrease in both fluorescence and cellular density as pollutant levels increased. This was indicative of the fact that cellular activity had decreased. More research into this specific application in order to determine the mechanism by which GFP acts as a pollutant marker. Similar results have been observed in zebrafish because zebrafish that were injected with GFP were approximately twenty times more susceptible to recognize cellular stresses than zebrafish that were not injected with GFP.
==== Advantages ====
The biggest advantage of GFP is that it can be heritable, depending on how it was introduced, allowing for continued study of cells and tissues it is expressed in. Visualizing GFP is noninvasive, requiring only illumination with blue light. GFP alone does not interfere with biological processes, but when fused to proteins of interest, careful design of linkers is required to maintain the function of the protein of interest. Moreover, if used with a monomer it is able to diffuse readily throughout cells.
=== Fluorescence microscopy ===
The availability of GFP and its derivatives has thoroughly redefined fluorescence microscopy and the way it is used in cell biology and other biological disciplines. While most small fluorescent molecules such as FITC (fluorescein isothiocyanate) are strongly phototoxic when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has triggered the development of highly automated live-cell fluorescence microscopy systems, which can be used to observe cells over time expressing one or more proteins tagged with fluorescent proteins.
There are many techniques to utilize GFP in a live cell imaging experiment. The most direct way of utilizing GFP is to directly attach it to a protein of interest. For example, GFP can be included in a plasmid expressing other genes to indicate a successful transfection of a gene of interest. Another method is to use a GFP that contains a mutation where the fluorescence will change from green to yellow over time, which is referred to as a fluorescent timer. With the fluorescent timer, researchers can study the state of protein production such as recently activated, continuously activated, or recently deactivated based on the color reported by the fluorescent protein. In yet another example, scientists have modified GFP to become active only after exposure to irradiation giving researchers a tool to selectively activate certain portions of a cell and observe where proteins tagged with the GFP move from the starting location. These are only two examples in a burgeoning field of fluorescent microcopy and a more complete review of biosensors utilizing GFP and other fluorescent proteins can be found here
For example, GFP had been widely used in labelling the spermatozoa of various organisms for identification purposes as in Drosophila melanogaster, where expression of GFP can be used as a marker for a particular characteristic. GFP can also be expressed in different structures enabling morphological distinction. In such cases, the gene for the production of GFP is incorporated into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that is, the gene's regulatory sequence now controls the production of GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. Analysis of such time lapse movies has redefined the understanding of many biological processes including protein folding, protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material. Obtained data are also used to calibrate mathematical models of intracellular systems and to estimate rates of gene expression. Similarly, GFP can be used as an indicator of protein expression in heterologous systems. In this scenario, fusion proteins containing GFP are introduced indirectly, using RNA of the construct, or directly, with the tagged protein itself. This method is useful for studying structural and functional characteristics of the tagged protein on a macromolecular or single-molecule scale with fluorescence microscopy.
The Vertico SMI microscope using the SPDM Phymod technology uses the so-called "reversible photobleaching" effect of fluorescent dyes like GFP and its derivatives to localize them as single molecules in an optical resolution of 10 nm. This can also be performed as a co-localization of two GFP derivatives (2CLM).
Another powerful use of GFP is to express the protein in small sets of specific cells. This allows researchers to optically detect specific types of cells in vitro (in a dish), or even in vivo (in the living organism). GFP is considered to be a reliable reporter of gene expression in eukaryotic cells when the fluorescence is measured by flow cytometry. Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry (Brainbow). Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of neuron membrane potential, tracking of AMPA receptors on cell membranes, viral entry and the infection of individual influenza viruses and lentiviral viruses, etc.
It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as regenerative medicine. By using "high-expresser" GFP, transgenic rats display high expression in most tissues, and many cells that have not been characterized or have been only poorly characterized in previous GFP-transgenic rats.
GFP has been shown to be useful in cryobiology as a viability assay. Correlation of viability as measured by trypan blue assays were 0.97. Another application is the use of GFP co-transfection as internal control for transfection efficiency in mammalian cells.
A novel possible use of GFP includes using it as a sensitive monitor of intracellular processes via an eGFP laser system made out of a human embryonic kidney cell line. The first engineered living laser is made by an eGFP expressing cell inside a reflective optical cavity and hitting it with pulses of blue light. At a certain pulse threshold, the eGFP's optical output becomes brighter and completely uniform in color of pure green with a wavelength of 516 nm. Before being emitted as laser light, the light bounces back and forth within the resonator cavity and passes the cell numerous times. By studying the changes in optical activity, researchers may better understand cellular processes.
GFP is used widely in cancer research to label and track cancer cells. GFP-labelled cancer cells have been used to model metastasis, the process by which cancer cells spread to distant organs.
=== Split GFP ===
GFP can be used to analyse the colocalization of proteins. This is achieved by "splitting" the protein into two fragments which are able to self-assemble, and then fusing each of these to the two proteins of interest. Alone, these incomplete GFP fragments are unable to fluoresce. However, if the two proteins of interest colocalize, then the two GFP fragments assemble together to form a GFP-like structure which is able to fluoresce. Therefore, by measuring the level of fluorescence it is possible to determine whether the two proteins of interest colocalize.
=== Macro-photography ===
Macro-scale biological processes, such as the spread of virus infections, can be followed using GFP labeling. In the past, mutagenic ultra violet light (UV) has been used to illuminate living organisms (e.g., see) to detect and photograph the GFP expression. Recently, a technique using non-mutagenic LED lights have been developed for macro-photography. The technique uses an epifluorescence camera attachment based on the same principle used in the construction of epifluorescence microscopes.
=== Transgenic pets ===
Alba, a green-fluorescent rabbit, was created by a French laboratory commissioned by Eduardo Kac using GFP for purposes of art and social commentary. The US company Yorktown Technologies markets to aquarium shops green fluorescent zebrafish (GloFish) that were initially developed to detect pollution in waterways. NeonPets, a US-based company has marketed green fluorescent mice to the pet industry as NeonMice. Green fluorescent pigs, known as Noels, were bred by a group of researchers led by Wu Shinn-Chih at the Department of Animal Science and Technology at National Taiwan University. A Japanese-American Team created green-fluorescent cats as proof of concept to use them potentially as model organisms for diseases, particularly HIV. In 2009 a South Korean team from Seoul National University bred the first transgenic beagles with fibroblast cells from sea anemones. The dogs give off a red fluorescent light, and they are meant to allow scientists to study the genes that cause human diseases like narcolepsy and blindness.
=== Art ===
Julian Voss-Andreae, a German-born artist specializing in "protein sculptures," created sculptures based on the structure of GFP, including the 1.70 metres (5 feet 7 inches) tall "Green Fluorescent Protein" (2004) and the 1.40 metres (4 feet 7 inches) tall "Steel Jellyfish" (2006). The latter sculpture is located at the place of GFP's discovery by Shimomura in 1962, the University of Washington's Friday Harbor Laboratories.
== See also ==
Protein tag
pGLO
Yellow fluorescent protein
Genetically encoded voltage indicator
== References ==
== Further reading ==
== External links ==
A comprehensive article on fluorescent proteins at Scholarpedia
Brief summary of landmark GFP papers
Interactive Java applet demonstrating the chemistry behind the formation of the GFP chromophore
Video of 2008 Nobel Prize lecture of Roger Tsien on fluorescent proteins
Excitation and emission spectra for various fluorescent proteins
Green Fluorescent Protein Chem Soc Rev themed issue dedicated to the 2008 Nobel Prize winners in Chemistry, Professors Osamu Shimomura, Martin Chalfie and Roger Y. Tsien
Molecule of the Month, June 2003: an illustrated overview of GFP by David Goodsell.
Molecule of the Month, June 2014: an illustrated overview of GFP-like variants by David Goodsell.
Green Fluorescent Protein on FPbase, a fluorescent protein database
Overview of all the structural information available in the PDB for UniProt: P42212 (Green fluorescent protein) at the PDBe-KB. | Wikipedia/Green_Fluorescent_Protein |
In medicine, proteinopathy ([pref. protein]; -pathy [suff. disease]; proteinopathies pl.; proteinopathic adj), or proteopathy, protein conformational disorder, or protein misfolding disease, is a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body.
Often the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way (a toxic gain-of-function) or they can lose their normal function. The proteinopathies include such diseases as Creutzfeldt–Jakob disease (and a variant associated with mad cow disease) and other prion diseases, Alzheimer's disease, Parkinson's disease, amyloidosis, multiple system atrophy, and a wide range of other disorders. The term proteopathy was first proposed in 2000 by Lary Walker and Harry LeVine.
The concept of proteopathy can trace its origins to the mid-19th century, when, in 1854, Rudolf Virchow coined the term amyloid ("starch-like") to describe a substance in cerebral corpora amylacea that exhibited a chemical reaction resembling that of cellulose. In 1859, Friedreich and Kekulé demonstrated that, rather than consisting of cellulose, "amyloid" actually is rich in protein. Subsequent research has shown that many different proteins can form amyloid, and that all amyloids show birefringence in cross-polarized light after staining with the dye Congo red, as well as a fibrillar ultrastructure when viewed with an electron microscope. However, some proteinaceous lesions lack birefringence and contain few or no classical amyloid fibrils, such as the diffuse deposits of amyloid beta (Aβ) protein in the brains of people with Alzheimer's. Furthermore, evidence has emerged that small, non-fibrillar protein aggregates known as oligomers are toxic to the cells of an affected organ, and that amyloidogenic proteins in their fibrillar form may be relatively benign.
== Pathophysiology ==
In most, if not all proteinopathies, a change in the 3-dimensional folding conformation increases the tendency of a specific protein to bind to itself. In this aggregated form, the protein is resistant to clearance and can interfere with the normal capacity of the affected organs. In some cases, misfolding of the protein results in a loss of its usual function. For example, cystic fibrosis is caused by a defective cystic fibrosis transmembrane conductance regulator (CFTR) protein, and in amyotrophic lateral sclerosis / frontotemporal lobar degeneration (FTLD), certain gene-regulating proteins inappropriately aggregate in the cytoplasm, and thus are unable to perform their normal tasks within the nucleus.
Because proteins share a common structural feature known as the polypeptide backbone, all proteins have the potential to misfold under some circumstances. However, only a relatively small number of proteins are linked to proteopathic disorders, possibly due to structural idiosyncrasies of the vulnerable proteins. For example, proteins that are normally unfolded or relatively unstable as monomers (that is, as single, unbound protein molecules) are more likely to misfold into an abnormal conformation. In nearly all instances, the disease-causing molecular configuration involves an increase in beta-sheet secondary structure of the protein.
The abnormal proteins in some proteopathies have been shown to fold into multiple 3-dimensional shapes; these variant, proteinaceous structures are defined by their different pathogenic, biochemical, and conformational properties. They have been most thoroughly studied with regard to prion disease, and are referred to as protein strains.
The likelihood that proteinopathy will develop is increased by certain risk factors that promote the self-assembly of a protein. These include destabilizing changes in the primary amino acid sequence of the protein, post-translational modifications (such as hyperphosphorylation), changes in temperature or pH, an increase in production of a protein, or a decrease in its clearance. Advancing age is a strong risk factor, as is traumatic brain injury. In the aging brain, multiple proteopathies can overlap. For example, in addition to tauopathy and Aβ-amyloidosis (which coexist as key pathologic features of Alzheimer's disease), many Alzheimer patients have concomitant synucleinopathy (Lewy bodies) in the brain.
It is hypothesized that chaperones and co-chaperones (proteins that assist protein folding) may antagonize proteotoxicity during aging and in protein misfolding-diseases to maintain proteostasis.
== Seeded induction ==
Some proteins can be induced to form abnormal assemblies by exposure to the same (or similar) protein that has folded into a disease-causing conformation, a process called 'seeding' or 'permissive templating'. In this way, the disease state can be brought about in a susceptible host by the introduction of diseased tissue extract from an affected donor. The best known forms of inducible proteopathy are prion diseases, which can be transmitted by exposure of a host organism to purified prion protein in a disease-causing conformation.
There is now evidence that other proteinopathies can be induced by a similar mechanism, including Aβ amyloidosis, amyloid A (AA) amyloidosis, and apolipoprotein AII amyloidosis, tauopathy, synucleinopathy, and the aggregation of superoxide dismutase-1 (SOD1), polyglutamine, and TAR DNA-binding protein-43 (TDP-43).
In all of these instances, an aberrant form of the protein itself appears to be the pathogenic agent. In some cases, the deposition of one type of protein can be experimentally induced by aggregated assemblies of other proteins that are rich in β-sheet structure, possibly because of structural complementarity of the protein molecules. For example, AA amyloidosis can be stimulated in mice by such diverse macromolecules as silk, the yeast amyloid Sup35, and curli fibrils from the bacterium Escherichia coli. AII amyloid can be induced in mice by a variety of β-sheet rich amyloid fibrils, and cerebral tauopathy can be induced by brain extracts that are rich in aggregated Aβ. There is also experimental evidence for cross-seeding between prion protein and Aβ. In general, such heterologous seeding is less efficient than is seeding by a corrupted form of the same protein.
== List of proteinopathies ==
== Management ==
The development of effective treatments for many proteopathies has been challenging. Because the proteopathies often involve different proteins arising from different sources, treatment strategies must be customized to each disorder; however, general therapeutic approaches include maintaining the function of affected organs, reducing the formation of the disease-causing proteins, preventing the proteins from misfolding and/or aggregating, or promoting their removal. For example, in Alzheimer's disease, researchers are seeking ways to reduce the production of the disease-associated protein Aβ by inhibiting the enzymes that free it from its parent protein. Another strategy is to use antibodies to neutralize specific proteins by active or passive immunization. In some proteopathies, inhibiting the toxic effects of protein oligomers might be beneficial.
For example, Amyloid A (AA) amyloidosis can be reduced by treating the inflammatory state that increases the amount of the protein in the blood (referred to as serum amyloid A, or SAA). In immunoglobulin light chain amyloidosis (AL amyloidosis), chemotherapy can be used to lower the number of the blood cells that make the light chain protein that forms amyloid in various bodily organs. Transthyretin (TTR) amyloidosis (ATTR) results from the deposition of misfolded TTR in multiple organs. Because TTR is mainly produced in the liver, TTR amyloidosis can be slowed in some hereditary cases by liver transplantation. TTR amyloidosis also can be treated by stabilizing the normal assemblies of the protein (called tetramers because they consist of four TTR molecules bound together). Stabilization prevents individual TTR molecules from escaping, misfolding, and aggregating into amyloid.
Several other treatment strategies for proteopathies are being investigated, including small molecules and biologic medicines such as small interfering RNAs, antisense oligonucleotides, peptides, and engineered immune cells. In some cases, multiple therapeutic agents may be combined to improve effectiveness.
== Additional images ==
== See also ==
Amyloidosis
Neurofibrillary tangles
Protein toxicity
Prion
Transmissible spongiform encephalopathy
== References ==
== External links ==
Amyloidosis
Prion-Related Diseases
Protein Misfolding Diseases Book | Wikipedia/Protein_misfolding |
A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may not correspond to individual proteins, are called capsomeres. The proteins making up the capsid are called capsid proteins or viral coat proteins (VCP). The virus genomic component inside the capsid, along with occasionally present virus core protein, is called the virus core. The capsid and core together are referred to as a nucleocapsid (cf. also virion).
Capsids are broadly classified according to their structure. The majority of the viruses have capsids with either helical or icosahedral structure. Some viruses, such as bacteriophages, have developed more complicated structures due to constraints of elasticity and electrostatics. The icosahedral shape, which has 20 equilateral triangular faces, approximates a sphere, while the helical shape resembles the shape of a spring, taking the space of a cylinder but not being a cylinder itself. The capsid faces may consist of one or more proteins. For example, the foot-and-mouth disease virus capsid has faces consisting of three proteins named VP1–3.
Some viruses are enveloped, meaning that the capsid is coated with a lipid membrane known as the viral envelope. The envelope is acquired by the capsid from an intracellular membrane in the virus' host; examples include the inner nuclear membrane, the Golgi membrane, and the cell's outer membrane.
Once the virus has infected a cell and begins replicating itself, new capsid subunits are synthesized using the protein biosynthesis mechanism of the cell. In some viruses, including those with helical capsids and especially those with RNA genomes, the capsid proteins co-assemble with their genomes. In other viruses, especially more complex viruses with double-stranded DNA genomes, the capsid proteins assemble into empty precursor procapsids that include a specialized portal structure at one vertex. Through this portal, viral DNA is translocated into the capsid.
Structural analyses of major capsid protein (MCP) architectures have been used to categorise viruses into lineages. For example, the bacteriophage PRD1, the algal virus Paramecium bursaria Chlorella virus-1 (PBCV-1), mimivirus and the mammalian adenovirus have been placed in the same lineage, whereas tailed, double-stranded DNA bacteriophages (Caudovirales) and herpesvirus belong to a second lineage.
== Specific shapes ==
=== Icosahedral ===
The icosahedral structure is extremely common among viruses. The icosahedron consists of 20 triangular faces delimited by 12 fivefold vertexes and consists of 60 asymmetric units. Thus, an icosahedral virus is made of 60N protein subunits. The number and arrangement of capsomeres in an icosahedral capsid can be classified using the "quasi-equivalence principle" proposed by Donald Caspar and Aaron Klug. Like the Goldberg polyhedra, an icosahedral structure can be regarded as being constructed from pentamers and hexamers. The structures can be indexed by two integers h and k, with
h
≥
1
{\displaystyle h\geq 1}
and
k
≥
0
{\displaystyle k\geq 0}
; the structure can be thought of as taking h steps from the edge of a pentamer, turning 60 degrees counterclockwise, then taking k steps to get to the next pentamer. The triangulation number T for the capsid is defined as:
T
=
h
2
+
h
⋅
k
+
k
2
{\displaystyle T=h^{2}+h\cdot k+k^{2}}
In this scheme, icosahedral capsids contain 12 pentamers plus 10(T − 1) hexamers. The T-number is representative of the size and complexity of the capsids. Geometric examples for many values of h, k, and T can be found at List of geodesic polyhedra and Goldberg polyhedra.
Many exceptions to this rule exist: For example, the polyomaviruses and papillomaviruses have pentamers instead of hexamers in hexavalent positions on a quasi T = 7 lattice. Members of the double-stranded RNA virus lineage, including reovirus, rotavirus and bacteriophage φ6 have capsids built of 120 copies of capsid protein, corresponding to a T = 2 capsid, or arguably a T = 1 capsid with a dimer in the asymmetric unit. Similarly, many small viruses have a pseudo T = 3 (or P = 3) capsid, which is organized according to a T = 3 lattice, but with distinct polypeptides occupying the three quasi-equivalent positions
=== Prolate ===
An elongated icosahedron is a common shape for the heads of bacteriophages. Such a structure is composed of a cylinder with a cap at either end. The cylinder is composed of 10 elongated triangular faces. The Q number (or Tmid), which can be any positive integer, specifies the number of triangles, composed of asymmetric subunits, that make up the 10 triangles of the cylinder. The caps are classified by the T (or Tend) number.
The bacterium E. coli is the host for bacteriophage T4 that has a prolate head structure. The bacteriophage encoded gp31 protein appears to be functionally homologous to E. coli chaperone protein GroES and able to substitute for it in the assembly of bacteriophage T4 virions during infection. Like GroES, gp31 forms a stable complex with GroEL chaperonin that is absolutely necessary for the folding and assembly in vivo of the bacteriophage T4 major capsid protein gp23.
=== Helical ===
Many rod-shaped and filamentous plant viruses have capsids with helical symmetry. The helical structure can be described as a set of n 1-D molecular helices related by an n-fold axial symmetry. The helical transformation are classified into two categories: one-dimensional and two-dimensional helical systems. Creating an entire helical structure relies on a set of translational and rotational matrices which are coded in the protein data bank. Helical symmetry is given by the formula P = μ x ρ, where μ is the number of structural units per turn of the helix, ρ is the axial rise per unit and P is the pitch of the helix. The structure is said to be open due to the characteristic that any volume can be enclosed by varying the length of the helix. The most understood helical virus is the tobacco mosaic virus. The virus is a single molecule of (+) strand RNA. Each coat protein on the interior of the helix binds three nucleotides of the RNA genome. Influenza A viruses differ by comprising multiple ribonucleoproteins, the viral NP protein organizes the RNA into a helical structure. The size is also different; the tobacco mosaic virus has a 16.33 protein subunits per helical turn, while the influenza A virus has a 28 amino acid tail loop.
== Functions ==
The functions of the capsid are to:
protect the genome,
deliver the genome, and
interact with the host.
The virus must assemble a stable, protective protein shell to protect the genome from lethal chemical and physical agents. These include extremes of pH or temperature and proteolytic and nucleolytic enzymes. For non-enveloped viruses, the capsid itself may be involved in interaction with receptors on the host cell, leading to penetration of the host cell membrane and internalization of the capsid. Delivery of the genome occurs by subsequent uncoating or disassembly of the capsid and release of the genome into the cytoplasm, or by ejection of the genome through a specialized portal structure directly into the host cell nucleus.
== Origin and evolution ==
It has been suggested that many viral capsid proteins have evolved on multiple occasions from functionally diverse cellular proteins. The recruitment of cellular proteins appears to have occurred at different stages of evolution so that some cellular proteins were captured and refunctionalized prior to the divergence of cellular organisms into the three contemporary domains of life, whereas others were hijacked relatively recently. As a result, some capsid proteins are widespread in viruses infecting distantly related organisms (e.g., capsid proteins with the jelly-roll fold), whereas others are restricted to a particular group of viruses (e.g., capsid proteins of alphaviruses).
A computational model (2015) has shown that capsids may have originated before viruses and that they served as a means of horizontal transfer between replicator communities since these communities could not survive if the number of gene parasites increased, with certain genes being responsible for the formation of these structures and those that favored the survival of self-replicating communities. The displacement of these ancestral genes between cellular organisms could favor the appearance of new viruses during evolution.
== See also ==
Geodesic polyhedron
Goldberg–Coxeter construction
Fullerene#Other buckyballs
== References ==
== Further reading ==
== External links ==
IRAM-Virus Capsid Database and Analysis Resource Archived 2019-10-23 at the Wayback Machine | Wikipedia/Virus_coat_protein |
Protein topology is a property of protein molecule that does not change under deformation (without cutting or breaking a bond).
== Frameworks ==
Two main topology frameworks have been developed and applied to protein molecules.
=== Knot Theory ===
Knot theory which categorises chain entanglements. The usage of knot theory is limited to a small percentage of proteins as most of them are unknot.
=== Circuit topology ===
Circuit topology categorises intra-chain contacts based on their arrangements. Circuit topology is a determinant of protein folding kinetics and stability.
== Other Uses ==
In biology literature, the term topology is also used to refer to mutual orientation of regular secondary structures, such as alpha-helices and beta strands in protein structure [1]. For example, two adjacent interacting alpha-helices or beta-strands can go in the same or in opposite directions. Topology diagrams of different proteins with known three-dimensional structure are provided by PDBsum (an example).
== See also ==
Circuit topology
Membrane topology
Protein folding
== References ==
== External links ==
Pro-origami: Protein structure cartoons
TOPS services at Glasgow University
PTGL
TOPDRAW | Wikipedia/Protein_topology |
Motor proteins are a class of molecular motors that can move along the cytoskeleton of cells. They do this by converting chemical energy into mechanical work by the hydrolysis of ATP.
== Cellular functions ==
Motor proteins are the driving force behind most active transport of proteins and vesicles in the cytoplasm. Kinesins and cytoplasmic dyneins play essential roles in intracellular transport such as axonal transport and in the formation of the spindle apparatus and the separation of the chromosomes during mitosis and meiosis. Axonemal dynein, found in cilia and flagella, is crucial to cell motility, for example in spermatozoa, and fluid transport, for example in trachea. The muscle protein myosin "motors" the contraction of muscle fibers in animals.
== Diseases associated with motor protein defects ==
The importance of motor proteins in cells becomes evident when they fail to fulfill their function. For example, kinesin deficiencies have been identified as the cause for Charcot-Marie-Tooth disease and some kidney diseases. Dynein deficiencies can lead to chronic infections of the respiratory tract as cilia fail to function without dynein. Numerous myosin deficiencies are related to disease states and genetic syndromes. Because myosin II is essential for muscle contraction, defects in muscular myosin predictably cause myopathies. Myosin is necessary in the process of hearing because of its role in the growth of stereocilia so defects in myosin protein structure can lead to Usher syndrome and non-syndromic deafness.
== Cytoskeletal motor proteins ==
Motor proteins utilizing the cytoskeleton for movement fall into two categories based on their substrate: microfilaments or microtubules. Actin-based motor proteins (myosin) move along microfilaments through interaction with actin, and microtubule motors (dynein and kinesin) move along microtubules through interaction with tubulin.
There are two basic types of microtubule motors: plus-end motors and minus-end motors, depending on the direction in which they "walk" along the microtubule cables within the cell.
=== Actin motors ===
==== Myosin ====
Myosins are a superfamily of actin motor proteins that convert chemical energy in the form of ATP to mechanical energy, thus generating force and movement. The first identified myosin, myosin II, is responsible for generating muscle contraction. Myosin II is an elongated protein that is formed from two heavy chains with motor heads and two light chains. Each myosin head contains actin and ATP binding site. The myosin heads bind and hydrolyze ATP, which provides the energy to walk toward the plus end of an actin filament. Myosin II are also vital in the process of cell division. For example, non-muscle myosin II bipolar thick filaments provide the force of contraction needed to divide the cell into two daughter cells during cytokinesis. In addition to myosin II, many other myosin types are responsible for variety of movement of non-muscle cells. For example, myosin is involved in intracellular organization and the protrusion of actin-rich structures at the cell surface. Myosin V is involved in vesicle and organelle transport. Myosin XI is involved in cytoplasmic streaming, wherein movement along microfilament networks in the cell allows organelles and cytoplasm to stream in a particular direction. Eighteen different classes of myosins are known.
Genomic representation of myosin motors:
Fungi (yeast): 5
Plants (Arabidopsis): 17
Insects (Drosophila): 13
Mammals (human): 40
Chromadorea ( nematode C. elegans): 15
=== Microtubule motors ===
==== Kinesin ====
Kinesins are a superfamily of related motor proteins that use a microtubule track in anterograde movement. They are vital to spindle formation in mitotic and meiotic chromosome separation during cell division and are also responsible for shuttling mitochondria, Golgi bodies, and vesicles within eukaryotic cells. Kinesins have two heavy chains and two light chains per active motor. The two globular head motor domains in heavy chains can convert the chemical energy of ATP hydrolysis into mechanical work to move along microtubules. The direction in which cargo is transported can be towards the plus-end or the minus-end, depending on the type of kinesin. In general, kinesins with N-terminal motor domains move their cargo towards the plus ends of microtubules located at the cell periphery, while kinesins with C-terminal motor domains move cargo towards the minus ends of microtubules located at the nucleus. Fourteen distinct kinesin families are known, with some additional kinesin-like proteins that cannot be classified into these families.
Genomic representation of kinesin motors:
Fungi (yeast): 6
Plants (Arabidopsis thaliana): 61
Insects (Drosophila melanogaster): 25
Mammals (human): 45
==== Dynein ====
Dyneins are microtubule motors capable of a retrograde sliding movement. Dynein complexes are much larger and more complex than kinesin and myosin motors. Dyneins are composed of two or three heavy chains and a large and variable number of associated light chains. Dyneins drive intracellular transport toward the minus end of microtubules which lies in the microtubule organizing center near the nucleus. The dynein family has two major branches. Axonemal dyneins facilitate the beating of cilia and flagella by rapid and efficient sliding movements of microtubules. Another branch is cytoplasmic dyneins which facilitate the transport of intracellular cargos. Compared to 15 types of axonemal dynein, only two cytoplasmic forms are known.
Genomic representation of dynein motors:
Fungi (yeast): 1
Plants (Arabidopsis thaliana): 0
Insects (Drosophila melanogaster): 13
Mammals (human): 14-15
=== Plant-specific motors ===
In contrast to animals, fungi and non-vascular plants, the cells of flowering plants lack dynein motors. However, they contain a larger number of different kinesins. Many of these plant-specific kinesin groups are specialized for functions during plant cell mitosis. Plant cells differ from animal cells in that they have a cell wall. During mitosis, the new cell wall is built by the formation of a cell plate starting in the center of the cell. This process is facilitated by a phragmoplast, a microtubule array unique to plant cell mitosis. The building of cell plate and ultimately the new cell wall requires kinesin-like motor proteins.
Another motor protein essential for plant cell division is kinesin-like calmodulin-binding protein (KCBP), which is unique to plants and part kinesin and part myosin.
== Other molecular motors ==
Besides the motor proteins above, there are many more types of proteins capable of generating forces and torque in the cell. Many of these molecular motors are ubiquitous in both prokaryotic and eukaryotic cells, although some, such as those involved with cytoskeletal elements or chromatin, are unique to eukaryotes. The motor protein prestin, expressed in mammalian cochlear outer hair cells, produces mechanical amplification in the cochlea. It is a direct voltage-to-force converter, which operates at the microsecond rate and possesses piezoelectric properties.
== See also ==
ATP synthase
Cytoskeleton
Protein dynamics
== References ==
== External links ==
MBInfo - What are Motor Proteins?
Ron Vale's Seminar: "Molecular Motor Proteins"
Biology of Motor Proteins Institute for Biophysical Chemistry, Göttingen
Jonathan Howard (2001), Mechanics of motor proteins and the cytoskeleton. ISBN 9780878933334 | Wikipedia/Motor_proteins |
In molecular biology, protein threading, also known as fold recognition, is a method of protein modeling which is used to model those proteins which have the same fold as proteins of known structures, but do not have homologous proteins with known structure.
It differs from the homology modeling method of structure prediction as it (protein threading) is used for proteins which do not have their homologous protein structures deposited in the Protein Data Bank (PDB), whereas homology modeling is used for those proteins which do. Threading works by using statistical knowledge of the relationship between the structures deposited in the PDB and the sequence of the protein which one wishes to model.
The prediction is made by "threading" (i.e. placing, aligning) each amino acid in the target sequence to a position in the template structure, and evaluating how well the target fits the template. After the best-fit template is selected, the structural model of the sequence is built based on the alignment with the chosen template. Protein threading is based on two basic observations: that the number of different folds in nature is fairly small (approximately 1300); and that 90% of the new structures submitted to the PDB in the past three years have similar structural folds to ones already in the PDB.
== Classification of protein structure ==
The Structural Classification of Proteins database (SCOP) provides a detailed and comprehensive description of the structural and evolutionary relationships of known structure. Proteins are classified to reflect both structural and evolutionary relatedness. Many levels exist in the hierarchy, but the principal levels are family, superfamily, and fold:
Family (clear evolutionary relationship): Proteins clustered together into families are clearly evolutionarily related. Generally, this means that pairwise residue identities between the proteins are 30% and greater. However, in some cases similar functions and structures provide definitive evidence of common descent in the absence of high sequence identity; for example, many globins form a family though some members have sequence identities of only 15%.
Superfamily (probable common evolutionary origin): Proteins that have low sequence identities, but whose structural and functional features suggest that a common evolutionary origin is probable, are placed together in superfamilies. For example, actin, the ATPase domain of the heat shock protein, and hexokinase together form a superfamily.
Fold (major structural similarity): Proteins are defined as having a common fold if they have the same major secondary structures in the same arrangement and with the same topological connections. Different proteins with the same fold often have peripheral elements of secondary structure and turn regions that differ in size and conformation. In some cases, these differing peripheral regions may comprise half the structure. Proteins placed together in the same fold category may not have a common evolutionary origin: the structural similarities could arise just from the physics and chemistry of proteins favoring certain packing arrangements and chain topologies.
=== Method ===
A general paradigm of protein threading consists of the following four steps:
The construction of a structure template database: Select protein structures from the protein structure databases as structural templates. This generally involves selecting protein structures from databases such as Protein Data Bank (PDB), Families of Structurally Similar Proteins database (FSSP), Structural Classification of Proteins database (SCOP), or CATH database, after removing protein structures with high sequence similarities.
The design of the scoring function: Design a good scoring function to measure the fitness between target sequences and templates based on the knowledge of the known relationships between the structures and the sequences. A good scoring function should contain mutation potential, environment fitness potential, pairwise potential, secondary structure compatibilities, and gap penalties. The quality of the energy function is closely related to the prediction accuracy, especially the alignment accuracy.
Threading alignment: Align the target sequence with each of the structure templates by optimizing the designed scoring function. This step is one of the major tasks of all threading-based structure prediction programs that take into account the pairwise contact potential; otherwise, a dynamic programming algorithm can fulfill it.
Threading prediction: Select the threading alignment that is statistically most probable as the threading prediction. Then construct a structure model for the target by placing the backbone atoms of the target sequence at their aligned backbone positions of the selected structural template.
=== Comparison with homology modeling ===
Homology modeling and protein threading are both template-based methods and there is no rigorous boundary between them in terms of prediction techniques. But the protein structures of their targets are different. Homology modeling is for those targets which have homologous proteins with known structure (usually/maybe of same family), while protein threading is for those targets with only fold-level homology found. In other words, homology modeling is for "easier" targets and protein threading is for "harder" targets.
Homology modeling treats the template in an alignment as a sequence, and only sequence homology is used for prediction. Protein threading treats the template in an alignment as a structure, and both sequence and structure information extracted from the alignment are used for prediction. When there is no significant homology found, protein threading can make a prediction based on the structure information. That also explains why protein threading may be more effective than homology modeling in many cases.
In practice, when the sequence identity in a sequence sequence alignment is low (i.e. <25%), homology modeling may not produce a significant prediction. In this case, if there is distant homology found for the target, protein threading can generate a good prediction.
=== More about threading ===
Fold recognition methods can be broadly divided into two types: those that derive a 1-D profile for each structure in the fold library and align the target sequence to these profiles; and those that consider the full 3-D structure of the protein template. A simple example of a profile representation would be to take each amino acid in the structure and simply label it according to whether it is buried in the core of the protein or exposed on the surface. More elaborate profiles might take into account the local secondary structure (e.g. whether the amino acid is part of an alpha helix) or even evolutionary information (how conserved the amino acid is). In the 3-D representation, the structure is modeled as a set of inter-atomic distances, i.e. the distances are calculated between some or all of the atom pairs in the structure. This is a much richer and far more flexible description of the structure, but is much harder to use in calculating an alignment. The profile-based fold recognition approach was first described by Bowie, Lüthy and David Eisenberg in 1991. The term threading was first coined by David Jones, William R. Taylor and Janet Thornton in 1992, and originally referred specifically to the use of a full 3-D structure atomic representation of the protein template in fold recognition. Today, the terms threading and fold recognition are frequently (though somewhat incorrectly) used interchangeably.
Fold recognition methods are widely used and effective because it is believed that there are a strictly limited number of different protein folds in nature, mostly as a result of evolution but also due to constraints imposed by the basic physics and chemistry of polypeptide chains. There is, therefore, a good chance (currently 70-80%) that a protein which has a similar fold to the target protein has already been studied by X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy and can be found in the PDB. Currently there are nearly 1300 different protein folds known, but new folds are still being discovered every year due in significant part to the ongoing structural genomics projects.
Many different algorithms have been proposed for finding the correct threading of a sequence onto a structure, though many make use of dynamic programming in some form. For full 3-D threading, the problem of identifying the best alignment is very difficult (it is an NP-hard problem for some models of threading). Researchers have made use of many combinatorial optimization methods such as conditional random fields, simulated annealing, branch and bound, and linear programming, searching to arrive at heuristic solutions. It is interesting to compare threading methods to methods which attempt to align two protein structures (protein structural alignment), and indeed many of the same algorithms have been applied to both problems.
== Protein threading software ==
HHpred is a popular threading server which runs HHsearch, a widely used software for remote homology detection based on pairwise comparison of hidden Markov models.
RAPTOR is an integer programming based protein threading software. It has been replaced by a new protein threading program RaptorX, which employs probabilistic graphical models and statistical inference to both single template and multi-template based protein threading. RaptorX significantly outperforms RAPTOR and is especially good at aligning proteins with sparse sequence profile. The RaptorX server is free to public.
Phyre is a popular threading server combining HHsearch with ab initio and multiple-template modelling.
MUSTER is a standard threading algorithm based on dynamic programming and sequence profile-profile alignment. It also combines multiple structural resources to assist the sequence profile alignment.
SPARKS X is a probabilistic-based sequence-to-structure matching between predicted one-dimensional structural properties of query and corresponding native properties of templates.
BioShell is a threading algorithm using optimized profile-to-profile dynamic programming algorithm combined with predicted secondary structure.
== See also ==
Homology modeling
Protein structure prediction
Protein structure prediction software
== References ==
== Further reading == | Wikipedia/Threading_(protein_sequence) |
Cdc4 (cell division control protein 4) is a substrate recognition component of the SCF (SKP1-CUL1-F-box protein) ubiquitin ligase complex, which acts as a mediator of ubiquitin transfer to target proteins, leading to their subsequent degradation via the ubiquitin-proteasome pathway. Cdc4 targets primarily cell cycle regulators for proteolysis. It serves the function of an adaptor that brings target molecules to the core SCF complex.
Cdc4 was originally identified in the model organism Saccharomyces cerevisiae.
CDC4 gene function is required at G1/S and G2/M transitions during mitosis and at various stages during meiosis.
== Homologues ==
The human homologue of the cdc4 gene is called FBXW7. The corresponding gene product is the F-box/WD repeat-containing protein 7.
In the nematode C. elegans, the homologue to Cdc4 is F-box/WD repeat-containing protein sel-10.
== Some general features ==
Cdc4 has a molecular weight of 86'089Da, an isoelectric point of 7.14, and consists of 779 amino acids. It resides exclusively in the nucleus because of a single monopartite nuclear localisation sequence (NLS) comprising amino acids 82-85 in the N-terminal domain.
== Structure ==
Cdc4 is one component of the E3 complex SCF (CDC4), which comprises CDC53, SKP1, RBX1, and CDC4.
Its 779 amino acids (in S. cerevisiae) are arranged into one F-box domain (approximately 40 amino acids ("F-box" motif)) and 7 WD repeats.
Cdc4 is a WD-40 repeat F-box protein. Like all members of this family, it contains a conserved dimerization motif called D domain. In yeast Cdc4, the D domain protomers arrange in a superhelical homodimeric manner. SCF (Cdc4) dimerization hardly affects the affinity for target molecules, but significantly increases ubiquitin conjugation. Cdc4 adapts a suprafacial configuration: The substrate-binding sites lie in the same plane AS the catalytic sites, with a separation of 64Å within and 102Å between each SCF monomer. In Cdc4, the substrate binding domain is built on WD40 domains, which use repeats of 40 amino acids), each forming four anti-parallel beta-strands, to assemble the blades of a so-called beta-propeller. Beta-propellers are a quite frequent form of adaptable surface for interaction between different proteins. This substrate interaction region is located C-terminally. There are three isoforms of Cdc4 in mammals: α, β, and γ. These are produced via alternative splicing of 3 unique 5’ exons to 10 common 3’ exons. This results in proteins that differ only at their N-termini.
Cdc4 protein interacts with Cdc34, an ubiquitin-conjugating enzyme, and Cdc53 in vivo. (There is a Cdc4p/Cdc53p-binding region on Cdc34p.) All three proteins are stable throughout the cell cycle.
== Function ==
Various cellular regulatory mechanisms heavily depend on ubiquitin-dependent degradation. The SCF (Cdc4) complex has a regulatory function in cell cycle progression, signal transduction, and transcription.
In order for the cell cycle to proceed, several inhibitory proteins, as well as cyclins, have to be eliminated at given time points. Cdc4 assists there by recruiting target molecules via its C-terminal substrate interaction domain (WD40 repeat domain) to the ubiquitination machinery. This causes transfer of ubiquitin molecules to the target, hence marks it for degradation.
Cdc4 recognizes and binds to phosphorylated target proteins.
Cdc4 can be essential, or nonessential, depending on the organism. For instance, it is essential in S. cerevisiae, while it is non-essential in C. albicans.
It is essential for initiation of DNA replication and separation of spindle pole bodies, hence for the formation of the poles of the mitotic spindle. In budding yeast it is also involved in bud development, fusion of zygotic nuclei (karyogamy) after conjugation, and several aspects of sporulation.
Roughly speaking, in the cell cycle Cdc4 function is required for G1/S and G2/M transition.
Some important interactions in which Cdc4 is involved are:
ubiquitination of the phosphorylated form of the cell cycle kinase inhibitor (CKI) SIC1
degradation of the CKI FAR1 in absence of pheromone; restriction of FAR1 degradation to the nucleus (since Cdc4 is exclusively nuclear)
transcription activation of the HTA1-HTB1 locus
degradation of the phosphorylated form of Cdc6
=== Onset of S-phase ===
Swi5 is a transcriptional activator of Sic1, which inhibits S-phase CDKs. Thus, Sic1 protein degradation is necessary to enter S-phase. SCF (Cdc4) complex’s regulatory function concerning S-phase entry comprises not only degradation of Sic1, but also degradation of Swi5.
In order for the substrate adapter unit Cdc4 to bind to Sic1, a minimum of any six of the nine cyclin-dependent kinase sites on Sic1 have to be phosphorylated. In other words: There is a threshold number of phosphorylation sites in order to achieve receptor-ligand binding. As recently stated, this "suggests that the ultrasensitivity in the Sic1-Cdc4 system may be driven at least in part by cumulative electrostatic interactions". In general, an ultrasensitive enzyme requires less than 81-fold increase in stimulus to drive it from 10% to 90% activity. "Ultrasensitivity" highlights that the upstroke of the stimulus/response curve is steeper than the one that is obtained for a hyperbolic Michaelis-Menten enzyme. Thus, ultrasensitivity allows a highly sensitive response: A graded input can be transformed into a sharply thresholded output. The development of B-type cyclin–cyclin-dependent kinase activity, as well as the onset of DNA replication, requires degradation of Sic1 in the late G1 phase of the cell cycle. The WD domain of Cdc4 binds to the phosphorylated form of Sic1. Each bond to a Sic1-Phosphate is weak, but together the binding is strong enough to enable Sic1-degradation via the pathway described before. Hence, in this case ultrasensitivity allows precise definition ("fine tuning") of the time point in which destruction of Sic1 occurs, leading to initiation of the next step in the cell cycle (-> DNA replication).
=== G2/M transition ===
Up until now it is not satisfyingly understood how Cdc4 triggers G2-M transition. In general, the second degradation complex involved in cell cycle progression, APC, is responsible for proteolysis at that stage.
However, experimental data suggests that Cdc4 function in G2/M transition may be linked to the degradation of Pds1 (anaphase inhibitor). And what is more, CDC4 and CDC20, an activator of APC, interact genetically.
Cdc4 recruits several other substrates than Sic1 to the SCF core complex, including the Cln-Cdc28 inhibitor / cytoskeletal scaffold protein Far1, the transcription factor Gcn4, and the replication protein Cdc6.
In addition to those functions mentioned above, Cdc4 is involved in some other degradation-dependent events in S. cerevisiae like for instance unfolded protein response.
== Clinical significance ==
In mammals, amongst others c-Myc, Src3, Cyclin E, and the Notch intracellular domain are substrates of Cdc4. Due to its involvement in degradation of various cell cycle regulators, as well as several compounds of signaling pathways (e.g. Notch), Cdc4 is a highly sensible component of every organism in which it functions.
The cdc4 gene is a haplo-insufficient tumor suppressor gene. Knock-out of this gene in mice leads to an embryonic lethal phenotype. CDC4 mutations occur in a number of cancer types. They are described best in colorectal tumors, and also have been found to be a mutational target in pancreatic cancer.
E3 has an additional function to its primary role in the degradation of certain cell cycle regulators: It is also involved in formation of the neural crest. Hence, Cdc4 is a protein "with separable but complementary functions in control of cell proliferation and differentiation". This evokes the assumption -beyond regulating cell cycle progression- Cdc4 as a tumor suppressor protein may extend its ability to directly regulate tissue differentiation. However, its concrete role in diseases is still to be elucidated.
== See also ==
ubiquitin ligase
ubiquitin proteasome system
cell cycle
== References == | Wikipedia/Cell_division_control_protein_4 |
A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multidomain enzymes, in which multiple catalytic domains are found in a single polypeptide chain.
Protein complexes are a form of quaternary structure. Proteins in a protein complex are linked by non-covalent protein–protein interactions. These complexes are a cornerstone of many (if not most) biological processes. The cell is seen to be composed of modular supramolecular complexes, each of which performs an independent, discrete biological function.
Through proximity, the speed and selectivity of binding interactions between enzymatic complex and substrates can be vastly improved, leading to higher cellular efficiency. Many of the techniques used to enter cells and isolate proteins are inherently disruptive to such large complexes, complicating the task of determining the components of a complex.
Examples of protein complexes include the proteasome for molecular degradation and most RNA polymerases. In stable complexes, large hydrophobic interfaces between proteins typically bury surface areas larger than 2500 square Ås.
== Function ==
Protein complex formation can activate or inhibit one or more of the complex members and in this way, protein complex formation can be similar to phosphorylation. Individual proteins can participate in a variety of protein complexes. Different complexes perform different functions, and the same complex can perform multiple functions depending on various factors. Factors include:
Cell compartment location
Cell cycle stage
Cell nutritional status
Many protein complexes are well understood, particularly in the model organism Saccharomyces cerevisiae (yeast). For this relatively simple organism, the study of protein complexes is now genome wide and the elucidation of most of its protein complexes is ongoing. In 2021, researchers used deep learning software RoseTTAFold along with AlphaFold to solve the structures of 712 eukaryote complexes. They compared 6000 yeast proteins to those from 2026 other fungi and 4325 other eukaryotes.
== Types of protein complexes ==
=== Obligate vs non-obligate protein complex ===
If a protein can form a stable well-folded structure on its own (without any other associated protein) in vivo, then the complexes formed by such proteins are termed "non-obligate protein complexes". However, some proteins can't be found to create a stable well-folded structure alone, but can be found as a part of a protein complex which stabilizes the constituent proteins. Such protein complexes are called "obligate protein complexes".
=== Transient vs permanent/stable protein complex ===
Transient protein complexes form and break down transiently in vivo, whereas permanent complexes have a relatively long half-life. Typically, the obligate interactions (protein–protein interactions in an obligate complex) are permanent, whereas non-obligate interactions have been found to be either permanent or transient. Note that there is no clear distinction between obligate and non-obligate interaction, rather there exist a continuum between them which depends on various conditions e.g. pH, protein concentration etc. However, there are important distinctions between the properties of transient and permanent/stable interactions: stable interactions are highly conserved but transient interactions are far less conserved, interacting proteins on the two sides of a stable interaction have more tendency of being co-expressed than those of a transient interaction (in fact, co-expression probability between two transiently interacting proteins is not higher than two random proteins), and transient interactions are much less co-localized than stable interactions. Though, transient by nature, transient interactions are very important for cell biology: the human interactome is enriched in such interactions, these interactions are the dominating players of gene regulation and signal transduction, and proteins with intrinsically disordered regions (IDR: regions in protein that show dynamic inter-converting structures in the native state) are found to be enriched in transient regulatory and signaling interactions.
=== Fuzzy complex ===
Fuzzy protein complexes have more than one structural form or dynamic structural disorder in the bound state. This means that proteins may not fold completely in either transient or permanent complexes. Consequently, specific complexes can have ambiguous interactions, which vary according to the environmental signals. Hence different ensembles of structures result in different (even opposite) biological functions. Post-translational modifications, protein interactions or alternative splicing modulate the conformational ensembles of fuzzy complexes, to fine-tune affinity or specificity of interactions. These mechanisms are often used for regulation within the eukaryotic transcription machinery.
== Essential proteins in protein complexes ==
Although some early studies suggested a strong correlation between essentiality and protein interaction degree (the "centrality-lethality" rule) subsequent analyses have shown that this correlation is weak for binary or transient interactions (e.g., yeast two-hybrid). However, the correlation is robust for networks of stable co-complex interactions. In fact, a disproportionate number of essential genes belong to protein complexes. This led to the conclusion that essentiality is a property of molecular machines (i.e. complexes) rather than individual components. Wang et al. (2009) noted that larger protein complexes are more likely to be essential, explaining why essential genes are more likely to have high co-complex interaction degree. Ryan et al. (2013) referred to the observation that entire complexes appear essential as "modular essentiality". These authors also showed that complexes tend to be composed of either essential or non-essential proteins rather than showing a random distribution (see Figure). However, this not an all or nothing phenomenon: only about 26% (105/401) of yeast complexes consist of solely essential or solely nonessential subunits.
In humans, genes whose protein products belong to the same complex are more likely to result in the same disease phenotype.
== Homomultimeric and heteromultimeric proteins ==
The subunits of a multimeric protein may be identical as in a homomultimeric (homooligomeric) protein or different as in a heteromultimeric protein. Many soluble and membrane proteins form homomultimeric complexes in a cell, majority of proteins in the Protein Data Bank are homomultimeric. Homooligomers are responsible for the diversity and specificity of many pathways, may mediate and regulate gene expression, activity of enzymes, ion channels, receptors, and cell adhesion processes.
The voltage-gated potassium channels in the plasma membrane of a neuron are heteromultimeric proteins composed of four of forty known alpha subunits. Subunits must be of the same subfamily to form the multimeric protein channel. The tertiary structure of the channel allows ions to flow through the hydrophobic plasma membrane. Connexons are an example of a homomultimeric protein composed of six identical connexins. A cluster of connexons forms the gap-junction in two neurons that transmit signals through an electrical synapse.
=== Intragenic complementation ===
When multiple copies of a polypeptide encoded by a gene form a complex, this protein structure is referred to as a multimer. When a multimer is formed from polypeptides produced by two different mutant alleles of a particular gene, the mixed multimer may exhibit greater functional activity than the unmixed multimers formed by each of the mutants alone. In such a case, the phenomenon is referred to as intragenic complementation (also called inter-allelic complementation). Intragenic complementation has been demonstrated in many different genes in a variety of organisms including the fungi Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe; the bacterium Salmonella typhimurium; the virus bacteriophage T4, an RNA virus and humans. In such studies, numerous mutations defective in the same gene were often isolated and mapped in a linear order on the basis of recombination frequencies to form a genetic map of the gene. Separately, the mutants were tested in pairwise combinations to measure complementation. An analysis of the results from such studies led to the conclusion that intragenic complementation, in general, arises from the interaction of differently defective polypeptide monomers to form a multimer. Genes that encode multimer-forming polypeptides appear to be common. One interpretation of the data is that polypeptide monomers are often aligned in the multimer in such a way that mutant polypeptides defective at nearby sites in the genetic map tend to form a mixed multimer that functions poorly, whereas mutant polypeptides defective at distant sites tend to form a mixed multimer that functions more effectively. The intermolecular forces likely responsible for self-recognition and multimer formation were discussed by Jehle.
== Structure determination ==
The molecular structure of protein complexes can be determined by experimental techniques such as X-ray crystallography, Single particle analysis or nuclear magnetic resonance. Increasingly the theoretical option of protein–protein docking is also becoming available. One method that is commonly used for identifying the meomplexes is immunoprecipitation. Recently, Raicu and coworkers developed a method to determine the quaternary structure of protein complexes in living cells. This method is based on the determination of pixel-level Förster resonance energy transfer (FRET) efficiency in conjunction with spectrally resolved two-photon microscope. The distribution of FRET efficiencies are simulated against different models to get the geometry and stoichiometry of the complexes.
== Assembly ==
Proper assembly of multiprotein complexes is important, since misassembly can lead to disastrous consequences. In order to study pathway assembly, researchers look at intermediate steps in the pathway. One such technique that allows one to do that is electrospray mass spectrometry, which can identify different intermediate states simultaneously. This has led to the discovery that most complexes follow an ordered assembly pathway. In the cases where disordered assembly is possible, the change from an ordered to a disordered state leads to a transition from function to dysfunction of the complex, since disordered assembly leads to aggregation.
The structure of proteins play a role in how the multiprotein complex assembles. The interfaces between proteins can be used to predict assembly pathways. The intrinsic flexibility of proteins also plays a role: more flexible proteins allow for a greater surface area available for interaction.
While assembly is a different process from disassembly, the two are reversible in both homomeric and heteromeric complexes. Thus, the overall process can be referred to as (dis)assembly.
=== Evolutionary significance of multiprotein complex assembly ===
In homomultimeric complexes, the homomeric proteins assemble in a way that mimics evolution. That is, an intermediate in the assembly process is present in the complex's evolutionary history.
The opposite phenomenon is observed in heteromultimeric complexes, where gene fusion occurs in a manner that preserves the original assembly pathway.
== See also ==
Heterotetramer
Biomolecular complex
Protein subunit
== References ==
== External links ==
Multiprotein+Complexes at the U.S. National Library of Medicine Medical Subject Headings (MeSH) | Wikipedia/Protein_complexes |
Biology is the scientific study of life and living organisms. It is a broad natural science that encompasses a wide range of fields and unifying principles that explain the structure, function, growth, origin, evolution, and distribution of life. Central to biology are five fundamental themes: the cell as the basic unit of life, genes and heredity as the basis of inheritance, evolution as the driver of biological diversity, energy transformation for sustaining life processes, and the maintenance of internal stability (homeostasis).
Biology examines life across multiple levels of organization, from molecules and cells to organisms, populations, and ecosystems. Subdisciplines include molecular biology, physiology, ecology, evolutionary biology, developmental biology, and systematics, among others. Each of these fields applies a range of methods to investigate biological phenomena, including observation, experimentation, and mathematical modeling. Modern biology is grounded in the theory of evolution by natural selection, first articulated by Charles Darwin, and in the molecular understanding of genes encoded in DNA. The discovery of the structure of DNA and advances in molecular genetics have transformed many areas of biology, leading to applications in medicine, agriculture, biotechnology, and environmental science.
Life on Earth is believed to have originated over 3.7 billion years ago. Today, it includes a vast diversity of organisms—from single-celled archaea and bacteria to complex multicellular plants, fungi, and animals. Biologists classify organisms based on shared characteristics and evolutionary relationships, using taxonomic and phylogenetic frameworks. These organisms interact with each other and with their environments in ecosystems, where they play roles in energy flow and nutrient cycling. As a constantly evolving field, biology incorporates new discoveries and technologies that enhance the understanding of life and its processes, while contributing to solutions for challenges such as disease, climate change, and biodiversity loss.
== Etymology ==
From Greek bios, life, (from Proto-Indo-European root *gwei-, to live) and logy, study of. The compound was coined in 1800 by Karl Friedrich Burdach and in 1802 used by both German naturalist Gottfried Reinhold Treviranus and Jean-Baptiste Lamarck.
== History ==
The earliest of roots of science, which included medicine, can be traced to ancient Egypt and Mesopotamia in around 3000 to 1200 BCE. Their contributions shaped ancient Greek natural philosophy. Ancient Greek philosophers such as Aristotle (384–322 BCE) contributed extensively to the development of biological knowledge. He explored biological causation and the diversity of life. His successor, Theophrastus, began the scientific study of plants. Scholars of the medieval Islamic world who wrote on biology included al-Jahiz (781–869), Al-Dīnawarī (828–896), who wrote on botany, and Rhazes (865–925) who wrote on anatomy and physiology. Medicine was especially well studied by Islamic scholars working in Greek philosopher traditions, while natural history drew heavily on Aristotelian thought.
Biology began to quickly develop with Anton van Leeuwenhoek's dramatic improvement of the microscope. It was then that scholars discovered spermatozoa, bacteria, infusoria and the diversity of microscopic life. Investigations by Jan Swammerdam led to new interest in entomology and helped to develop techniques of microscopic dissection and staining. Advances in microscopy had a profound impact on biological thinking. In the early 19th century, biologists pointed to the central importance of the cell. In 1838, Schleiden and Schwann began promoting the now universal ideas that (1) the basic unit of organisms is the cell and (2) that individual cells have all the characteristics of life, although they opposed the idea that (3) all cells come from the division of other cells, continuing to support spontaneous generation. However, Robert Remak and Rudolf Virchow were able to reify the third tenet, and by the 1860s most biologists accepted all three tenets which consolidated into cell theory.
Meanwhile, taxonomy and classification became the focus of natural historians. Carl Linnaeus published a basic taxonomy for the natural world in 1735, and in the 1750s introduced scientific names for all his species. Georges-Louis Leclerc, Comte de Buffon, treated species as artificial categories and living forms as malleable—even suggesting the possibility of common descent.
Serious evolutionary thinking originated with the works of Jean-Baptiste Lamarck, who presented a coherent theory of evolution. The British naturalist Charles Darwin, combining the biogeographical approach of Humboldt, the uniformitarian geology of Lyell, Malthus's writings on population growth, and his own morphological expertise and extensive natural observations, forged a more successful evolutionary theory based on natural selection; similar reasoning and evidence led Alfred Russel Wallace to independently reach the same conclusions.
The basis for modern genetics began with the work of Gregor Mendel in 1865. This outlined the principles of biological inheritance. However, the significance of his work was not realized until the early 20th century when evolution became a unified theory as the modern synthesis reconciled Darwinian evolution with classical genetics. In the 1940s and early 1950s, a series of experiments by Alfred Hershey and Martha Chase pointed to DNA as the component of chromosomes that held the trait-carrying units that had become known as genes. A focus on new kinds of model organisms such as viruses and bacteria, along with the discovery of the double-helical structure of DNA by James Watson and Francis Crick in 1953, marked the transition to the era of molecular genetics. From the 1950s onwards, biology has been vastly extended in the molecular domain. The genetic code was cracked by Har Gobind Khorana, Robert W. Holley and Marshall Warren Nirenberg after DNA was understood to contain codons. The Human Genome Project was launched in 1990 to map the human genome.
== Chemical basis ==
=== Atoms and molecules ===
All organisms are made up of chemical elements; oxygen, carbon, hydrogen, and nitrogen account for most (96%) of the mass of all organisms, with calcium, phosphorus, sulfur, sodium, chlorine, and magnesium constituting essentially all the remainder. Different elements can combine to form compounds such as water, which is fundamental to life. Biochemistry is the study of chemical processes within and relating to living organisms. Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including molecular synthesis, modification, mechanisms, and interactions.
=== Water ===
Life arose from the Earth's first ocean, which formed some 3.8 billion years ago. Since then, water continues to be the most abundant molecule in every organism. Water is important to life because it is an effective solvent, capable of dissolving solutes such as sodium and chloride ions or other small molecules to form an aqueous solution. Once dissolved in water, these solutes are more likely to come in contact with one another and therefore take part in chemical reactions that sustain life. In terms of its molecular structure, water is a small polar molecule with a bent shape formed by the polar covalent bonds of two hydrogen (H) atoms to one oxygen (O) atom (H2O). Because the O–H bonds are polar, the oxygen atom has a slight negative charge and the two hydrogen atoms have a slight positive charge. This polar property of water allows it to attract other water molecules via hydrogen bonds, which makes water cohesive. Surface tension results from the cohesive force due to the attraction between molecules at the surface of the liquid. Water is also adhesive as it is able to adhere to the surface of any polar or charged non-water molecules. Water is denser as a liquid than it is as a solid (or ice). This unique property of water allows ice to float above liquid water such as ponds, lakes, and oceans, thereby insulating the liquid below from the cold air above. Water has the capacity to absorb energy, giving it a higher specific heat capacity than other solvents such as ethanol. Thus, a large amount of energy is needed to break the hydrogen bonds between water molecules to convert liquid water into water vapor. As a molecule, water is not completely stable as each water molecule continuously dissociates into hydrogen and hydroxyl ions before reforming into a water molecule again. In pure water, the number of hydrogen ions balances (or equals) the number of hydroxyl ions, resulting in a pH that is neutral.
=== Organic compounds ===
Organic compounds are molecules that contain carbon bonded to another element such as hydrogen. With the exception of water, nearly all the molecules that make up each organism contain carbon. Carbon can form covalent bonds with up to four other atoms, enabling it to form diverse, large, and complex molecules. For example, a single carbon atom can form four single covalent bonds such as in methane, two double covalent bonds such as in carbon dioxide (CO2), or a triple covalent bond such as in carbon monoxide (CO). Moreover, carbon can form very long chains of interconnecting carbon–carbon bonds such as octane or ring-like structures such as glucose.
The simplest form of an organic molecule is the hydrocarbon, which is a large family of organic compounds that are composed of hydrogen atoms bonded to a chain of carbon atoms. A hydrocarbon backbone can be substituted by other elements such as oxygen (O), hydrogen (H), phosphorus (P), and sulfur (S), which can change the chemical behavior of that compound. Groups of atoms that contain these elements (O-, H-, P-, and S-) and are bonded to a central carbon atom or skeleton are called functional groups. There are six prominent functional groups that can be found in organisms: amino group, carboxyl group, carbonyl group, hydroxyl group, phosphate group, and sulfhydryl group.
In 1953, the Miller–Urey experiment showed that organic compounds could be synthesized abiotically within a closed system mimicking the conditions of early Earth, thus suggesting that complex organic molecules could have arisen spontaneously in early Earth (see abiogenesis).
=== Macromolecules ===
Macromolecules are large molecules made up of smaller subunits or monomers. Monomers include sugars, amino acids, and nucleotides. Carbohydrates include monomers and polymers of sugars.
Lipids are the only class of macromolecules that are not made up of polymers. They include steroids, phospholipids, and fats, largely nonpolar and hydrophobic (water-repelling) substances.
Proteins are the most diverse of the macromolecules. They include enzymes, transport proteins, large signaling molecules, antibodies, and structural proteins. The basic unit (or monomer) of a protein is an amino acid. Twenty amino acids are used in proteins.
Nucleic acids are polymers of nucleotides. Their function is to store, transmit, and express hereditary information.
== Cells ==
Cell theory states that cells are the fundamental units of life, that all living things are composed of one or more cells, and that all cells arise from preexisting cells through cell division. Most cells are very small, with diameters ranging from 1 to 100 micrometers and are therefore only visible under a light or electron microscope. There are generally two types of cells: eukaryotic cells, which contain a nucleus, and prokaryotic cells, which do not. Prokaryotes are single-celled organisms such as bacteria, whereas eukaryotes can be single-celled or multicellular. In multicellular organisms, every cell in the organism's body is derived ultimately from a single cell in a fertilized egg.
=== Cell structure ===
Every cell is enclosed within a cell membrane that separates its cytoplasm from the extracellular space. A cell membrane consists of a lipid bilayer, including cholesterols that sit between phospholipids to maintain their fluidity at various temperatures. Cell membranes are semipermeable, allowing small molecules such as oxygen, carbon dioxide, and water to pass through while restricting the movement of larger molecules and charged particles such as ions. Cell membranes also contain membrane proteins, including integral membrane proteins that go across the membrane serving as membrane transporters, and peripheral proteins that loosely attach to the outer side of the cell membrane, acting as enzymes shaping the cell. Cell membranes are involved in various cellular processes such as cell adhesion, storing electrical energy, and cell signalling and serve as the attachment surface for several extracellular structures such as a cell wall, glycocalyx, and cytoskeleton.
Within the cytoplasm of a cell, there are many biomolecules such as proteins and nucleic acids. In addition to biomolecules, eukaryotic cells have specialized structures called organelles that have their own lipid bilayers or are spatially units. These organelles include the cell nucleus, which contains most of the cell's DNA, or mitochondria, which generate adenosine triphosphate (ATP) to power cellular processes. Other organelles such as endoplasmic reticulum and Golgi apparatus play a role in the synthesis and packaging of proteins, respectively. Biomolecules such as proteins can be engulfed by lysosomes, another specialized organelle. Plant cells have additional organelles that distinguish them from animal cells such as a cell wall that provides support for the plant cell, chloroplasts that harvest sunlight energy to produce sugar, and vacuoles that provide storage and structural support as well as being involved in reproduction and breakdown of plant seeds. Eukaryotic cells also have cytoskeleton that is made up of microtubules, intermediate filaments, and microfilaments, all of which provide support for the cell and are involved in the movement of the cell and its organelles. In terms of their structural composition, the microtubules are made up of tubulin (e.g., α-tubulin and β-tubulin) whereas intermediate filaments are made up of fibrous proteins. Microfilaments are made up of actin molecules that interact with other strands of proteins.
=== Metabolism ===
All cells require energy to sustain cellular processes. Metabolism is the set of chemical reactions in an organism. The three main purposes of metabolism are: the conversion of food to energy to run cellular processes; the conversion of food/fuel to monomer building blocks; and the elimination of metabolic wastes. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolic reactions may be categorized as catabolic—the breaking down of compounds (for example, the breaking down of glucose to pyruvate by cellular respiration); or anabolic—the building up (synthesis) of compounds (such as proteins, carbohydrates, lipids, and nucleic acids). Usually, catabolism releases energy, and anabolism consumes energy. The chemical reactions of metabolism are organized into metabolic pathways, in which one chemical is transformed through a series of steps into another chemical, each step being facilitated by a specific enzyme. Enzymes are crucial to metabolism because they allow organisms to drive desirable reactions that require energy that will not occur by themselves, by coupling them to spontaneous reactions that release energy. Enzymes act as catalysts—they allow a reaction to proceed more rapidly without being consumed by it—by reducing the amount of activation energy needed to convert reactants into products. Enzymes also allow the regulation of the rate of a metabolic reaction, for example in response to changes in the cell's environment or to signals from other cells.
=== Cellular respiration ===
Cellular respiration is a set of metabolic reactions and processes that take place in cells to convert chemical energy from nutrients into adenosine triphosphate (ATP), and then release waste products. The reactions involved in respiration are catabolic reactions, which break large molecules into smaller ones, releasing energy. Respiration is one of the key ways a cell releases chemical energy to fuel cellular activity. The overall reaction occurs in a series of biochemical steps, some of which are redox reactions. Although cellular respiration is technically a combustion reaction, it clearly does not resemble one when it occurs in a cell because of the slow, controlled release of energy from the series of reactions.
Sugar in the form of glucose is the main nutrient used by animal and plant cells in respiration. Cellular respiration involving oxygen is called aerobic respiration, which has four stages: glycolysis, citric acid cycle (or Krebs cycle), electron transport chain, and oxidative phosphorylation. Glycolysis is a metabolic process that occurs in the cytoplasm whereby glucose is converted into two pyruvates, with two net molecules of ATP being produced at the same time. Each pyruvate is then oxidized into acetyl-CoA by the pyruvate dehydrogenase complex, which also generates NADH and carbon dioxide. Acetyl-CoA enters the citric acid cycle, which takes places inside the mitochondrial matrix. At the end of the cycle, the total yield from 1 glucose (or 2 pyruvates) is 6 NADH, 2 FADH2, and 2 ATP molecules. Finally, the next stage is oxidative phosphorylation, which in eukaryotes, occurs in the mitochondrial cristae. Oxidative phosphorylation comprises the electron transport chain, which is a series of four protein complexes that transfer electrons from one complex to another, thereby releasing energy from NADH and FADH2 that is coupled to the pumping of protons (hydrogen ions) across the inner mitochondrial membrane (chemiosmosis), which generates a proton motive force. Energy from the proton motive force drives the enzyme ATP synthase to synthesize more ATPs by phosphorylating ADPs. The transfer of electrons terminates with molecular oxygen being the final electron acceptor.
If oxygen were not present, pyruvate would not be metabolized by cellular respiration but undergoes a process of fermentation. The pyruvate is not transported into the mitochondrion but remains in the cytoplasm, where it is converted to waste products that may be removed from the cell. This serves the purpose of oxidizing the electron carriers so that they can perform glycolysis again and removing the excess pyruvate. Fermentation oxidizes NADH to NAD+ so it can be re-used in glycolysis. In the absence of oxygen, fermentation prevents the buildup of NADH in the cytoplasm and provides NAD+ for glycolysis. This waste product varies depending on the organism. In skeletal muscles, the waste product is lactic acid. This type of fermentation is called lactic acid fermentation. In strenuous exercise, when energy demands exceed energy supply, the respiratory chain cannot process all of the hydrogen atoms joined by NADH. During anaerobic glycolysis, NAD+ regenerates when pairs of hydrogen combine with pyruvate to form lactate. Lactate formation is catalyzed by lactate dehydrogenase in a reversible reaction. Lactate can also be used as an indirect precursor for liver glycogen. During recovery, when oxygen becomes available, NAD+ attaches to hydrogen from lactate to form ATP. In yeast, the waste products are ethanol and carbon dioxide. This type of fermentation is known as alcoholic or ethanol fermentation. The ATP generated in this process is made by substrate-level phosphorylation, which does not require oxygen.
=== Photosynthesis ===
Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that can later be released to fuel the organism's metabolic activities via cellular respiration. This chemical energy is stored in carbohydrate molecules, such as sugars, which are synthesized from carbon dioxide and water. In most cases, oxygen is released as a waste product. Most plants, algae, and cyanobacteria perform photosynthesis, which is largely responsible for producing and maintaining the oxygen content of the Earth's atmosphere, and supplies most of the energy necessary for life on Earth.
Photosynthesis has four stages: Light absorption, electron transport, ATP synthesis, and carbon fixation. Light absorption is the initial step of photosynthesis whereby light energy is absorbed by chlorophyll pigments attached to proteins in the thylakoid membranes. The absorbed light energy is used to remove electrons from a donor (water) to a primary electron acceptor, a quinone designated as Q. In the second stage, electrons move from the quinone primary electron acceptor through a series of electron carriers until they reach a final electron acceptor, which is usually the oxidized form of NADP+, which is reduced to NADPH, a process that takes place in a protein complex called photosystem I (PSI). The transport of electrons is coupled to the movement of protons (or hydrogen) from the stroma to the thylakoid membrane, which forms a pH gradient across the membrane as hydrogen becomes more concentrated in the lumen than in the stroma. This is analogous to the proton-motive force generated across the inner mitochondrial membrane in aerobic respiration.
During the third stage of photosynthesis, the movement of protons down their concentration gradients from the thylakoid lumen to the stroma through the ATP synthase is coupled to the synthesis of ATP by that same ATP synthase. The NADPH and ATPs generated by the light-dependent reactions in the second and third stages, respectively, provide the energy and electrons to drive the synthesis of glucose by fixing atmospheric carbon dioxide into existing organic carbon compounds, such as ribulose bisphosphate (RuBP) in a sequence of light-independent (or dark) reactions called the Calvin cycle.
=== Cell signaling ===
Cell signaling (or communication) is the ability of cells to receive, process, and transmit signals with its environment and with itself. Signals can be non-chemical such as light, electrical impulses, and heat, or chemical signals (or ligands) that interact with receptors, which can be found embedded in the cell membrane of another cell or located deep inside a cell. There are generally four types of chemical signals: autocrine, paracrine, juxtacrine, and hormones. In autocrine signaling, the ligand affects the same cell that releases it. Tumor cells, for example, can reproduce uncontrollably because they release signals that initiate their own self-division. In paracrine signaling, the ligand diffuses to nearby cells and affects them. For example, brain cells called neurons release ligands called neurotransmitters that diffuse across a synaptic cleft to bind with a receptor on an adjacent cell such as another neuron or muscle cell. In juxtacrine signaling, there is direct contact between the signaling and responding cells. Finally, hormones are ligands that travel through the circulatory systems of animals or vascular systems of plants to reach their target cells. Once a ligand binds with a receptor, it can influence the behavior of another cell, depending on the type of receptor. For instance, neurotransmitters that bind with an inotropic receptor can alter the excitability of a target cell. Other types of receptors include protein kinase receptors (e.g., receptor for the hormone insulin) and G protein-coupled receptors. Activation of G protein-coupled receptors can initiate second messenger cascades. The process by which a chemical or physical signal is transmitted through a cell as a series of molecular events is called signal transduction.
=== Cell cycle ===
The cell cycle is a series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA and some of its organelles, and the subsequent partitioning of its cytoplasm into two daughter cells in a process called cell division. In eukaryotes (i.e., animal, plant, fungal, and protist cells), there are two distinct types of cell division: mitosis and meiosis. Mitosis is part of the cell cycle, in which replicated chromosomes are separated into two new nuclei. Cell division gives rise to genetically identical cells in which the total number of chromosomes is maintained. In general, mitosis (division of the nucleus) is preceded by the S stage of interphase (during which the DNA is replicated) and is often followed by telophase and cytokinesis; which divides the cytoplasm, organelles and cell membrane of one cell into two new cells containing roughly equal shares of these cellular components. The different stages of mitosis all together define the mitotic phase of an animal cell cycle—the division of the mother cell into two genetically identical daughter cells. The cell cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed. After cell division, each of the daughter cells begin the interphase of a new cycle. In contrast to mitosis, meiosis results in four haploid daughter cells by undergoing one round of DNA replication followed by two divisions. Homologous chromosomes are separated in the first division (meiosis I), and sister chromatids are separated in the second division (meiosis II). Both of these cell division cycles are used in the process of sexual reproduction at some point in their life cycle. Both are believed to be present in the last eukaryotic common ancestor.
Prokaryotes (i.e., archaea and bacteria) can also undergo cell division (or binary fission). Unlike the processes of mitosis and meiosis in eukaryotes, binary fission in prokaryotes takes place without the formation of a spindle apparatus on the cell. Before binary fission, DNA in the bacterium is tightly coiled. After it has uncoiled and duplicated, it is pulled to the separate poles of the bacterium as it increases the size to prepare for splitting. Growth of a new cell wall begins to separate the bacterium (triggered by FtsZ polymerization and "Z-ring" formation). The new cell wall (septum) fully develops, resulting in the complete split of the bacterium. The new daughter cells have tightly coiled DNA rods, ribosomes, and plasmids.
=== Sexual reproduction and meiosis ===
Meiosis is a central feature of sexual reproduction in eukaryotes, and the most fundamental function of meiosis appears to be conservation of the integrity of the genome that is passed on to progeny by parents. Two aspects of sexual reproduction, meiotic recombination and outcrossing, are likely maintained respectively by the adaptive advantages of recombinational repair of genomic DNA damage and genetic complementation which masks the expression of deleterious recessive mutations.
The beneficial effect of genetic complementation, derived from outcrossing (cross-fertilization) is also referred to as hybrid vigor or heterosis. Charles Darwin in his 1878 book The Effects of Cross and Self-Fertilization in the Vegetable Kingdom at the start of chapter XII noted “The first and most important of the conclusions which may be drawn from the observations given in this volume, is that generally cross-fertilisation is beneficial and self-fertilisation often injurious, at least with the plants on which I experimented.” Genetic variation, often produced as a byproduct of sexual reproduction, may provide long-term advantages to those sexual lineages that engage in outcrossing.
== Genetics ==
=== Inheritance ===
Genetics is the scientific study of inheritance. Mendelian inheritance, specifically, is the process by which genes and traits are passed on from parents to offspring. It has several principles. The first is that genetic characteristics, alleles, are discrete and have alternate forms (e.g., purple vs. white or tall vs. dwarf), each inherited from one of two parents. Based on the law of dominance and uniformity, which states that some alleles are dominant while others are recessive; an organism with at least one dominant allele will display the phenotype of that dominant allele. During gamete formation, the alleles for each gene segregate, so that each gamete carries only one allele for each gene. Heterozygotic individuals produce gametes with an equal frequency of two alleles. Finally, the law of independent assortment, states that genes of different traits can segregate independently during the formation of gametes, i.e., genes are unlinked. An exception to this rule would include traits that are sex-linked. Test crosses can be performed to experimentally determine the underlying genotype of an organism with a dominant phenotype. A Punnett square can be used to predict the results of a test cross. The chromosome theory of inheritance, which states that genes are found on chromosomes, was supported by Thomas Morgans's experiments with fruit flies, which established the sex linkage between eye color and sex in these insects.
=== Genes and DNA ===
A gene is a unit of heredity that corresponds to a region of deoxyribonucleic acid (DNA) that carries genetic information that controls form or function of an organism. DNA is composed of two polynucleotide chains that coil around each other to form a double helix. It is found as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell is collectively known as its genome. In eukaryotes, DNA is mainly in the cell nucleus. In prokaryotes, the DNA is held within the nucleoid. The genetic information is held within genes, and the complete assemblage in an organism is called its genotype.
DNA replication is a semiconservative process whereby each strand serves as a template for a new strand of DNA. Mutations are heritable changes in DNA. They can arise spontaneously as a result of replication errors that were not corrected by proofreading or can be induced by an environmental mutagen such as a chemical (e.g., nitrous acid, benzopyrene) or radiation (e.g., x-ray, gamma ray, ultraviolet radiation, particles emitted by unstable isotopes). Mutations can lead to phenotypic effects such as loss-of-function, gain-of-function, and conditional mutations.
Some mutations are beneficial, as they are a source of genetic variation for evolution. Others are harmful if they were to result in a loss of function of genes needed for survival.
=== Gene expression ===
Gene expression is the molecular process by which a genotype encoded in DNA gives rise to an observable phenotype in the proteins of an organism's body. This process is summarized by the central dogma of molecular biology, which was formulated by Francis Crick in 1958. According to the Central Dogma, genetic information flows from DNA to RNA to protein. There are two gene expression processes: transcription (DNA to RNA) and translation (RNA to protein).
=== Gene regulation ===
The regulation of gene expression by environmental factors and during different stages of development can occur at each step of the process such as transcription, RNA splicing, translation, and post-translational modification of a protein. Gene expression can be influenced by positive or negative regulation, depending on which of the two types of regulatory proteins called transcription factors bind to the DNA sequence close to or at a promoter. A cluster of genes that share the same promoter is called an operon, found mainly in prokaryotes and some lower eukaryotes (e.g., Caenorhabditis elegans). In positive regulation of gene expression, the activator is the transcription factor that stimulates transcription when it binds to the sequence near or at the promoter. Negative regulation occurs when another transcription factor called a repressor binds to a DNA sequence called an operator, which is part of an operon, to prevent transcription. Repressors can be inhibited by compounds called inducers (e.g., allolactose), thereby allowing transcription to occur. Specific genes that can be activated by inducers are called inducible genes, in contrast to constitutive genes that are almost constantly active. In contrast to both, structural genes encode proteins that are not involved in gene regulation. In addition to regulatory events involving the promoter, gene expression can also be regulated by epigenetic changes to chromatin, which is a complex of DNA and protein found in eukaryotic cells.
=== Genes, development, and evolution ===
Development is the process by which a multicellular organism (plant or animal) goes through a series of changes, starting from a single cell, and taking on various forms that are characteristic of its life cycle. There are four key processes that underlie development: Determination, differentiation, morphogenesis, and growth. Determination sets the developmental fate of a cell, which becomes more restrictive during development. Differentiation is the process by which specialized cells arise from less specialized cells such as stem cells. Stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. Cellular differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals, which are largely due to highly controlled modifications in gene expression and epigenetics. With a few exceptions, cellular differentiation almost never involves a change in the DNA sequence itself. Thus, different cells can have very different physical characteristics despite having the same genome. Morphogenesis, or the development of body form, is the result of spatial differences in gene expression. A small fraction of the genes in an organism's genome called the developmental-genetic toolkit control the development of that organism. These toolkit genes are highly conserved among phyla, meaning that they are ancient and very similar in widely separated groups of animals. Differences in deployment of toolkit genes affect the body plan and the number, identity, and pattern of body parts. Among the most important toolkit genes are the Hox genes. Hox genes determine where repeating parts, such as the many vertebrae of snakes, will grow in a developing embryo or larva.
== Evolution ==
=== Evolutionary processes ===
Evolution is a central organizing concept in biology. It is the change in heritable characteristics of populations over successive generations. In artificial selection, animals were selectively bred for specific traits.
Given that traits are inherited, populations contain a varied mix of traits, and reproduction is able to increase any population, Darwin argued that in the natural world, it was nature that played the role of humans in selecting for specific traits. Darwin inferred that individuals who possessed heritable traits better adapted to their environments are more likely to survive and produce more offspring than other individuals. He further inferred that this would lead to the accumulation of favorable traits over successive generations, thereby increasing the match between the organisms and their environment.
=== Speciation ===
A species is a group of organisms that mate with one another and speciation is the process by which one lineage splits into two lineages as a result of having evolved independently from each other. For speciation to occur, there has to be reproductive isolation. Reproductive isolation can result from incompatibilities between genes as described by Bateson–Dobzhansky–Muller model. Reproductive isolation also tends to increase with genetic divergence. Speciation can occur when there are physical barriers that divide an ancestral species, a process known as allopatric speciation.
=== Phylogeny ===
A phylogeny is an evolutionary history of a specific group of organisms or their genes. It can be represented using a phylogenetic tree, a diagram showing lines of descent among organisms or their genes. Each line drawn on the time axis of a tree represents a lineage of descendants of a particular species or population. When a lineage divides into two, it is represented as a fork or split on the phylogenetic tree. Phylogenetic trees are the basis for comparing and grouping different species. Different species that share a feature inherited from a common ancestor are described as having homologous features (or synapomorphy). Phylogeny provides the basis of biological classification. This classification system is rank-based, with the highest rank being the domain followed by kingdom, phylum, class, order, family, genus, and species. All organisms can be classified as belonging to one of three domains: Archaea (originally Archaebacteria), Bacteria (originally eubacteria), or Eukarya (includes the fungi, plant, and animal kingdoms).
=== History of life ===
The history of life on Earth traces how organisms have evolved from the earliest emergence of life to present day. Earth formed about 4.5 billion years ago and all life on Earth, both living and extinct, descended from a last universal common ancestor that lived about 3.5 billion years ago. Geologists have developed a geologic time scale that divides the history of the Earth into major divisions, starting with four eons (Hadean, Archean, Proterozoic, and Phanerozoic), the first three of which are collectively known as the Precambrian, which lasted approximately 4 billion years. Each eon can be divided into eras, with the Phanerozoic eon that began 539 million years ago being subdivided into Paleozoic, Mesozoic, and Cenozoic eras. These three eras together comprise eleven periods (Cambrian, Ordovician, Silurian, Devonian, Carboniferous, Permian, Triassic, Jurassic, Cretaceous, Tertiary, and Quaternary).
The similarities among all known present-day species indicate that they have diverged through the process of evolution from their common ancestor. Biologists regard the ubiquity of the genetic code as evidence of universal common descent for all bacteria, archaea, and eukaryotes. Microbial mats of coexisting bacteria and archaea were the dominant form of life in the early Archean eon and many of the major steps in early evolution are thought to have taken place in this environment. The earliest evidence of eukaryotes dates from 1.85 billion years ago, and while they may have been present earlier, their diversification accelerated when they started using oxygen in their metabolism. Later, around 1.7 billion years ago, multicellular organisms began to appear, with differentiated cells performing specialised functions.
Algae-like multicellular land plants are dated back to about 1 billion years ago, although evidence suggests that microorganisms formed the earliest terrestrial ecosystems, at least 2.7 billion years ago. Microorganisms are thought to have paved the way for the inception of land plants in the Ordovician period. Land plants were so successful that they are thought to have contributed to the Late Devonian extinction event.
Ediacara biota appear during the Ediacaran period, while vertebrates, along with most other modern phyla originated about 525 million years ago during the Cambrian explosion. During the Permian period, synapsids, including the ancestors of mammals, dominated the land, but most of this group became extinct in the Permian–Triassic extinction event 252 million years ago. During the recovery from this catastrophe, archosaurs became the most abundant land vertebrates; one archosaur group, the dinosaurs, dominated the Jurassic and Cretaceous periods. After the Cretaceous–Paleogene extinction event 66 million years ago killed off the non-avian dinosaurs, mammals increased rapidly in size and diversity. Such mass extinctions may have accelerated evolution by providing opportunities for new groups of organisms to diversify.
== Diversity ==
=== Bacteria and Archaea ===
Bacteria are a type of cell that constitute a large domain of prokaryotic microorganisms. Typically a few micrometers in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs, radioactive waste, and the deep biosphere of the Earth's crust. Bacteria also live in symbiotic and parasitic relationships with plants and animals. Most bacteria have not been characterised, and only about 27 percent of the bacterial phyla have species that can be grown in the laboratory.
Archaea constitute the other domain of prokaryotic cells and were initially classified as bacteria, receiving the name archaebacteria (in the Archaebacteria kingdom), a term that has fallen out of use. Archaeal cells have unique properties separating them from the other two domains, Bacteria and Eukaryota. Archaea are further divided into multiple recognized phyla. Archaea and bacteria are generally similar in size and shape, although a few archaea have very different shapes, such as the flat and square cells of Haloquadratum walsbyi. Despite this morphological similarity to bacteria, archaea possess genes and several metabolic pathways that are more closely related to those of eukaryotes, notably for the enzymes involved in transcription and translation. Other aspects of archaeal biochemistry are unique, such as their reliance on ether lipids in their cell membranes, including archaeols. Archaea use more energy sources than eukaryotes: these range from organic compounds, such as sugars, to ammonia, metal ions or even hydrogen gas. Salt-tolerant archaea (the Haloarchaea) use sunlight as an energy source, and other species of archaea fix carbon, but unlike plants and cyanobacteria, no known species of archaea does both. Archaea reproduce asexually by binary fission, fragmentation, or budding; unlike bacteria, no known species of Archaea form endospores.
The first observed archaea were extremophiles, living in extreme environments, such as hot springs and salt lakes with no other organisms. Improved molecular detection tools led to the discovery of archaea in almost every habitat, including soil, oceans, and marshlands. Archaea are particularly numerous in the oceans, and the archaea in plankton may be one of the most abundant groups of organisms on the planet.
Archaea are a major part of Earth's life. They are part of the microbiota of all organisms. In the human microbiome, they are important in the gut, mouth, and on the skin. Their morphological, metabolic, and geographical diversity permits them to play multiple ecological roles: carbon fixation; nitrogen cycling; organic compound turnover; and maintaining microbial symbiotic and syntrophic communities, for example.
=== Eukaryotes ===
Eukaryotes are hypothesized to have split from archaea, which was followed by their endosymbioses with bacteria (or symbiogenesis) that gave rise to mitochondria and chloroplasts, both of which are now part of modern-day eukaryotic cells. The major lineages of eukaryotes diversified in the Precambrian about 1.5 billion years ago and can be classified into eight major clades: alveolates, excavates, stramenopiles, plants, rhizarians, amoebozoans, fungi, and animals. Five of these clades are collectively known as protists, which are mostly microscopic eukaryotic organisms that are not plants, fungi, or animals. While it is likely that protists share a common ancestor (the last eukaryotic common ancestor), protists by themselves do not constitute a separate clade as some protists may be more closely related to plants, fungi, or animals than they are to other protists. Like groupings such as algae, invertebrates, or protozoans, the protist grouping is not a formal taxonomic group but is used for convenience. Most protists are unicellular; these are called microbial eukaryotes.
Plants are mainly multicellular organisms, predominantly photosynthetic eukaryotes of the kingdom Plantae, which would exclude fungi and some algae. Plant cells were derived by endosymbiosis of a cyanobacterium into an early eukaryote about one billion years ago, which gave rise to chloroplasts. The first several clades that emerged following primary endosymbiosis were aquatic and most of the aquatic photosynthetic eukaryotic organisms are collectively described as algae, which is a term of convenience as not all algae are closely related. Algae comprise several distinct clades such as glaucophytes, which are microscopic freshwater algae that may have resembled in form to the early unicellular ancestor of Plantae. Unlike glaucophytes, the other algal clades such as red and green algae are multicellular. Green algae comprise three major clades: chlorophytes, coleochaetophytes, and stoneworts.
Fungi are eukaryotes that digest foods outside their bodies, secreting digestive enzymes that break down large food molecules before absorbing them through their cell membranes. Many fungi are also saprobes, feeding on dead organic matter, making them important decomposers in ecological systems.
Animals are multicellular eukaryotes. With few exceptions, animals consume organic material, breathe oxygen, are able to move, can reproduce sexually, and grow from a hollow sphere of cells, the blastula, during embryonic development. Over 1.5 million living animal species have been described—of which around 1 million are insects—but it has been estimated there are over 7 million animal species in total. They have complex interactions with each other and their environments, forming intricate food webs.
=== Viruses ===
Viruses are submicroscopic infectious agents that replicate inside the cells of organisms. Viruses infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. More than 6,000 virus species have been described in detail. Viruses are found in almost every ecosystem on Earth and are the most numerous type of biological entity.
The origins of viruses in the evolutionary history of life are unclear: some may have evolved from plasmids—pieces of DNA that can move between cells—while others may have evolved from bacteria. In evolution, viruses are an important means of horizontal gene transfer, which increases genetic diversity in a way analogous to sexual reproduction. Because viruses possess some but not all characteristics of life, they have been described as "organisms at the edge of life", and as self-replicators.
== Ecology ==
Ecology is the study of the distribution and abundance of life, the interaction between organisms and their environment.
=== Ecosystems ===
The community of living (biotic) organisms in conjunction with the nonliving (abiotic) components (e.g., water, light, radiation, temperature, humidity, atmosphere, acidity, and soil) of their environment is called an ecosystem. These biotic and abiotic components are linked together through nutrient cycles and energy flows. Energy from the sun enters the system through photosynthesis and is incorporated into plant tissue. By feeding on plants and on one another, animals move matter and energy through the system. They also influence the quantity of plant and microbial biomass present. By breaking down dead organic matter, decomposers release carbon back to the atmosphere and facilitate nutrient cycling by converting nutrients stored in dead biomass back to a form that can be readily used by plants and other microbes.
=== Populations ===
A population is the group of organisms of the same species that occupies an area and reproduce from generation to generation. Population size can be estimated by multiplying population density by the area or volume. The carrying capacity of an environment is the maximum population size of a species that can be sustained by that specific environment, given the food, habitat, water, and other resources that are available. The carrying capacity of a population can be affected by changing environmental conditions such as changes in the availability of resources and the cost of maintaining them. In human populations, new technologies such as the Green revolution have helped increase the Earth's carrying capacity for humans over time, which has stymied the attempted predictions of impending population decline, the most famous of which was by Thomas Malthus in the 18th century.
=== Communities ===
A community is a group of populations of species occupying the same geographical area at the same time. A biological interaction is the effect that a pair of organisms living together in a community have on each other. They can be either of the same species (intraspecific interactions), or of different species (interspecific interactions). These effects may be short-term, like pollination and predation, or long-term; both often strongly influence the evolution of the species involved. A long-term interaction is called a symbiosis. Symbioses range from mutualism, beneficial to both partners, to competition, harmful to both partners. Every species participates as a consumer, resource, or both in consumer–resource interactions, which form the core of food chains or food webs. There are different trophic levels within any food web, with the lowest level being the primary producers (or autotrophs) such as plants and algae that convert energy and inorganic material into organic compounds, which can then be used by the rest of the community. At the next level are the heterotrophs, which are the species that obtain energy by breaking apart organic compounds from other organisms. Heterotrophs that consume plants are primary consumers (or herbivores) whereas heterotrophs that consume herbivores are secondary consumers (or carnivores). And those that eat secondary consumers are tertiary consumers and so on. Omnivorous heterotrophs are able to consume at multiple levels. Finally, there are decomposers that feed on the waste products or dead bodies of organisms.
On average, the total amount of energy incorporated into the biomass of a trophic level per unit of time is about one-tenth of the energy of the trophic level that it consumes. Waste and dead material used by decomposers as well as heat lost from metabolism make up the other ninety percent of energy that is not consumed by the next trophic level.
=== Biosphere ===
In the global ecosystem or biosphere, matter exists as different interacting compartments, which can be biotic or abiotic as well as accessible or inaccessible, depending on their forms and locations. For example, matter from terrestrial autotrophs are both biotic and accessible to other organisms whereas the matter in rocks and minerals are abiotic and inaccessible. A biogeochemical cycle is a pathway by which specific elements of matter are turned over or moved through the biotic (biosphere) and the abiotic (lithosphere, atmosphere, and hydrosphere) compartments of Earth. There are biogeochemical cycles for nitrogen, carbon, and water.
=== Conservation ===
Conservation biology is the study of the conservation of Earth's biodiversity with the aim of protecting species, their habitats, and ecosystems from excessive rates of extinction and the erosion of biotic interactions. It is concerned with factors that influence the maintenance, loss, and restoration of biodiversity and the science of sustaining evolutionary processes that engender genetic, population, species, and ecosystem diversity. The concern stems from estimates suggesting that up to 50% of all species on the planet will disappear within the next 50 years, which has contributed to poverty, starvation, and will reset the course of evolution on this planet. Biodiversity affects the functioning of ecosystems, which provide a variety of services upon which people depend. Conservation biologists research and educate on the trends of biodiversity loss, species extinctions, and the negative effect these are having on our capabilities to sustain the well-being of human society. Organizations and citizens are responding to the current biodiversity crisis through conservation action plans that direct research, monitoring, and education programs that engage concerns at local through global scales.
== See also ==
== References ==
== Further reading ==
== External links ==
OSU's Phylocode
Biology Online – Wiki Dictionary
MIT video lecture series on biology
OneZoom Tree of Life
Journal of the History of Biology (springer.com)
Journal links
PLOS ONE
PLOS Biology A peer-reviewed, open-access journal published by the Public Library of Science
Current Biology: General journal publishing original research from all areas of biology
Biology Letters: A high-impact Royal Society journal publishing peer-reviewed biology papers of general interest
Science: Internationally renowned AAAS science journal – see sections of the life sciences
International Journal of Biological Sciences: A biological journal publishing significant peer-reviewed scientific papers
Perspectives in Biology and Medicine: An interdisciplinary scholarly journal publishing essays of broad relevance | Wikipedia/biology |
Deoxyribonucleic acid ( ; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.
The two DNA strands are known as polynucleotides as they are composed of simpler monomeric units called nucleotides. Each nucleotide is composed of one of four nitrogen-containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine [T]), a sugar called deoxyribose, and a phosphate group. The nucleotides are joined to one another in a chain by covalent bonds (known as the phosphodiester linkage) between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The nitrogenous bases of the two separate polynucleotide strands are bound together, according to base pairing rules (A with T and C with G), with hydrogen bonds to make double-stranded DNA. The complementary nitrogenous bases are divided into two groups, the single-ringed pyrimidines and the double-ringed purines. In DNA, the pyrimidines are thymine and cytosine; the purines are adenine and guanine.
Both strands of double-stranded DNA store the same biological information. This information is replicated when the two strands separate. A large part of DNA (more than 98% for humans) is non-coding, meaning that these sections do not serve as patterns for protein sequences. The two strands of DNA run in opposite directions to each other and are thus antiparallel. Attached to each sugar is one of four types of nucleobases (or bases). It is the sequence of these four nucleobases along the backbone that encodes genetic information. RNA strands are created using DNA strands as a template in a process called transcription, where DNA bases are exchanged for their corresponding bases except in the case of thymine (T), for which RNA substitutes uracil (U). Under the genetic code, these RNA strands specify the sequence of amino acids within proteins in a process called translation.
Within eukaryotic cells, DNA is organized into long structures called chromosomes. Before typical cell division, these chromosomes are duplicated in the process of DNA replication, providing a complete set of chromosomes for each daughter cell. Eukaryotic organisms (animals, plants, fungi and protists) store most of their DNA inside the cell nucleus as nuclear DNA, and some in the mitochondria as mitochondrial DNA or in chloroplasts as chloroplast DNA. In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm, in circular chromosomes. Within eukaryotic chromosomes, chromatin proteins, such as histones, compact and organize DNA. These compacting structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.
== Properties ==
DNA is a long polymer made from repeating units called nucleotides. The structure of DNA is dynamic along its length, being capable of coiling into tight loops and other shapes. In all species it is composed of two helical chains, bound to each other by hydrogen bonds. Both chains are coiled around the same axis, and have the same pitch of 34 ångströms (3.4 nm). The pair of chains have a radius of 10 Å (1.0 nm). According to another study, when measured in a different solution, the DNA chain measured 22–26 Å (2.2–2.6 nm) wide, and one nucleotide unit measured 3.3 Å (0.33 nm) long. The buoyant density of most DNA is 1.7g/cm3.
DNA does not usually exist as a single strand, but instead as a pair of strands that are held tightly together. These two long strands coil around each other, in the shape of a double helix. The nucleotide contains both a segment of the backbone of the molecule (which holds the chain together) and a nucleobase (which interacts with the other DNA strand in the helix). A nucleobase linked to a sugar is called a nucleoside, and a base linked to a sugar and to one or more phosphate groups is called a nucleotide. A biopolymer comprising multiple linked nucleotides (as in DNA) is called a polynucleotide.
The backbone of the DNA strand is made from alternating phosphate and sugar groups. The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These are known as the 3′-end (three prime end), and 5′-end (five prime end) carbons, the prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond.
Therefore, any DNA strand normally has one end at which there is a phosphate group attached to the 5′ carbon of a ribose (the 5′ phosphoryl) and another end at which there is a free hydroxyl group attached to the 3′ carbon of a ribose (the 3′ hydroxyl). The orientation of the 3′ and 5′ carbons along the sugar-phosphate backbone confers directionality (sometimes called polarity) to each DNA strand. In a nucleic acid double helix, the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are said to have a directionality of five prime end (5′ ), and three prime end (3′), with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the related pentose sugar ribose in RNA.
The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases. The four bases found in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar-phosphate to form the complete nucleotide, as shown for adenosine monophosphate. Adenine pairs with thymine and guanine pairs with cytosine, forming A-T and G-C base pairs.
=== Nucleobase classification ===
The nucleobases are classified into two types: the purines, A and G, which are fused five- and six-membered heterocyclic compounds, and the pyrimidines, the six-membered rings C and T. A fifth pyrimidine nucleobase, uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. In addition to RNA and DNA, many artificial nucleic acid analogues have been created to study the properties of nucleic acids, or for use in biotechnology.
=== Non-canonical bases ===
Modified bases occur in DNA. The first of these recognized was 5-methylcytosine, which was found in the genome of Mycobacterium tuberculosis in 1925. The reason for the presence of these noncanonical bases in bacterial viruses (bacteriophages) is to avoid the restriction enzymes present in bacteria. This enzyme system acts at least in part as a molecular immune system protecting bacteria from infection by viruses. Modifications of the bases cytosine and adenine, the more common and modified DNA bases, play vital roles in the epigenetic control of gene expression in plants and animals.
A number of noncanonical bases are known to occur in DNA. Most of these are modifications of the canonical bases plus uracil.
Modified Adenine
N6-carbamoyl-methyladenine
N6-methyadenine
Modified Guanine
7-Deazaguanine
7-Methylguanine
Modified Cytosine
N4-Methylcytosine
5-Carboxylcytosine
5-Formylcytosine
5-Glycosylhydroxymethylcytosine
5-Hydroxycytosine
5-Methylcytosine
Modified Thymidine
α-Glutamythymidine
α-Putrescinylthymine
Uracil and modifications
Base J
Uracil
5-Dihydroxypentauracil
5-Hydroxymethyldeoxyuracil
Others
Deoxyarchaeosine
2,6-Diaminopurine (2-Aminoadenine)
=== Grooves ===
Twin helical strands form the DNA backbone. Another double helix may be found tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site. As the strands are not symmetrically located with respect to each other, the grooves are unequally sized. The major groove is 22 ångströms (2.2 nm) wide, while the minor groove is 12 Å (1.2 nm) in width. Due to the larger width of the major groove, the edges of the bases are more accessible in the major groove than in the minor groove. As a result, proteins such as transcription factors that can bind to specific sequences in double-stranded DNA usually make contact with the sides of the bases exposed in the major groove. This situation varies in unusual conformations of DNA within the cell (see below), but the major and minor grooves are always named to reflect the differences in width that would be seen if the DNA was twisted back into the ordinary B form.
=== Base pairing ===
In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other strand. This is called complementary base pairing. Purines form hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds. This arrangement of two nucleotides binding together across the double helix (from six-carbon ring to six-carbon ring) is called a Watson-Crick base pair. DNA with high GC-content is more stable than DNA with low GC-content. A Hoogsteen base pair (hydrogen bonding the 6-carbon ring to the 5-carbon ring) is a rare variation of base-pairing. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can thus be pulled apart like a zipper, either by a mechanical force or high temperature. As a result of this base pair complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. This reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in organisms.
==== ssDNA vs. dsDNA ====
Most DNA molecules are actually two polymer strands, bound together in a helical fashion by noncovalent bonds; this double-stranded (dsDNA) structure is maintained largely by the intrastrand base stacking interactions, which are strongest for G,C stacks. The two strands can come apart—a process known as melting—to form two single-stranded DNA (ssDNA) molecules. Melting occurs at high temperatures, low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH is rarely used).
The stability of the dsDNA form depends not only on the GC-content (% G,C basepairs) but also on sequence (since stacking is sequence specific) and also length (longer molecules are more stable). The stability can be measured in various ways; a common way is the melting temperature (also called Tm value), which is the temperature at which 50% of the double-strand molecules are converted to single-strand molecules; melting temperature is dependent on ionic strength and the concentration of DNA. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determines the strength of the association between the two strands of DNA. Long DNA helices with a high GC-content have more strongly interacting strands, while short helices with high AT content have more weakly interacting strands. In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters, tend to have a high AT content, making the strands easier to pull apart.
In the laboratory, the strength of this interaction can be measured by finding the melting temperature Tm necessary to break half of the hydrogen bonds. When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others.
=== Amount ===
In humans, the total female diploid nuclear genome per cell extends for 6.37 Gigabase pairs (Gbp), is 208.23 cm long and weighs 6.51 picograms (pg). Male values are 6.27 Gbp, 205.00 cm, 6.41 pg. Each DNA polymer can contain hundreds of millions of nucleotides, such as in chromosome 1. Chromosome 1 is the largest human chromosome with approximately 220 million base pairs, and would be 85 mm long if straightened.
In eukaryotes, in addition to nuclear DNA, there is also mitochondrial DNA (mtDNA) which encodes certain proteins used by the mitochondria. The mtDNA is usually relatively small in comparison to the nuclear DNA. For example, the human mitochondrial DNA forms closed circular molecules, each of which contains 16,569 DNA base pairs, with each such molecule normally containing a full set of the mitochondrial genes. Each human mitochondrion contains, on average, approximately 5 such mtDNA molecules. Each human cell contains approximately 100 mitochondria, giving a total number of mtDNA molecules per human cell of approximately 500. However, the amount of mitochondria per cell also varies by cell type, and an egg cell can contain 100,000 mitochondria, corresponding to up to 1,500,000 copies of the mitochondrial genome (constituting up to 90% of the DNA of the cell).
=== Sense and antisense ===
A DNA sequence is called a "sense" sequence if it is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands can contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear. One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.
A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes. In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription, while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome.
=== Supercoiling ===
DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound. If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases. These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.
=== Alternative DNA structures ===
DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although only B-DNA and Z-DNA have been directly observed in functional organisms. The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, and the presence of polyamines in solution.
The first published reports of A-DNA X-ray diffraction patterns—and also B-DNA—used analyses based on Patterson functions that provided only a limited amount of structural information for oriented fibers of DNA. An alternative analysis was proposed by Wilkins et al. in 1953 for the in vivo B-DNA X-ray diffraction-scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions. In the same journal, James Watson and Francis Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double helix.
Although the B-DNA form is most common under the conditions found in cells, it is not a well-defined conformation but a family of related DNA conformations that occur at the high hydration levels present in cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder.
Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a narrower, deeper major groove. The A form occurs under non-physiological conditions in partly dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, and in enzyme-DNA complexes. Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form. Here, the strands turn about the helical axis in a left-handed spiral, the opposite of the more common B form. These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription.
=== Alternative DNA chemistry ===
For many years, exobiologists have proposed the existence of a shadow biosphere, a postulated microbial biosphere of Earth that uses radically different biochemical and molecular processes than currently known life. One of the proposals was the existence of lifeforms that use arsenic instead of phosphorus in DNA. A report in 2010 of the possibility in the bacterium GFAJ-1 was announced, though the research was disputed, and evidence suggests the bacterium actively prevents the incorporation of arsenic into the DNA backbone and other biomolecules.
=== Quadruplex structures ===
At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes. These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected. In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence.
These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases, known as a guanine tetrad, form a flat plate. These flat four-base units then stack on top of each other to form a stable G-quadruplex structure. These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit. Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure.
In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins. At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop or D-loop.
=== Branched DNA ===
In DNA, fraying occurs when non-complementary regions exist at the end of an otherwise complementary double-strand of DNA. However, branched DNA can occur if a third strand of DNA is introduced and contains adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Although the simplest example of branched DNA involves only three strands of DNA, complexes involving additional strands and multiple branches are also possible. Branched DNA can be used in nanotechnology to construct geometric shapes, see the section on uses in technology below.
=== Artificial bases ===
Several artificial nucleobases have been synthesized, and successfully incorporated in the eight-base DNA analogue named Hachimoji DNA. Dubbed S, B, P, and Z, these artificial bases are capable of bonding with each other in a predictable way (S–B and P–Z), maintain the double helix structure of DNA, and be transcribed to RNA. Their existence could be seen as an indication that there is nothing special about the four natural nucleobases that evolved on Earth. On the other hand, DNA is tightly related to RNA which does not only act as a transcript of DNA but also performs as molecular machines many tasks in cells. For this purpose it has to fold into a structure. It has been shown that to allow to create all possible structures at least four bases are required for the corresponding RNA, while a higher number is also possible but this would be against the natural principle of least effort.
=== Acidity ===
The phosphate groups of DNA give it similar acidic properties to phosphoric acid and it can be considered as a strong acid. It will be fully ionized at a normal cellular pH, releasing protons which leave behind negative charges on the phosphate groups. These negative charges protect DNA from breakdown by hydrolysis by repelling nucleophiles which could hydrolyze it.
=== Macroscopic appearance ===
Pure DNA extracted from cells forms white, stringy clumps.
== Chemical modifications and altered DNA packaging ==
=== Base modifications and DNA packaging ===
The expression of genes is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin. Base modifications can be involved in packaging, with regions that have low or no gene expression usually containing high levels of methylation of cytosine bases. DNA packaging and its influence on gene expression can also occur by covalent modifications of the histone protein core around which DNA is wrapped in the chromatin structure or else by remodeling carried out by chromatin remodeling complexes (see Chromatin remodeling). There is, further, crosstalk between DNA methylation and histone modification, so they can coordinately affect chromatin and gene expression.
For one example, cytosine methylation produces 5-methylcytosine, which is important for X-inactivation of chromosomes. The average level of methylation varies between organisms—the worm Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing 5-methylcytosine. Despite the importance of 5-methylcytosine, it can deaminate to leave a thymine base, so methylated cytosines are particularly prone to mutations. Other base modifications include adenine methylation in bacteria, the presence of 5-hydroxymethylcytosine in the brain, and the glycosylation of uracil to produce the "J-base" in kinetoplastids.
=== Damage ===
DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Mutagens include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers, which are cross-links between pyrimidine bases. On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, and double-strand breaks. A typical human cell contains about 150,000 bases that have suffered oxidative damage. Of these oxidative lesions, the most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations, insertions, deletions from the DNA sequence, and chromosomal translocations. These mutations can cause cancer. Because of inherent limits in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer. DNA damages that are naturally occurring, due to normal cellular processes that produce reactive oxygen species, the hydrolytic activities of cellular water, etc., also occur frequently. Although most of these damages are repaired, in any cell some DNA damage may remain despite the action of repair processes. These remaining DNA damages accumulate with age in mammalian postmitotic tissues. This accumulation appears to be an important underlying cause of aging.
Many mutagens fit into the space between two adjacent base pairs, this is called intercalation. Most intercalators are aromatic and planar molecules; examples include ethidium bromide, acridines, daunomycin, and doxorubicin. For an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen. Others such as benzo[a]pyrene diol epoxide and aflatoxin form DNA adducts that induce errors in replication. Nevertheless, due to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to inhibit rapidly growing cancer cells.
== Biological functions ==
DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation, which depends on the same interaction between RNA nucleotides. In an alternative fashion, a cell may copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles; here the focus is on the interactions between DNA and other molecules that mediate the function of the genome.
=== Genes and genomes ===
Genomic DNA is tightly and orderly packed in the process called DNA condensation, to fit the small available volumes of the cell. In eukaryotes, DNA is located in the cell nucleus, with small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid. The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, and regulatory sequences such as promoters and enhancers, which control transcription of the open reading frame.
In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences. The reasons for the presence of so much noncoding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species, represent a long-standing puzzle known as the "C-value enigma". However, some DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.
Some noncoding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes but are important for the function and stability of chromosomes. An abundant form of noncoding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation. These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence.
=== Transcription and translation ===
A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).
In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (43 combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAG, TAA, and TGA codons, (UAG, UAA, and UGA on the mRNA).
=== Replication ===
Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Here, the two strands are separated and then each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix. In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.
=== Extracellular nucleic acids ===
Naked extracellular DNA (eDNA), most of it released by cell death, is nearly ubiquitous in the environment. Its concentration in soil may be as high as 2 μg/L, and its concentration in natural aquatic environments may be as high at 88 μg/L. Various possible functions have been proposed for eDNA: it may be involved in horizontal gene transfer; it may provide nutrients; and it may act as a buffer to recruit or titrate ions or antibiotics. Extracellular DNA acts as a functional extracellular matrix component in the biofilms of several bacterial species. It may act as a recognition factor to regulate the attachment and dispersal of specific cell types in the biofilm; it may contribute to biofilm formation; and it may contribute to the biofilm's physical strength and resistance to biological stress.
Cell-free fetal DNA is found in the blood of the mother, and can be sequenced to determine a great deal of information about the developing fetus.
Under the name of environmental DNA eDNA has seen increased use in the natural sciences as a survey tool for ecology, monitoring the movements and presence of species in water, air, or on land, and assessing an area's biodiversity.
=== Neutrophil extracellular traps ===
Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of DNA, which allow neutrophils, a type of white blood cell, to kill extracellular pathogens while minimizing damage to the host cells.
== Interactions with proteins ==
All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.
=== DNA-binding proteins ===
Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes, this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones, making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are thus largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation, phosphorylation, and acetylation. These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA. These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.
A distinct group of DNA-binding proteins is the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination, and DNA repair. These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.
In contrast, other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription. Alternatively, transcription factors can bind enzymes that modify the histones at the promoter. This changes the accessibility of the DNA template to the polymerase.
As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes. Consequently, these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.
=== DNA-modifying enzymes ===
==== Nucleases and ligases ====
Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GATATC-3′ and makes a cut at the horizontal line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system. In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.
Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly important in lagging strand DNA replication, as they join the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.
==== Topoisomerases and helicases ====
Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the DNA break. Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix. Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.
Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly adenosine triphosphate (ATP), to break hydrogen bonds between bases and unwind the DNA double helix into single strands. These enzymes are essential for most processes where enzymes need to access the DNA bases.
==== Polymerases ====
Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products is created based on existing polynucleotide chains—which are called templates. These enzymes function by repeatedly adding a nucleotide to the 3′ hydroxyl group at the end of the growing polynucleotide chain. As a consequence, all polymerases work in a 5′ to 3′ direction. In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.
In DNA replication, DNA-dependent DNA polymerases make copies of DNA polynucleotide chains. To preserve biological information, it is essential that the sequence of bases in each copy are precisely complementary to the sequence of bases in the template strand. Many DNA polymerases have a proofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed. In most organisms, DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases.
RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres. For example, HIV reverse transcriptase is an enzyme for AIDS virus replication. Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure. It synthesizes telomeres at the ends of chromosomes. Telomeres prevent fusion of the ends of neighboring chromosomes and protect chromosome ends from damage.
Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits.
== Genetic recombination ==
A DNA helix usually does not interact with other segments of DNA, and in human cells, the different chromosomes even occupy separate areas in the nucleus called "chromosome territories". This physical separation of different chromosomes is important for the ability of DNA to function as a stable repository for information, as one of the few times chromosomes interact is in chromosomal crossover which occurs during sexual reproduction, when genetic recombination occurs. Chromosomal crossover is when two DNA helices break, swap a section and then rejoin.
Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of new proteins. Genetic recombination can also be involved in DNA repair, particularly in the cell's response to double-strand breaks.
The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51. The first step in recombination is a double-stranded break caused by either an endonuclease or damage to the DNA. A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA. Only strands of like polarity exchange DNA during recombination. There are two types of cleavage: east-west cleavage and north–south cleavage. The north–south cleavage nicks both strands of DNA, while the east–west cleavage has one strand of DNA intact. The formation of a Holliday junction during recombination makes it possible for genetic diversity, genes to exchange on chromosomes, and expression of wild-type viral genomes.
== Evolution ==
DNA contains the genetic information that allows all forms of life to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material. RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes. This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes. However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible because DNA survives in the environment for less than one million years, and slowly degrades into short fragments in solution. Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old, but these claims are controversial.
Building blocks of DNA (adenine, guanine, and related organic molecules) may have been formed extraterrestrially in outer space. Complex DNA and RNA organic compounds of life, including uracil, cytosine, and thymine, have also been formed in the laboratory under conditions mimicking those found in outer space, using starting chemicals, such as pyrimidine, found in meteorites. Pyrimidine, like polycyclic aromatic hydrocarbons (PAHs), the most carbon-rich chemical found in the universe, may have been formed in red giants or in interstellar cosmic dust and gas clouds.
Ancient DNA has been recovered from ancient organisms at a timescale where genome evolution can be directly observed, including from extinct organisms up to millions of years old, such as the woolly mammoth.
== Uses in technology ==
=== Genetic engineering ===
Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction, and to manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant DNA technology. Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector. The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research, or be grown in agriculture.
=== DNA profiling ===
Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual, such as a perpetrator. This process is formally termed DNA profiling, also called DNA fingerprinting. In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a matching DNA. However, identification can be complicated if the scene is contaminated with DNA from several people. DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.
The development of forensic science and the ability to now obtain genetic matching on minute samples of blood, skin, saliva, or hair has led to re-examining many cases. Evidence can now be uncovered that was scientifically impossible at the time of the original examination. Combined with the removal of the double jeopardy law in some places, this can allow cases to be reopened where prior trials have failed to produce sufficient evidence to convince a jury. People charged with serious crimes may be required to provide a sample of DNA for matching purposes. The most obvious defense to DNA matches obtained forensically is to claim that cross-contamination of evidence has occurred. This has resulted in meticulous strict handling procedures with new cases of serious crime.
DNA profiling is also used successfully to positively identify victims of mass casualty incidents, bodies or body parts in serious accidents, and individual victims in mass war graves, via matching to family members.
DNA profiling is also used in DNA paternity testing to determine if someone is the biological parent or grandparent of a child with the probability of parentage is typically 99.99% when the alleged parent is biologically related to the child. Normal DNA sequencing methods happen after birth, but there are new methods to test paternity while a mother is still pregnant.
=== DNA enzymes or catalytic DNA ===
Deoxyribozymes, also called DNAzymes or catalytic DNA, were first discovered in 1994. They are mostly single stranded DNA sequences isolated from a large pool of random DNA sequences through a combinatorial approach called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). DNAzymes catalyze variety of chemical reactions including RNA-DNA cleavage, RNA-DNA ligation, amino acids phosphorylation-dephosphorylation, carbon-carbon bond formation, etc. DNAzymes can enhance catalytic rate of chemical reactions up to 100,000,000,000-fold over the uncatalyzed reaction. The most extensively studied class of DNAzymes is RNA-cleaving types which have been used to detect different metal ions and designing therapeutic agents. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific), the CA1-3 DNAzymes (copper-specific), the 39E DNAzyme (uranyl-specific) and the NaA43 DNAzyme (sodium-specific). The NaA43 DNAzyme, which is reported to be more than 10,000-fold selective for sodium over other metal ions, was used to make a real-time sodium sensor in cells.
=== Bioinformatics ===
Bioinformatics involves the development of techniques to store, data mine, search and manipulate biological data, including DNA nucleic acid sequence data. These have led to widely applied advances in computer science, especially string searching algorithms, machine learning, and database theory. String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides. The DNA sequence may be aligned with other DNA sequences to identify homologous sequences and locate the specific mutations that make them distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function. Data sets representing entire genomes' worth of DNA sequences, such as those produced by the Human Genome Project, are difficult to use without the annotations that identify the locations of genes and regulatory elements on each chromosome. Regions of DNA sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products and their possible functions in an organism even before they have been isolated experimentally. Entire genomes may also be compared, which can shed light on the evolutionary history of particular organism and permit the examination of complex evolutionary events.
=== DNA nanotechnology ===
DNA nanotechnology uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties. DNA is thus used as a structural material rather than as a carrier of biological information. This has led to the creation of two-dimensional periodic lattices (both tile-based and using the DNA origami method) and three-dimensional structures in the shapes of polyhedra. Nanomechanical devices and algorithmic self-assembly have also been demonstrated, and these DNA structures have been used to template the arrangement of other molecules such as gold nanoparticles and streptavidin proteins. DNA and other nucleic acids are the basis of aptamers, synthetic oligonucleotide ligands for specific target molecules used in a range of biotechnology and biomedical applications.
=== History and anthropology ===
Because DNA collects mutations over time, which are then inherited, it contains historical information, and, by comparing DNA sequences, geneticists can infer the evolutionary history of organisms, their phylogeny. This field of phylogenetics is a powerful tool in evolutionary biology. If DNA sequences within a species are compared, population geneticists can learn the history of particular populations. This can be used in studies ranging from ecological genetics to anthropology.
=== Information storage ===
DNA as a storage device for information has enormous potential since it has much higher storage density compared to electronic devices. However, high costs, slow read and write times (memory latency), and insufficient reliability has prevented its practical use.
== History ==
DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein". In 1878, Albrecht Kossel isolated the non-protein component of "nuclein", nucleic acid, and later isolated its five primary nucleobases.
In 1909, Phoebus Levene identified the base, sugar, and phosphate nucleotide unit of RNA (then named "yeast nucleic acid"). In 1929, Levene identified deoxyribose sugar in "thymus nucleic acid" (DNA). Levene suggested that DNA consisted of a string of four nucleotide units linked together through the phosphate groups ("tetranucleotide hypothesis"). Levene thought the chain was short and the bases repeated in a fixed order. In 1927, Nikolai Koltsov proposed that inherited traits would be inherited via a "giant hereditary molecule" made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template". In 1928, Frederick Griffith in his experiment discovered that traits of the "smooth" form of Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form. This system provided the first clear suggestion that DNA carries genetic information.
In 1933, while studying virgin sea urchin eggs, Jean Brachet suggested that DNA is found in the cell nucleus and that RNA is present exclusively in the cytoplasm. At the time, "yeast nucleic acid" (RNA) was thought to occur only in plants, while "thymus nucleic acid" (DNA) only in animals. The latter was thought to be a tetramer, with the function of buffering cellular pH.
In 1937, William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure.
In 1943, Oswald Avery, along with co-workers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle, supporting Griffith's suggestion (Avery–MacLeod–McCarty experiment). Erwin Chargaff developed and published observations now known as Chargaff's rules, stating that in DNA from any species of any organism, the amount of guanine should be equal to cytosine and the amount of adenine should be equal to thymine.
Late in 1951, Francis Crick started working with James Watson at the Cavendish Laboratory within the University of Cambridge. DNA's role in heredity was confirmed in 1952 when Alfred Hershey and Martha Chase in the Hershey–Chase experiment showed that DNA is the genetic material of the enterobacteria phage T2.
In May 1952, Raymond Gosling, a graduate student working under the supervision of Rosalind Franklin, took an X-ray diffraction image, labeled as "Photo 51", at high hydration levels of DNA. This photo was given to Watson and Crick by Maurice Wilkins and was critical to their obtaining the correct structure of DNA. Franklin told Crick and Watson that the backbones had to be on the outside. Before then, Linus Pauling, and Watson and Crick, had erroneous models with the chains inside and the bases pointing outwards. Franklin's identification of the space group for DNA crystals revealed to Crick that the two DNA strands were antiparallel. In February 1953, Linus Pauling and Robert Corey proposed a model for nucleic acids containing three intertwined chains, with the phosphates near the axis, and the bases on the outside. Watson and Crick completed their model, which is now accepted as the first correct model of the double helix of DNA. On 28 February 1953 Crick interrupted patrons' lunchtime at The Eagle pub in Cambridge, England to announce that he and Watson had "discovered the secret of life".
The 25 April 1953 issue of the journal Nature published a series of five articles giving the Watson and Crick double-helix structure DNA and evidence supporting it. The structure was reported in a letter titled "MOLECULAR STRUCTURE OF NUCLEIC ACIDS A Structure for Deoxyribose Nucleic Acid", in which they said, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." This letter was followed by a letter from Franklin and Gosling, which was the first publication of their own X-ray diffraction data and of their original analysis method. Then followed a letter by Wilkins and two of his colleagues, which contained an analysis of in vivo B-DNA X-ray patterns, and which supported the presence in vivo of the Watson and Crick structure.
In April 2023, scientists, based on new evidence, concluded that Rosalind Franklin was a contributor and "equal player" in the discovery process of DNA, rather than otherwise, as may have been presented subsequently after the time of the discovery.
In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine. Nobel Prizes are awarded only to living recipients. A debate continues about who should receive credit for the discovery.
In an influential presentation in 1957, Crick laid out the central dogma of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis". Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson–Stahl experiment. Further work by Crick and co-workers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley, and Marshall Warren Nirenberg to decipher the genetic code. These findings represent the birth of molecular biology.
In 1986, DNA analysis was first used in a criminal investigation when police in the UK requested Alec Jeffreys of the University of Leicester to prove or disprove the involvement in a particular case of a suspect who claimed innocence in the matter. Although the suspect had already confessed to committing a recent rape-murder, he was denying any involvement in a similar crime committed three years earlier. Yet the details of the two cases were so alike that the police concluded both crimes had been committed by the same person. However, all charges against the suspect were dropped when Jeffreys' DNA testing exonerated the suspect — from both the earlier murder and the one to which he'd confessed. Further such DNA profiling led to positive identification of another suspect who, in 1988, was found guilty of both rape-murders.
== See also ==
== References ==
== Further reading ==
== External links ==
DNA binding site prediction on protein
DNA the Double Helix Game From the official Nobel Prize web site
DNA under electron microscope
Dolan DNA Learning Center
Double Helix: 50 years of DNA, Nature
Proteopedia DNA
Proteopedia Forms_of_DNA
ENCODE threads explorer ENCODE home page at Nature
Double Helix 1953–2003 National Centre for Biotechnology Education
Genetic Education Modules for Teachers – DNA from the Beginning Study Guide
PDB Molecule of the Month DNA
"Clue to chemistry of heredity found". The New York Times, June 1953. First American newspaper coverage of the discovery of the DNA structure
DNA from the Beginning Another DNA Learning Center site on DNA, genes, and heredity from Mendel to the human genome project.
The Register of Francis Crick Personal Papers 1938 – 2007 at Mandeville Special Collections Library, University of California, San Diego
Seven-page, handwritten letter that Crick sent to his 12-year-old son Michael in 1953 describing the structure of DNA. See Crick's medal goes under the hammer, Nature, 5 April 2013. | Wikipedia/Structure_of_DNA |
The Boveri–Sutton chromosome theory (also known as the chromosome theory of inheritance or the Sutton–Boveri theory) is a fundamental unifying theory of genetics which identifies chromosomes as the carriers of genetic material. It correctly explains the mechanism underlying the laws of Mendelian inheritance by identifying chromosomes with the paired factors (particles) required by Mendel's laws. It also states that chromosomes are linear structures with genes located at specific sites called loci along them.
It states simply that chromosomes, which are seen in all dividing cells and pass from one generation to the next, are the basis for all genetic inheritance.
Over a period of time random mutation
creates changes in the DNA sequence of a gene. Genes are located on chromosomes.
== Background ==
The chromosome theory of inheritance is credited to papers by Walter Sutton in 1902 and 1903, as well as to independent work by Theodor Boveri during roughly the same period. Boveri was studying sea urchins, in which he found that all the chromosomes had to be present for proper embryonic development to take place. Sutton's work with grasshoppers showed that chromosomes occur in matched pairs of maternal and paternal chromosomes which separate during meiosis and "may constitute the physical basis of the Mendelian law of heredity".
This groundbreaking work led E.B. Wilson in his classic text to name the chromosome theory of inheritance the "Sutton-Boveri Theory". Wilson was close to both men since the young Sutton was his student and the prominent Boveri was his friend (in fact, Wilson dedicated the aforementioned book to Boveri). Although the naming precedence is now often reversed to "Boveri-Sutton", there are some who argue that Boveri did not actually articulate the theory until 1904.
== Verification ==
The proposal that chromosomes carried the factors of Mendelian inheritance was initially controversial, but in 1905 it gained
strong support when Nettie Stevens showed that the "accessory chromosome" of mealworms' sperm cells was decisive in the sex identity of the progeny, a discovery supported by her mentor E.B. Wilson. Later, Eleanor Carothers documented definitive evidence of independent assortment of chromosomes in a species of grasshopper. Debate continued, however, until 1915 when Thomas Hunt Morgan's work on inheritance and genetic linkage in the fruit fly Drosophila melanogaster provided incontrovertible evidence for the proposal. The unifying theory stated that inheritance patterns may be generally explained by assuming that genes are located in specific sites on chromosomes.
== References ==
== External links ==
Each Organism's Traits Are Inherited from a Parent through Transmission of DNA SciTable by Nature Education. | Wikipedia/Boveri–Sutton_chromosome_theory |
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity.
A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are rarely considered to be proteins and are commonly called peptides. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids; but in certain organisms the genetic code can include selenocysteine and—in certain archaea—pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by post-translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Some proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can work together to achieve a particular function, and they often associate to form stable protein complexes.
Once formed, proteins only exist for a certain period and are then degraded and recycled by the cell's machinery through the process of protein turnover. A protein's lifespan is measured in terms of its half-life and covers a wide range. They can exist for minutes or years with an average lifespan of 1–2 days in mammalian cells. Abnormal or misfolded proteins are degraded more rapidly either due to being targeted for destruction or due to being unstable.
Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism. Some proteins have structural or mechanical functions, such as actin and myosin in muscle, and the cytoskeleton's scaffolding proteins that maintain cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. In animals, proteins are needed in the diet to provide the essential amino acids that cannot be synthesized. Digestion breaks the proteins down for metabolic use.
== History and etymology ==
=== Discovery and early studies ===
Proteins have been studied and recognized since the 1700s by Antoine Fourcroy and others, who often collectively called them "albumins", or "albuminous materials" (Eiweisskörper, in German). Gluten, for example, was first separated from wheat in published research around 1747, and later determined to exist in many plants. In 1789, Antoine Fourcroy recognized three distinct varieties of animal proteins: albumin, fibrin, and gelatin. Vegetable (plant) proteins studied in the late 1700s and early 1800s included gluten, plant albumin, gliadin, and legumin.
Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius in 1838. Mulder carried out elemental analysis of common proteins and found that nearly all proteins had the same empirical formula, C400H620N100O120P1S1. He came to the erroneous conclusion that they might be composed of a single type of (very large) molecule. The term "protein" to describe these molecules was proposed by Mulder's associate Berzelius; protein is derived from the Greek word πρώτειος (proteios), meaning "primary", "in the lead", or "standing in front", + -in. Mulder went on to identify the products of protein degradation such as the amino acid leucine for which he found a (nearly correct) molecular weight of 131 Da.
Early nutritional scientists such as the German Carl von Voit believed that protein was the most important nutrient for maintaining the structure of the body, because it was generally believed that "flesh makes flesh." Around 1862, Karl Heinrich Ritthausen isolated the amino acid glutamic acid. Thomas Burr Osborne compiled a detailed review of the vegetable proteins at the Connecticut Agricultural Experiment Station. Osborne, alongside Lafayette Mendel, established several nutritionally essential amino acids in feeding experiments with laboratory rats. Diets lacking an essential amino acid stunts the rats' growth, consistent with Liebig's law of the minimum. The final essential amino acid to be discovered, threonine, was identified by William Cumming Rose.
The difficulty in purifying proteins impeded work by early protein biochemists. Proteins could be obtained in large quantities from blood, egg whites, and keratin, but individual proteins were unavailable. In the 1950s, the Armour Hot Dog Company purified 1 kg of bovine pancreatic ribonuclease A and made it freely available to scientists. This gesture helped ribonuclease A become a major target for biochemical study for the following decades.
=== Polypeptides ===
The understanding of proteins as polypeptides, or chains of amino acids, came through the work of Franz Hofmeister and Hermann Emil Fischer in 1902. The central role of proteins as enzymes in living organisms that catalyzed reactions was not fully appreciated until 1926, when James B. Sumner showed that the enzyme urease was in fact a protein.
Linus Pauling is credited with the successful prediction of regular protein secondary structures based on hydrogen bonding, an idea first put forth by William Astbury in 1933. Later work by Walter Kauzmann on denaturation, based partly on previous studies by Kaj Linderstrøm-Lang, contributed an understanding of protein folding and structure mediated by hydrophobic interactions.
The first protein to have its amino acid chain sequenced was insulin, by Frederick Sanger, in 1949. Sanger correctly determined the amino acid sequence of insulin, thus conclusively demonstrating that proteins consisted of linear polymers of amino acids rather than branched chains, colloids, or cyclols. He won the Nobel Prize for this achievement in 1958. Christian Anfinsen's studies of the oxidative folding process of ribonuclease A, for which he won the nobel prize in 1972, solidified the thermodynamic hypothesis of protein folding, according to which the folded form of a protein represents its free energy minimum.
=== Structure ===
With the development of X-ray crystallography, it became possible to determine protein structures as well as their sequences. The first protein structures to be solved were hemoglobin by Max Perutz and myoglobin by John Kendrew, in 1958. The use of computers and increasing computing power has supported the sequencing of complex proteins. In 1999, Roger Kornberg sequenced the highly complex structure of RNA polymerase using high intensity X-rays from synchrotrons.
Since then, cryo-electron microscopy (cryo-EM) of large macromolecular assemblies has been developed. Cryo-EM uses protein samples that are frozen rather than crystals, and beams of electrons rather than X-rays. It causes less damage to the sample, allowing scientists to obtain more information and analyze larger structures. Computational protein structure prediction of small protein structural domains has helped researchers to approach atomic-level resolution of protein structures.
As of April 2024, the Protein Data Bank contains 181,018 X-ray, 19,809 EM and 12,697 NMR protein structures.
== Classification ==
Proteins are primarily classified by sequence and structure, although other classifications are commonly used. Especially for enzymes the EC number system provides a functional classification scheme. Similarly, gene ontology classifies both genes and proteins by their biological and biochemical function, and by their intracellular location.
Sequence similarity is used to classify proteins both in terms of evolutionary and functional similarity. This may use either whole proteins or protein domains, especially in multi-domain proteins. Protein domains allow protein classification by a combination of sequence, structure and function, and they can be combined in many ways. In an early study of 170,000 proteins, about two-thirds were assigned at least one domain, with larger proteins containing more domains (e.g. proteins larger than 600 amino acids having an average of more than 5 domains).
== Biochemistry ==
Most proteins consist of linear polymers built from series of up to 20 L-α-amino acids. All proteinogenic amino acids have a common structure where an α-carbon is bonded to an amino group, a carboxyl group, and a variable side chain. Only proline differs from this basic structure as its side chain is cyclical, bonding to the amino group, limiting protein chain flexibility. The side chains of the standard amino acids have a variety of chemical structures and properties, and it is the combined effect of all amino acids that determines its three-dimensional structure and chemical reactivity.
The amino acids in a polypeptide chain are linked by peptide bonds between amino and carboxyl group. An individual amino acid in a chain is called a residue, and the linked series of carbon, nitrogen, and oxygen atoms are known as the main chain or protein backbone.: 19 The peptide bond has two resonance forms that confer some double-bond character to the backbone. The alpha carbons are roughly coplanar with the nitrogen and the carbonyl (C=O) group. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone. One conseqence of the N-C(O) double bond character is that proteins are somewhat rigid.: 31 A polypeptide chain ends with a free amino group, known as the N-terminus or amino terminus, and a free carboxyl group, known as the C-terminus or carboxy terminus. By convention, peptide sequences are written N-terminus to C-terminus, correlating with the order in which proteins are synthesized by ribosomes.
The words protein, polypeptide, and peptide are a little ambiguous and can overlap in meaning. Protein is generally used to refer to the complete biological molecule in a stable conformation, whereas peptide is generally reserved for a short amino acid oligomers often lacking a stable 3D structure. But the boundary between the two is not well defined and usually lies near 20–30 residues.
Proteins can interact with many types of molecules and ions, including with other proteins, with lipids, with carbohydrates, and with DNA.
=== Abundance in cells ===
A typical bacterial cell, e.g. E. coli and Staphylococcus aureus, is estimated to contain about 2 million proteins. Smaller bacteria, such as Mycoplasma or spirochetes contain fewer molecules, on the order of 50,000 to 1 million. By contrast, eukaryotic cells are larger and thus contain much more protein. For instance, yeast cells have been estimated to contain about 50 million proteins and human cells on the order of 1 to 3 billion. The concentration of individual protein copies ranges from a few molecules per cell up to 20 million. Not all genes coding proteins are expressed in most cells and their number depends on, for example, cell type and external stimuli. For instance, of the 20,000 or so proteins encoded by the human genome, only 6,000 are detected in lymphoblastoid cells. The most abundant protein in nature is thought to be RuBisCO, an enzyme that catalyzes the incorporation of carbon dioxide into organic matter in photosynthesis. Plants can consist of as much as 1% by weight of this enzyme.
== Synthesis ==
=== Biosynthesis ===
Proteins are assembled from amino acids using information encoded in genes. Each protein has its own unique amino acid sequence that is specified by the nucleotide sequence of the gene encoding this protein. The genetic code is a set of three-nucleotide sets called codons and each three-nucleotide combination designates an amino acid, for example AUG (adenine–uracil–guanine) is the code for methionine. Because DNA contains four nucleotides, the total number of possible codons is 64; hence, there is some redundancy in the genetic code, with some amino acids specified by more than one codon.: 1002–42 Genes encoded in DNA are first transcribed into pre-messenger RNA (mRNA) by proteins such as RNA polymerase. Most organisms then process the pre-mRNA (a primary transcript) using various forms of post-transcriptional modification to form the mature mRNA, which is then used as a template for protein synthesis by the ribosome. In prokaryotes the mRNA may either be used as soon as it is produced, or be bound by a ribosome after having moved away from the nucleoid. In contrast, eukaryotes make mRNA in the cell nucleus and then translocate it across the nuclear membrane into the cytoplasm, where protein synthesis then takes place. The rate of protein synthesis is higher in prokaryotes than eukaryotes and can reach up to 20 amino acids per second.
The process of synthesizing a protein from an mRNA template is known as translation. The mRNA is loaded onto the ribosome and is read three nucleotides at a time by matching each codon to its base pairing anticodon located on a transfer RNA molecule, which carries the amino acid corresponding to the codon it recognizes. The enzyme aminoacyl tRNA synthetase "charges" the tRNA molecules with the correct amino acids. The growing polypeptide is often termed the nascent chain. Proteins are always biosynthesized from N-terminus to C-terminus.: 1002–42
The size of a synthesized protein can be measured by the number of amino acids it contains and by its total molecular mass, which is normally reported in units of daltons (synonymous with atomic mass units), or the derivative unit kilodalton (kDa). The average size of a protein increases from Archaea to Bacteria to Eukaryote (283, 311, 438 residues and 31, 34, 49 kDa respectively) due to a bigger number of protein domains constituting proteins in higher organisms. For instance, yeast proteins are on average 466 amino acids long and 53 kDa in mass. The largest known proteins are the titins, a component of the muscle sarcomere, with a molecular mass of almost 3,000 kDa and a total length of almost 27,000 amino acids.
=== Chemical synthesis ===
Short proteins can be synthesized chemically by a family of peptide synthesis methods. These rely on organic synthesis techniques such as chemical ligation to produce peptides in high yield. Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains, such as attachment of fluorescent probes to amino acid side chains. These methods are useful in laboratory biochemistry and cell biology, though generally not for commercial applications. Chemical synthesis is inefficient for polypeptides longer than about 300 amino acids, and the synthesized proteins may not readily assume their native tertiary structure. Most chemical synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction.
== Structure ==
Most proteins fold into unique 3D structures. The shape into which a protein naturally folds is known as its native conformation.: 36 Although many proteins can fold unassisted, simply through the chemical properties of their amino acids, others require the aid of molecular chaperones to fold into their native states.: 37 Biochemists often refer to four distinct aspects of a protein's structure:: 30–34
Primary structure: the amino acid sequence. A protein is a polyamide.
Secondary structure: regularly repeating local structures stabilized by hydrogen bonds. The most common examples are the α-helix, β-sheet and turns. Because secondary structures are local, many regions of distinct secondary structure can be present in the same protein molecule.
Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another. Tertiary structure is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulfide bonds, and even post-translational modifications. The term "tertiary structure" is often used as synonymous with the term fold. The tertiary structure is what controls the basic function of the protein.
Quaternary structure: the structure formed by several protein molecules (polypeptide chains), usually called protein subunits in this context, which function as a single protein complex.
Quinary structure: the signatures of protein surface that organize the crowded cellular interior. Quinary structure is dependent on transient, yet essential, macromolecular interactions that occur inside living cells.
Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift between several related structures while they perform their functions. In the context of these functional rearrangements, these tertiary or quaternary structures are usually referred to as "conformations", and transitions between them are called conformational changes. Such changes are often induced by the binding of a substrate molecule to an enzyme's active site, or the physical region of the protein that participates in chemical catalysis. In solution, protein structures vary because of thermal vibration and collisions with other molecules.: 368–75
Proteins can be informally divided into three main classes, which correlate with typical tertiary structures: globular proteins, fibrous proteins, and membrane proteins. Almost all globular proteins are soluble and many are enzymes. Fibrous proteins are often structural, such as collagen, the major component of connective tissue, or keratin, the protein component of hair and nails. Membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane.: 165–85
A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their own dehydration, are called dehydrons.
=== Protein domains ===
Many proteins are composed of several protein domains, i.e. segments of a protein that fold into distinct structural units.: 134 Domains usually have specific functions, such as enzymatic activities (e.g. kinase) or they serve as binding modules.: 155–156
=== Sequence motif ===
Short amino acid sequences within proteins often act as recognition sites for other proteins. For instance, SH3 domains typically bind to short PxxP motifs (i.e. 2 prolines [P], separated by two unspecified amino acids [x], although the surrounding amino acids may determine the exact binding specificity). Many such motifs has been collected in the Eukaryotic Linear Motif (ELM) database.
== Cellular functions ==
Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes. With the exception of certain types of RNA, most other biological molecules are relatively inert elements upon which proteins act. Proteins make up half the dry weight of an Escherichia coli cell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively. The set of proteins expressed in a particular cell or cell type is known as its proteome.: 120
The chief characteristic of proteins that allows their diverse set of functions is their ability to bind other molecules specifically and tightly. The region of the protein responsible for binding another molecule is known as the binding site and is often a depression or "pocket" on the molecular surface. This binding ability is mediated by the tertiary structure of the protein, which defines the binding site pocket, and by the chemical properties of the surrounding amino acids' side chains. Protein binding can be extraordinarily tight and specific; for example, the ribonuclease inhibitor protein binds to human angiogenin with a sub-femtomolar dissociation constant (<10−15 M) but does not bind at all to its amphibian homolog onconase (> 1 M). Extremely minor chemical changes such as the addition of a single methyl group to a binding partner can sometimes suffice to nearly eliminate binding; for example, the aminoacyl tRNA synthetase specific to the amino acid valine discriminates against the very similar side chain of the amino acid isoleucine.
Proteins can bind to other proteins as well as to small-molecule substrates. When proteins bind specifically to other copies of the same molecule, they can oligomerize to form fibrils; this process occurs often in structural proteins that consist of globular monomers that self-associate to form rigid fibers. Protein–protein interactions regulate enzymatic activity, control progression through the cell cycle, and allow the assembly of large protein complexes that carry out many closely related reactions with a common biological function. Proteins can bind to, or be integrated into, cell membranes. The ability of binding partners to induce conformational changes in proteins allows the construction of enormously complex signaling networks.: 830–49
As interactions between proteins are reversible and depend heavily on the availability of different groups of partner proteins to form aggregates that are capable to carry out discrete sets of function, study of the interactions between specific proteins is a key to understand important aspects of cellular function, and ultimately the properties that distinguish particular cell types.
=== Enzymes ===
The best-known role of proteins in the cell is as enzymes, which catalyse chemical reactions. Enzymes are usually highly specific and accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions involved in metabolism, as well as manipulating DNA in processes such as DNA replication, DNA repair, and transcription. Some enzymes act on other proteins to add or remove chemical groups in a process known as posttranslational modification. About 4,000 reactions are known to be catalysed by enzymes. The rate acceleration conferred by enzymatic catalysis is often enormous—as much as 1017-fold increase in rate over the uncatalysed reaction in the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the enzyme).
The molecules bound and acted upon by enzymes are called substrates. Although enzymes can consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in contact with the substrate, and an even smaller fraction—three to four residues on average—that are directly involved in catalysis. The region of the enzyme that binds the substrate and contains the catalytic residues is known as the active site.: 389
Dirigent proteins are members of a class of proteins that dictate the stereochemistry of a compound synthesized by other enzymes.
=== Cell signaling and ligand binding ===
Many proteins are involved in the process of cell signaling and signal transduction. Some proteins, such as insulin, are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distant tissues. Others are membrane proteins that act as receptors whose main function is to bind a signaling molecule and induce a biochemical response in the cell. Many receptors have a binding site exposed on the cell surface and an effector domain within the cell, which may have enzymatic activity or may undergo a conformational change detected by other proteins within the cell.: 251–81
Antibodies are protein components of an adaptive immune system whose main function is to bind antigens, or foreign substances in the body, and target them for destruction. Antibodies can be secreted into the extracellular environment or anchored in the membranes of specialized B cells known as plasma cells. Whereas enzymes are limited in their binding affinity for their substrates by the necessity of conducting their reaction, antibodies have no such constraints. An antibody's binding affinity to its target is extraordinarily high.: 275–50
Many ligand transport proteins bind particular small biomolecules and transport them to other locations in the body of a multicellular organism. These proteins must have a high binding affinity when their ligand is present in high concentrations, and release the ligand when it is present at low concentrations in the target tissues. The canonical example of a ligand-binding protein is haemoglobin, which transports oxygen from the lungs to other organs and tissues in all vertebrates and has close homologs in every biological kingdom.: 222–29 Lectins are sugar-binding proteins which are highly specific for their sugar moieties. Lectins typically play a role in biological recognition phenomena involving cells and proteins. Receptors and hormones are highly specific binding proteins.
Transmembrane proteins can serve as ligand transport proteins that alter the permeability of the cell membrane to small molecules and ions. The membrane alone has a hydrophobic core through which polar or charged molecules cannot diffuse. Membrane proteins contain internal channels that allow such molecules to enter and exit the cell. Many ion channel proteins are specialized to select for only a particular ion; for example, potassium and sodium channels often discriminate for only one of the two ions.: 232–34
=== Structural proteins ===
Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most structural proteins are fibrous proteins; for example, collagen and elastin are critical components of connective tissue such as cartilage, and keratin is found in hard or filamentous structures such as hair, nails, feathers, hooves, and some animal shells.: 178–81 Some globular proteins can play structural functions, for example, actin and tubulin are globular and soluble as monomers, but polymerize to form long, stiff fibers that make up the cytoskeleton, which allows the cell to maintain its shape and size.: 490
Other proteins that serve structural functions are motor proteins such as myosin, kinesin, and dynein, which are capable of generating mechanical forces. These proteins are crucial for cellular motility of single celled organisms and the sperm of many multicellular organisms which reproduce sexually. They generate the forces exerted by contracting muscles: 258–64, 272 and play essential roles in intracellular transport.: 481, 490
== Methods of study ==
Methods commonly used to study protein structure and function include immunohistochemistry, site-directed mutagenesis, X-ray crystallography, nuclear magnetic resonance and mass spectrometry. The activities and structures of proteins may be examined in vitro, in vivo, and in silico. In vitro studies of purified proteins in controlled environments are useful for learning how a protein carries out its function: for example, enzyme kinetics studies explore the chemical mechanism of an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. By contrast, in vivo experiments can provide information about the physiological role of a protein in the context of a cell or even a whole organism, and can often provide more information about protein behavior in different contexts. In silico studies use computational methods to study proteins.
=== Protein purification ===
Proteins may be purified from other cellular components using a variety of techniques such as ultracentrifugation, precipitation, electrophoresis, and chromatography;: 21–24 the advent of genetic engineering has made possible a number of methods to facilitate purification.
To perform in vitro analysis, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; membrane lipids and proteins; cellular organelles, and nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity.: 21–24 The level of purification can be monitored using various types of gel electrophoresis if the desired protein's molecular weight and isoelectric point are known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Additionally, proteins can be isolated according to their charge using electrofocusing.
For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded. A number of tags have been developed to help researchers purify specific proteins from complex mixtures.
=== Cellular localization ===
The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the cytoplasm and membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins are targeted to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion protein or chimera consisting of the natural protein of interest linked to a "reporter" such as green fluorescent protein (GFP). The fused protein's position within the cell can then be cleanly and efficiently visualized using microscopy.
Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER, the Golgi, lysosomes or vacuoles, mitochondria, chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions of these markers or of antibodies to known markers, it becomes much simpler to identify the localization of a protein of interest. For example, indirect immunofluorescence will allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose.
Other possibilities exist, as well. For example, immunohistochemistry usually uses an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be compared between samples, allowing for localization information. Another applicable technique is cofractionation in sucrose (or other material) gradients using isopycnic centrifugation. While this technique does not prove colocalization of a compartment of known density and the protein of interest, it indicates an increased likelihood.
Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This technique uses an antibody to the protein of interest, along with classical electron microscopy techniques. The sample is prepared for normal electron microscopic examination, and then treated with an antibody to the protein of interest that is conjugated to an extremely electro-dense material, usually gold. This allows for the localization of both ultrastructural details as well as the protein of interest.
Through another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation. This technique even allows the incorporation of unnatural amino acids into proteins, using modified tRNAs, and may allow the rational design of new proteins with novel properties.
=== Proteomics ===
The total complement of proteins present at a time in a cell or cell type is known as its proteome, and the study of such large-scale data sets defines the field of proteomics, named by analogy to the related field of genomics. Key experimental techniques in proteomics include 2D electrophoresis, which allows the separation of many proteins, mass spectrometry, which allows rapid high-throughput identification of proteins and sequencing of peptides (most often after in-gel digestion), protein microarrays, which allow the detection of the relative levels of the various proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of protein–protein interactions. The total complement of biologically possible such interactions is known as the interactome. A systematic attempt to determine the structures of proteins representing every possible fold is known as structural genomics.
=== Structure determination ===
Discovering the tertiary structure of a protein, or the quaternary structure of its complexes, can provide important clues about how the protein performs its function and how it can be affected, i.e. in drug design. As proteins are too small to be seen under a light microscope, other methods have to be employed to determine their structure. Common experimental methods include X-ray crystallography and NMR spectroscopy, both of which can produce structural information at atomic resolution. However, NMR experiments are able to provide information from which a subset of distances between pairs of atoms can be estimated, and the final possible conformations for a protein are determined by solving a distance geometry problem. Dual polarisation interferometry is a quantitative analytical method for measuring the overall protein conformation and conformational changes due to interactions or other stimulus. Circular dichroism is another laboratory technique for determining internal β-sheet / α-helical composition of proteins. Cryoelectron microscopy is used to produce lower-resolution structural information about very large protein complexes, including assembled viruses;: 340–41 a variant known as electron crystallography can produce high-resolution information in some cases, especially for two-dimensional crystals of membrane proteins. Solved structures are usually deposited in the Protein Data Bank (PDB), a freely available resource from which structural data about thousands of proteins can be obtained in the form of Cartesian coordinates for each atom in the protein.
Many more gene sequences are known than protein structures. Further, the set of solved structures is biased toward proteins that can be easily subjected to the conditions required in X-ray crystallography, one of the major structure determination methods. In particular, globular proteins are comparatively easy to crystallize in preparation for X-ray crystallography. Membrane proteins and large protein complexes, by contrast, are difficult to crystallize and are underrepresented in the PDB. Structural genomics initiatives have attempted to remedy these deficiencies by systematically solving representative structures of major fold classes. Protein structure prediction methods attempt to provide a means of generating a plausible structure for proteins whose structures have not been experimentally determined.
=== Structure prediction ===
Complementary to the field of structural genomics, protein structure prediction develops efficient mathematical models of proteins to computationally predict the molecular formations in theory, instead of detecting structures with laboratory observation. The most successful type of structure prediction, known as homology modeling, relies on the existence of a "template" structure with sequence similarity to the protein being modeled; structural genomics' goal is to provide sufficient representation in solved structures to model most of those that remain. Although producing accurate models remains a challenge when only distantly related template structures are available, it has been suggested that sequence alignment is the bottleneck in this process, as quite accurate models can be produced if a "perfect" sequence alignment is known. Many structure prediction methods have served to inform the emerging field of protein engineering, in which novel protein folds have already been designed. Many proteins (in eukaryotes ~33%) contain large unstructured but biologically functional segments and can be classified as intrinsically disordered proteins. Predicting and analysing protein disorder is an important part of protein structure characterisation.
=== In silico simulation of dynamical processes ===
A more complex computational problem is the prediction of intermolecular interactions, such as in molecular docking, protein folding, protein–protein interaction and chemical reactivity. Mathematical models to simulate these dynamical processes involve molecular mechanics, in particular, molecular dynamics. In this regard, in silico simulations discovered the folding of small α-helical protein domains such as the villin headpiece, the HIV accessory protein and hybrid methods combining standard molecular dynamics with quantum mechanical mathematics have explored the electronic states of rhodopsins.
Beyond classical molecular dynamics, quantum dynamics methods allow the simulation of proteins in atomistic detail with an accurate description of quantum mechanical effects. Examples include the multi-layer multi-configuration time-dependent Hartree method and the hierarchical equations of motion approach, which have been applied to plant cryptochromes and bacteria light-harvesting complexes, respectively. Both quantum and classical mechanical simulations of biological-scale systems are extremely computationally demanding, so distributed computing initiatives such as the Folding@home project facilitate the molecular modeling by exploiting advances in GPU parallel processing and Monte Carlo techniques.
=== Chemical analysis ===
The total nitrogen content of organic matter is mainly formed by the amino groups in proteins. The Total Kjeldahl Nitrogen (TKN) is a measure of nitrogen widely used in the analysis of (waste) water, soil, food, feed and organic matter in general. As the name suggests, the Kjeldahl method is applied. More sensitive methods are available.
== Digestion ==
In the absence of catalysts, proteins are slow to hydrolyze. The breakdown of proteins to small peptides and amino acids (proteolysis) is a step in digestion; these breakdown products are then absorbed in the small intestine. The hydrolysis of proteins relies on enzymes called proteases or peptidases. Proteases, which are themselves proteins, come in several types according to the particular peptide bonds that they cleave as well as their tendency to cleave peptide bonds at the terminus of a protein (exopeptidases) vs peptide bonds at the interior of the protein (endopeptidases). Pepsin is an endopeptidase in the stomach. Subsequent to the stomach, the pancreas secretes other proteases to complete the hydrolysis, these include trypsin and chymotrypsin.
Protein hydrolysis is employed commercially as a means of producing amino acids from bulk sources of protein, such as blood meal, feathers, keratin. Such materials are treated with hot hydrochloric acid, which effects the hydrolysis of the peptide bonds.
== Mechanical properties ==
The mechanical properties of proteins are highly diverse and are often central to their biological function, as in the case of proteins like keratin and collagen. For instance, the ability of muscle tissue to continually expand and contract is directly tied to the elastic properties of their underlying protein makeup. Beyond fibrous proteins, the conformational dynamics of enzymes and the structure of biological membranes, among other biological functions, are governed by the mechanical properties of the proteins. Outside of their biological context, the unique mechanical properties of many proteins, along with their relative sustainability when compared to synthetic polymers, have made them desirable targets for next-generation materials design.
Young's modulus, E, is calculated as the axial stress σ over the resulting strain ε. It is a measure of the relative stiffness of a material. In the context of proteins, this stiffness often directly correlates to biological function. For example, collagen, found in connective tissue, bones, and cartilage, and keratin, found in nails, claws, and hair, have observed stiffnesses that are several orders of magnitude higher than that of elastin, which is though to give elasticity to structures such as blood vessels, pulmonary tissue, and bladder tissue, among others. In comparison to this, globular proteins, such as Bovine Serum Albumin, which float relatively freely in the cytosol and often function as enzymes (and thus undergoing frequent conformational changes) have comparably much lower Young's moduli.
The Young's modulus of a single protein can be found through molecular dynamics simulation. Using either atomistic force-fields, such as CHARMM or GROMOS, or coarse-grained forcefields like Martini, a single protein molecule can be stretched by a uniaxial force while the resulting extension is recorded in order to calculate the strain. Experimentally, methods such as atomic force microscopy can be used to obtain similar data. The internal dynamics of proteins involve subtle elastic and plastic deformations induced by viscoelastic forces, which can be probed by nano-rheology techniques. These estimates yield typical spring constants around k ≈ 100 pN/nm, equivalent to Yonung's moduli of E ≈ 100 MPa, and typical friction coefficients of γ ≈ 0.1 pN·s/nm, corresponding to viscosity of η ≈ 0.01 pN·s/nm2 = 107cP (that is, 107 more viscous than water).
At the macroscopic level, the Young's modulus of cross-linked protein networks can be obtained through more traditional mechanical testing. Experimentally observed values for a few proteins can be seen below.
== See also ==
== References ==
== Further reading ==
Textbooks
History
Tanford C, Reynolds JA (2001). Nature's Robots: A History of Proteins. Oxford New York: Oxford University Press, USA. ISBN 978-0-19-850466-5.
== External links ==
=== Databases and projects ===
NCBI Entrez Protein database
NCBI Protein Structure database
Human Protein Reference Database
Human Proteinpedia
Folding@Home (Stanford University) Archived 2012-09-08 at the Wayback Machine
Protein Databank in Europe (see also PDBeQuips, short articles and tutorials on interesting PDB structures)
Research Collaboratory for Structural Bioinformatics (see also Molecule of the Month Archived 2020-07-24 at the Wayback Machine, presenting short accounts on selected proteins from the PDB)
Proteopedia – Life in 3D: rotatable, zoomable 3D model with wiki annotations for every known protein molecular structure.
UniProt the Universal Protein Resource
=== Tutorials and educational websites ===
"An Introduction to Proteins" from HOPES (Huntington's Disease Outreach Project for Education at Stanford)
Proteins: Biogenesis to Degradation – The Virtual Library of Biochemistry and Cell Biology | Wikipedia/Structural_proteins |
Terrestrial ecosystems are ecosystems that are found on land. Examples include tundra, taiga, temperate deciduous forest, tropical rain forest, grassland, deserts.
Terrestrial ecosystems differ from aquatic ecosystems by the predominant presence of soil rather than water at the surface and by the extension of plants above this soil/water surface in terrestrial ecosystems. There is a wide range of water availability among terrestrial ecosystems (including water scarcity in some cases), whereas water is seldom a limiting factor to organisms in aquatic ecosystems. Because water buffers temperature fluctuations, terrestrial ecosystems usually experience greater diurnal and seasonal temperature fluctuations than do aquatic ecosystems in similar climates.
Terrestrial ecosystems are of particular importance especially in meeting Sustainable Development Goal 15 that targets the conservation-restoration and sustainable use of terrestrial ecosystems.
== Organisms and processes ==
Organisms in terrestrial ecosystems have adaptations that allow them to obtain water when the entire body is no longer bathed in that fluid, means of transporting the water from limited sites of acquisition to the rest of the body, and means of preventing the evaporation of water from body surfaces. They also have traits that provide body support in the atmosphere, a much less buoyant medium than water, and other traits that render them capable of withstanding the extremes of temperature, wind, and humidity that characterize terrestrial ecosystems. Finally, the organisms in terrestrial ecosystems have evolved many methods of transporting gametes in environments where fluid flow is much less effective as a transport medium. This is terrestrial ecosystems.
Common Types of Terrestrial Plants
Four main groupings for terrestrial plants are bryophytes, pteridophytes, gymnosperms, and angiosperms, have been existing for many years and have allowed diversity into our ecosystems .
== Size and plants ==
Terrestrial ecosystems occupy 55,660,000 mi2 (144,150,000 km2), or 28.26% of Earth's surface. Major plant taxa in terrestrial ecosystems are members of the division Magnoliophyta (flowering plants), of which there are about 275,000 species, and the division Pinophyta (conifers), of which there are about 500 species. Members of the division Bryophyta (mosses and liverworts), of which there are about 24,000 species, are also important in some terrestrial ecosystems. Major animal taxa in terrestrial ecosystems include the classes Insecta (insects) with about 900,000 species, Aves (birds) with 8,500 species, and Mammalia (mammals) with approximately 4,100 species.
== See also ==
Aquatic-terrestrial subsidies
Colonization of land - history of terrestrial life
Soil ecology
== References ==
[1]
[2]
[3] | Wikipedia/Terrestrial_ecosystem |
Peripheral membrane proteins, or extrinsic membrane proteins, are membrane proteins that adhere only temporarily to the biological membrane with which they are associated. These proteins attach to integral membrane proteins, or penetrate the peripheral regions of the lipid bilayer. The regulatory protein subunits of many ion channels and transmembrane receptors, for example, may be defined as peripheral membrane proteins. In contrast to integral membrane proteins, peripheral membrane proteins tend to collect in the water-soluble component, or fraction, of all the proteins extracted during a protein purification procedure. Proteins with GPI anchors are an exception to this rule and can have purification properties similar to those of integral membrane proteins.
The reversible attachment of proteins to biological membranes has shown to regulate cell signaling and many other important cellular events, through a variety of mechanisms. For example, the close association between many enzymes and biological membranes may bring them into close proximity with their lipid substrate(s). Membrane binding may also promote rearrangement, dissociation, or conformational changes within many protein structural domains, resulting in an activation of their biological activity. Additionally, the positioning of many proteins are localized to either the inner or outer surfaces or leaflets of their resident membrane.
This facilitates the assembly of multi-protein complexes by increasing the probability of any appropriate protein–protein interactions.
== Binding to the lipid bilayer ==
Peripheral membrane proteins may interact with other proteins or directly with the lipid bilayer. In the latter case, they are then known as amphitropic proteins.
Some proteins, such as G-proteins and certain protein kinases, interact with transmembrane proteins and the lipid bilayer simultaneously. Some polypeptide hormones, antimicrobial peptides, and neurotoxins accumulate at the membrane surface prior to locating and interacting with their cell surface receptor targets, which may themselves be peripheral membrane proteins.
The phospholipid bilayer that forms the cell surface membrane consists of a hydrophobic inner core region sandwiched between two regions of hydrophilicity, one at the inner surface and one at the outer surface of the cell membrane (see lipid bilayer article for a more detailed structural description of the cell membrane). The inner and outer surfaces, or interfacial regions, of model phospholipid bilayers have been shown to have a thickness of around 8 to 10 Å, although this may be wider in biological membranes that include large amounts of gangliosides or lipopolysaccharides.
The hydrophobic inner core region of typical biological membranes may have a thickness of around 27 to 32 Å, as estimated by Small angle X-ray scattering (SAXS).
The boundary region between the hydrophobic inner core and the hydrophilic interfacial regions is very narrow, at around 3 Å, (see lipid bilayer article for a description of its component chemical groups). Moving outwards away from the hydrophobic core region and into the interfacial hydrophilic region, the effective concentration of water rapidly changes across this boundary layer, from nearly zero to a concentration of around 2 M.
The phosphate groups within phospholipid bilayers are fully hydrated or saturated with water and are situated around 5 Å outside the boundary of the hydrophobic core region.
Some water-soluble proteins associate with lipid bilayers irreversibly and can form transmembrane alpha-helical or beta-barrel channels. Such transformations occur in pore forming toxins such as colicin A, alpha-hemolysin, and others. They may also occur in BcL-2 like protein , in some amphiphilic antimicrobial peptides , and in certain annexins . These proteins are usually described as peripheral as one of their conformational states is water-soluble or only loosely associated with a membrane.
== Membrane binding mechanisms ==
The association of a protein with a lipid bilayer may involve significant changes within tertiary structure of a protein. These may include the folding of regions of protein structure that were previously unfolded or a re-arrangement in the folding or a refolding of the membrane-associated part of the proteins. It also may involve the formation or dissociation of protein quaternary structures or oligomeric complexes, and specific binding of ions, ligands, or regulatory lipids.
Typical amphitropic proteins must interact strongly with the lipid bilayer in order to perform their biological functions. These include the enzymatic processing of lipids and other hydrophobic substances, membrane anchoring, and the binding and transfer of small nonpolar compounds between different cellular membranes. These proteins may be anchored to the bilayer as a result of hydrophobic interactions between the bilayer and exposed nonpolar residues at the surface of a protein, by specific non-covalent binding interactions with regulatory lipids , or through their attachment to covalently bound lipid anchors.
It has been shown that the membrane binding affinities of many peripheral proteins depend on the specific lipid composition of the membrane with which they are associated.
=== Non-specific hydrophobic association ===
Amphitropic proteins associate with lipid bilayers via various hydrophobic anchor structures. Such as amphiphilic α-helixes, exposed nonpolar loops, post-translationally acylated or lipidated amino acid residues, or acyl chains of specifically bound regulatory lipids such as phosphatidylinositol phosphates. Hydrophobic interactions have been shown to be important even for highly cationic peptides and proteins, such as the polybasic domain of the MARCKS protein or histactophilin, when their natural hydrophobic anchors are present.
=== Covalently bound lipid anchors ===
Lipid anchored proteins are covalently attached to different fatty acid acyl chains on the cytoplasmic side of the cell membrane via palmitoylation, myristoylation, or prenylation. On the exoplasmic face of the cell membrane, lipid anchored proteins are covalently attached to the lipids glycosylphosphatidylinositol (GPI) and cholesterol. Protein association with membranes through the use of acylated residues is a reversible process, as the acyl chain can be buried in a protein's hydrophobic binding pocket after dissociation from the membrane. This process occurs within the beta-subunits of G-proteins. Perhaps because of this additional need for structural flexibility, lipid anchors are usually bound to the highly flexible segments of proteins tertiary structure that are not well resolved by protein crystallographic studies.
=== Specific protein–lipid binding ===
Some cytosolic proteins are recruited to different cellular membranes by recognizing certain types of lipid found within a given membrane. Binding of a protein to a specific lipid occurs via specific membrane-targeting structural domains that occur within the protein and have specific binding pockets for the lipid head groups of the lipids to which they bind. This is a typical biochemical protein–ligand interaction, and is stabilized by the formation of intermolecular hydrogen bonds, van der Waals interactions, and hydrophobic interactions between the protein and lipid ligand. Such complexes are also stabilized by the formation of ionic bridges between the aspartate or glutamate residues of the protein and lipid phosphates via intervening calcium ions (Ca2+). Such ionic bridges can occur and are stable when ions (such as Ca2+) are already bound to a protein in solution, prior to lipid binding. The formation of ionic bridges is seen in the protein–lipid interaction between both protein C2 type domains and annexins..
=== Protein–lipid electrostatic interactions ===
Any positively charged protein will be attracted to a negatively charged membrane by nonspecific electrostatic interactions. However, not all peripheral peptides and proteins are cationic, and only certain sides of membrane are negatively charged. These include the cytoplasmic side of plasma membranes, the outer leaflet of bacterial outer membranes and mitochondrial membranes. Therefore, electrostatic interactions play an important role in membrane targeting of electron carriers such as cytochrome c, cationic toxins such as charybdotoxin, and specific membrane-targeting domains such as some PH domains, C1 domains, and C2 domains.
Electrostatic interactions are strongly dependent on the ionic strength of the solution. These interactions are relatively weak at the physiological ionic strength (0.14M NaCl): ~3 to 4 kcal/mol for small cationic proteins, such as cytochrome c, charybdotoxin or hisactophilin.
== Spatial position in membrane ==
Orientations and penetration depths of many amphitropic proteins and peptides in membranes are studied using site-directed spin labeling, chemical labeling, measurement of membrane binding affinities of protein mutants, fluorescence spectroscopy, solution or solid-state NMR spectroscopy,
ATR FTIR spectroscopy, X-ray or neutron diffraction, and computational methods.
Two distinct membrane-association modes of proteins have been identified. Typical water-soluble proteins have no exposed nonpolar residues or any other hydrophobic anchors. Therefore, they remain completely in aqueous solution and do not penetrate into the lipid bilayer, which would be energetically costly. Such proteins interact with bilayers only electrostatically, for example, ribonuclease and poly-lysine interact with membranes in this mode. However, typical amphitropic proteins have various hydrophobic anchors that penetrate the interfacial region and reach the hydrocarbon interior of the membrane. Such proteins "deform" the lipid bilayer, decreasing the temperature of lipid fluid-gel transition. The binding is usually a strongly exothermic reaction. Association of amphiphilic α-helices with membranes occurs similarly. Intrinsically unstructured or unfolded peptides with nonpolar residues or lipid anchors can also penetrate the interfacial region of the membrane and reach the hydrocarbon core, especially when such peptides are cationic and interact with negatively charged membranes.
== Categories ==
=== Enzymes ===
Peripheral enzymes participate in metabolism of different membrane components, such as lipids (phospholipases and cholesterol oxidases), cell wall oligosaccharides (glycosyltransferase and transglycosidases), or proteins (signal peptidase and palmitoyl protein thioesterases). Lipases can also digest lipids that form micelles or nonpolar droplets in water.
=== Membrane-targeting domains ("lipid clamps") ===
Membrane-targeting domains associate specifically with head groups of their lipid ligands embedded into the membrane. These lipid ligands are present in different concentrations in distinct types of biological membranes (for example, PtdIns3P can be found mostly in membranes of early endosomes, PtdIns(3,5)P2 in late endosomes, and PtdIns4P in the Golgi). Hence, each domain is targeted to a specific membrane.
C1 domains and phorbol esters.
C2 domains bind phosphatidylserine, phosphatidylcholine or PtdIns(3,4)P2 or PtdIns(4,5)P2.
Pleckstrin homology domains, PX domains, and Tubby domains bind different phosphoinositides
FYVE domains are more specific for PtdIns3P.
ENTH domains bind PtdIns(3,4)P2 or PtdIns(4,5)P2.
ANTH domain binds PtdIns(4,5)P2.
Proteins from ERM (ezrin/radixin/moesin) family bind PtdIns(4,5)P2.
Other phosphoinositide-binding proteins include phosphotyrosine-binding domain and certain PDZ domains. They bind PtdIns(4,5)P2.
Discoidin domains of blood coagulation factors
ENTH, VHS and ANTH domains
=== Structural domains ===
Structural domains mediate attachment of other proteins to membranes. Their binding to membranes can be mediated by calcium ions (Ca2+) that form bridges between the acidic protein residues and phosphate groups of lipids, as in annexins or GLA domains.
=== Transporters of small hydrophobic molecules ===
These peripheral proteins function as carriers of non-polar compounds between different types of cell membranes or between membranes and cytosolic protein complexes. The transported substances are phosphatidylinositol, tocopherol, gangliosides, glycolipids, sterol derivatives, retinol, fatty acids, water, macromolecules, red blood cells, phospholipids, and nucleotides.
Glycolipid transfer proteins
Lipocalins including retinol binding proteins and fatty acid-binding proteins
Polyisoprenoid-binding protein, such as YceI protein domain
Ganglioside GM2 activator proteins
CRAL-TRIO domain (α-Tocopherol and phosphatidylinositol sec14p transfer proteins)
Sterol carrier proteins
Phosphatidylinositol transfer proteins and STAR domains
Oxysterol-binding protein
=== Electron carriers ===
These proteins are involved in electron transport chains. They include cytochrome c, cupredoxins, high potential iron protein, adrenodoxin reductase, some flavoproteins, and others.
=== Polypeptide hormones, toxins, and antimicrobial peptides ===
Many hormones, toxins, inhibitors, or antimicrobial peptides interact specifically with transmembrane protein complexes. They can also accumulate at the lipid bilayer surface, prior to binding their protein targets. Such polypeptide ligands are often positively charged and interact electrostatically with anionic membranes.
Some water-soluble proteins and peptides can also form transmembrane channels. They usually undergo oligomerization, significant conformational changes, and associate with membranes irreversibly. 3D structure of one such transmembrane channel, α-hemolysin, has been determined. In other cases, the experimental structure represents a water-soluble conformation that interacts with the lipid bilayer peripherally, although some of the channel-forming peptides are rather hydrophobic and therefore were studied by NMR spectroscopy in organic solvents or in the presence of micelles.
== See also ==
Lipoproteins
Membrane proteins
== References ==
== Further reading ==
== External links ==
Peripheral membrane proteins in OPM database
DOLOP Genomics-oriented database of bacterial lipoproteins
Peptaibol database
Antimicrobial Peptide Database Archived 2011-07-20 at the Wayback Machine | Wikipedia/Peripheral_protein |
Ecology: From Individuals to Ecosystems is a 2006 higher education textbook on general ecology written by Michael Begon, Colin R. Townsend and John L. Harper. Published by Blackwell Publishing, it is now in its fourth edition. The first three editions were published by Blackwell Science under the title Ecology: Individuals, Populations and Communities. Since it first became available it has had a positive reception, and has long been one of the leading textbooks on ecology.
== Background and history ==
The book is written by Michael Begon of the University of Liverpool's School of Biosciences, Colin Townsend, from the Department of Zoology of New Zealand's University of Otago, and the University of Exeter's John L. Harper. The first edition was published in 1986. This was followed in 1990 with a second edition. The third edition became available in 1996. The most recent edition appeared in 2006 under the new subtitle From Individuals to Ecosystems.
One of the book's authors, John L. Harper, is now deceased. The fourth edition cover is an image of a mural on a Wellington street created by Christopher Meech and a group of urban artists to generate thought about the topic of environmental degradation. It reads "we did not inherit the earth from our ancestors, we borrowed it from our children."
== Contents ==
Part 1. ORGANISMS
1. Organisms in their environments: the evolutionary backdrop
2. Conditions
3. Resources
4. Life, death and life histories
5. Intraspecific competition
6. Dispersal, dormancy and metapopulations
7. Ecological applications at the level of organisms and single-species populations
Part 2. SPECIES INTERACTIONS
8. Interspecific competition
9. The nature of predation
10. The population dynamics of predation
11. Decomposers and detritivores
12. Parasitism and disease
13. Symbiosis and mutualism
14. Abundance
15. Ecological applications at the level of population interactions
Part 3. COMMUNITIES AND ECOSYSTEMS
16. The nature of the community
17. The flux of energy through ecosystems
18. The flux of matter through ecosystems
19. The influence of population interactions on community structure
20. Food webs
21. Patterns in species richness
22. Ecological applications at the level of communities and ecosystems
== References ==
== External links ==
Book Information from publisher | Wikipedia/Ecology:_From_Individuals_to_Ecosystems |
Vertebrate Palaeontology is a basic textbook on vertebrate paleontology by Michael J. Benton, published by Blackwell's. It has so far appeared in five editions, published in 1990, 1997, 2005, 2014, and 2024. It is designed for paleontology graduate courses in biology and geology as well as for the interested layman.
The book is widely used, and has received excellent reviews:
"This book is a ′must′ for a biology or geology student and researcher concerned by palaeontology. It perfectly succeeds in showing how palaeobiological information is obtained". Review of 3rd edition, Zentrallblatt fur Geologie und Palaontologie, 2007.
"One anticipates that Benton's Vertebrate Palaeontology will become the 'industry standard', and as such it should occupy space on the shelves of all involved in undergraduate teaching". Ivan Sansom, School of Earth Sciences, University of Birmingham. Review of the 2nd edition for the Micropalaeontological Society.
"... his expertise in a range of problems of vertebrate paleontology is amazing. As a result the contents of his book [are] very well balanced". Jerzy Dzik, Instytut Palaeobiologii PAN, Warsaw. Review of the 3rd edition for the Journal of Sedimentary Research.
The book gives an overall account of every major group of living and fossil vertebrate. At the rear of the book is a phylogenetic classification which combines both the Linnaean hierarchy and the cladistic arrangement, and has been used as a guideline for the Wikipedia pages on living and extinct vertebrate taxa. However, some of Benton's classification differs from that in the Tree of Life Web Project, especially as regards the relationship of early amphibian groups (Batrachomorpha and Reptiliomorpha).
== Bibliography ==
Benton, M. J. (2005), Vertebrate Palaeontology, 3rd ed. Blackwell Science Ltd
Benton, M. J. (2014), Vertebrate Palaeontology, 4th ed. Wiley-Blackwell
Publisher's Website and Book Overview: 3rd edition, edition
Benton, M. J. (1995) Paleontología y evolución de los Vertebrados. Ed. Perfils. ISBN 978-84-87695-16-2. Spanish translation by Aurora Grandal-d'Anglade upon the first edition.
Benton, M. J. (2007), Paläontologie der Wirbeltiere, Verlag Dr. Friedrich Pfeil. German translation by Hans-Ulrich Pfretzschner based upon the third edition.
== References == | Wikipedia/Vertebrate_Palaeontology_(Benton) |
Gain-of-function research (GoF research or GoFR) is medical research that genetically alters an organism in a way that may enhance the biological functions of gene products. This may include an altered pathogenesis, transmissibility, or host range, i.e., the types of hosts that a microorganism can infect. This research is intended to reveal targets to better predict emerging infectious diseases and to develop vaccines and therapeutics. For example, influenza B can infect only humans and harbor seals. Introducing a mutation that would allow influenza B to infect rabbits in a controlled laboratory situation would be considered a gain-of-function experiment, as the virus did not previously have that function. That type of experiment could then help reveal which parts of the virus's genome correspond to the species that it can infect, enabling the creation of antiviral medicines which block this function.
In virology, gain-of-function research is usually employed with the intention of better understanding current and future pandemics. In vaccine development, gain-of-function research is conducted in the hope of gaining a head start on a virus and being able to develop a vaccine or therapeutic before it emerges. The term "gain of function" is sometimes applied more narrowly to refer to "research which could enable a pandemic-potential pathogen to replicate more quickly or cause more harm in humans or other closely-related mammals."
Some forms of gain-of-function research (specifically work which involves certain select agent pathogens) carry inherent biosafety and biosecurity risks, and are thus also referred to as dual use research of concern (DURC). To mitigate these risks while allowing the benefits of such research, various governments have mandated that DURC experiments be regulated under additional oversight by institutions (so-called institutional "DURC" committees) and government agencies (such as the NIH's recombinant DNA advisory committee). A mirrored approach can be seen in the European Union's Dual Use Coordination Group (DUCG).
Importantly, regulations in the United States and European Union both mandate that at least one unaffiliated member of the public should be an active participant in the oversight process. Significant debate has taken place in the scientific community on how to assess the risks and benefit of gain-of-function research, how to publish such research responsibly, and how to engage the public in an open and honest review. In January 2020, the National Science Advisory Board for Biosecurity convened an expert panel to revisit the rules for gain-of-function research and provide more clarity in how such experiments are approved, and when they should be disclosed to the public.
== Experiments that have been referred to as "gain-of-function" ==
In early 2011, two groups were investigating how flu viruses specific to birds could possibly cross over and create pandemics in humans: one led by Yoshihiro Kawaoka at the University of Wisconsin–Madison in Madison, Wisconsin, and another led by Ron Fouchier at Erasmus University Medical Center in the Netherlands. Both groups had serially passaged the H5N1 avian influenza in ferrets, manually taking the virus from one ferret to another, until it was capable of spreading via respiratory droplets. The normally bird-specific virus, through replication over time in the ferrets' lungs, had adopted several amino acid changes that enabled it to replicate in the mammalian lungs, which are notably colder than those found in birds. This small change also allowed the virus to transmit via droplets in the air made when the ferrets coughed or sneezed.
Proponents of the Kawaoka and Fouchier experiments cited several benefits: these answered the question of how a virus like H5N1 could possibly become airborne in humans, allowed other researchers to develop vaccines and therapeutics which specifically targeted these amino acid changes, and also demonstrated that there was a linkage in avian viruses between transmissibility and lethality: while the virus had become more transmissible, it had also become significantly less deadly. Various critics of the research (including members of Congress) responded to the publications with alarm. Others called the experiments an "engineered doomsday". Questions were raised by other scientists including Marc Lipsitch of the T. H. Chan School of Public Health at Harvard University about the relative risks and benefits of this research.
At an international technical consultation convened by the WHO, it was concluded that this work was an important contribution to public health surveillance of H5N1 viruses and to a better understanding of the properties of these viruses, but that broader global discussions were needed. The European Academies of Science Advisory Council (EASAC) concluded that all required laws, rules, regulations, and codes of conduct are in place in several EU countries to continue this type of work responsibly. In the US, where regulations were previously less strict than in the EU, a new governmental policy and review mechanism was launched for "Potential Pandemic Pathogen Care and Oversight" (P3CO).
In May 2013, a group led by Hualan Chen, director of China's National Avian Influenza Reference Laboratory, published several experiments they had conducted at the BSL3+ laboratory of the Harbin Veterinary Research Institute, investigating what would happen if a 2009 H1N1 circulating in humans infected the same cell as an avian influenza H5N1. Importantly, the experiments had been conducted before a research pause on H5N1 experiments had been agreed upon by the broader virologist community. They used these experiments to determine that certain genes, if reassorted in such a dual-infection scenario in the wild, would allow transmission of the H5N1 virus more easily in mammals (notably guinea pigs as a model organism for rodent species), proving that certain agricultural scenarios carry the risk of allowing H5N1 to cross over into mammals. As in the Fouchier and Kawaoka experiments above, the viruses in this study were also significantly less lethal after the modification.
Critics of the 2013 Chen group study (including Simon Wain-Hobson of the Pasteur Institute and former Royal Society President Robert May) decried this as an unsafe experiment that was unnecessary to prove the intended conclusions, calling Chen's work "appallingly irresponsible" and also raising concerns about the biosafety of the laboratory itself. Others (including the director of the WHO Collaborating Centre on Influenza in Tokyo, Masato Tashiro) praised Chen's laboratory as "state of the art". Jeremy Farrar, director of the Oxford University Clinical Research Unit in Ho Chi Minh City, described the work as "remarkable" and said that it demonstrated the "very real threat" that "continued circulation of H5N1 strains in Asia and Egypt" posed.
A preprint by Boston University researchers, published on 14 October 2022, described their experiments splicing the SARS-CoV-2 BA.1 Omicron's spike protein into an ancestral SARS-CoV-2 variant isolated in the early days of the pandemic, creating a new chimeric version of the virus. All of the six mice exposed to the ancestral variant died; eight of the ten mice exposed to the chimeric variant died; and none of the ten mice exposed to Omicron died. This suggests that "mutations outside of spike are major determinants of the attenuated pathogenicity of Omicron in K18-hACE2 mice". According to the preprint, the work was supported by grants from various branches of the NIH, but the NIH later denied funding the experiments and the researchers stated the NIH did not fund the experiments directly. On 17 October, the Daily Mail ran the headline "Boston University CREATES a new COVID strain that has an 80% kill rate—echoing dangerous experiments feared to have started the pandemic". The headline was later flagged "as part of Facebook's efforts to combat false news and misinformation". PolitiFact noted the "lab leak" theory was unproven, and also stated "citing the 80% figure alone leaves out key context, including that the resulting strain was less fatal than the original, which killed 100% of mice. Experts say this kind of research is not unusual and the experiment was conducted in accordance with accepted safety procedures." All research funded by the NIH that can make COVID more virulent or transmissible must undergo an extra gain-of-function review. Critics charged that, because the chimera could have combined Omicron's high transmissibility with the ancestral strain's lethality, the experiment should have undergone the extra review. The researchers denied that the experiment qualified as gain-of-function in the first place.
== Gain-of-function research of concern ==
Significant debate has taken place in the scientific community on how to assess the risks and benefits of gain-of-function research, and how to engage the public in deliberations for policymaking. These concerns encompass biosafety, relating to the accidental release of a pathogen into the population, biosecurity relating to the intentional release of a pathogen into the population, and bioethics, the principles of biorisk management and research review procedures.
== Academic symposia ==
=== Gain-of-Function Research: A Symposium ===
In December 2014, the National Research Council and the Institute of Medicine organized a two-day symposium to discuss the potential risks and benefits of gain-of-function research. The event was attended by scientists from around the world, including George Gao, Gabriel Leung and Michael Selgelid, Baruch Fischhoff, Alta Charo, Harvey Fineberg, Jonathan Moreno, Ralph Cicerone, Margaret Hamburg, Jo Handelsman, Samuel Stanley, Kenneth Berns, Ralph Baric, Robert Lamb, Silja Vöneky, Keiji Fukuda, David Relman, and Marc Lipsitch. Shortly thereafter, the US government granted exceptions to the GoFR moratorium to 7 out of 18 research projects that had been affected.
=== Gain-of-Function Research: A Second Symposium ===
On March 10–11, 2016, the National Academies of Sciences, Engineering, and Medicine held its second public symposium to discuss potential U.S. government policies for the oversight of gain-of-function research. The symposium was held at the request of the U.S. government to provide a mechanism to engage the life sciences community and the broader public and solicit feedback on optimal approaches to ensure effective federal oversight of GoFR as part of a broader U.S. government deliberative process.
== Academic advocacy groups ==
=== Cambridge Working Group ===
The Cambridge Working Group was formed by Harvard epidemiologist Marc Lipsitch with fellow scientists at a meeting held in Cambridge, Massachusetts, following a "trifecta" of biosecurity incidents involving the CDC, including the accidental exposure of viable anthrax to personnel at CDC's Roybal Campus, the discovery of six vials containing viable smallpox from the 1950s, labeled as Variola but in a box with other samples poorly labeled, at the FDA's White Oak campus, and the accidental shipping of H9N2 vials contaminated with H5N1 from the CDC lab to a USDA lab.
On July 14, 2014, the group published a Consensus Statement authored by 18 founding members, including Amir Attaran, Barry Bloom, Arturo Casadevall, Richard H. Ebright, Alison Galvani, Edward Hammond, Thomas Inglesby, Michael Osterholm, David Relman, Richard Roberts, Marcel Salathé and Silja Vöneky. Since its initial publication, over 300 scientists, academics, and physicians have added their signature.
The statement advocates for all work involving potential pandemic pathogens to be halted until a quantitative and objective assessment of the risks has been undertaken. It then argues that alternative approaches that do not involve such risks should be used instead.
The group engaged in public advocacy, influencing the US government's decision in December 2014 to suspend funding of research that would create certain types of novel potential pandemic pathogens.
=== Scientists for Science ===
Shortly after the Cambridge Working Group released its position statement, Scientists for Science was formed by 37 signatories taking an alternative position, that "biomedical research on potentially dangerous pathogens can be performed safely and is essential for a comprehensive understanding of microbial disease pathogenesis, prevention and treatment." Since its publication, the SfS statement has received 200+ signatures from working scientists, academics, and biosafety professionals.
One of the group's founding members, University of Pittsburgh virologist W. Paul Duprex, has argued (c. 2014) that the then-recent few events were exceptions to an overall good record of lab safety, and that these exceptions should not have been a reason for shutting down experiments that may have been of tangible benefit to public health. He and other SfS signatories have argued that these pathogens are already subject to extensive regulations and that it would be more advantageous and effective to focus on improving lab safety and oversight, ensuring that experiments are conducted in the public interest.
Notable signatories are Constance Cepko, Dickson Despommier, Erica Ollmann Saphire, Geoffrey Smith, Karla Kirkegaard, Sean Whelan, Vincent Racaniello and Yoshihiro Kawaoka. Columbia University virologist Ian Lipkin, who signed both statements, said "there has to be a coming together of what should be done".
Founders of both groups published a series of letters detailing their discussions and viewpoints. All authors, however, agreed that more education of the public and open discussion of the risks and benefits was necessary. Several also wrote that sensationalized headlines and framings of the ongoing process as a "debate" with "opposing sides" had negatively affected the process, while the reality is much more collegial.
== International policies and regulations ==
International outlook and engagement on gain-of-function research policy and regulation vary by country and region. Due to the potential effect on the global community at large, the ethical acceptability of such experiments depends on the extent to which it is accepted internationally. In 2010, the World Health Organization developed a non-binding guidance document for DURC, summarizing the positions of many different nations as "self-governing" and others as strictly following oversight based on the International Health Regulations, the Biological and Toxin Weapons Convention (BTWC), and the Center for International Security Studies' Biological Research Security System. The document also recommended the aforementioned as potential resources for countries to develop their own policies and procedures for DURC.
=== European Union ===
The European Academies Science Advisory Council has formed a working group to examine the issues raised by gain-of-function research and to make recommendations for the management of such research and its outputs. The possibility for developing common approaches between the United States and Europe has been explored.
In May 2014, the German National Ethics Council presented a report to the Bundestag on proposed guidance for governance of GoFR. The report called for national legislation on DURC. As of May 2021, the German government has not passed the endorsed legislation. The NEC also proposed a national code-of-conduct for researchers to consent, endorsing which experiments qualify as misconduct and which do not, based on founding principles of public benefit. The German Research Foundation and German National Academy of Sciences made a joint suggestion to expand the role of existing research ethics committees to also evaluate proposals of DURC.
=== United States ===
==== Gain-of-function research moratorium ====
From 2014 to 2017, the White House Office of Science and Technology Policy and the Department of Health and Human Services instituted a gain-of-function research moratorium and funding pause on any dual-use research into specific pandemic-potential pathogens (influenza, MERS, and SARS) while the regulatory environment and review process were reconsidered and overhauled. Under the moratorium, any laboratory who conducted such research would put their future funding (for any project, not just the indicated pathogens) in jeopardy. The NIH has said 18 studies were affected by the moratorium.
The moratorium was a response to laboratory biosecurity incidents that occurred in 2014, including not properly inactivating anthrax samples, the discovery of unlogged smallpox samples, and injecting a chicken with the wrong strain of influenza. These incidents were not related to gain-of-function research. One of the goals of the moratorium was to reduce the handling of dangerous pathogens by all laboratories until safety procedures were evaluated and improved.
Subsequently, symposia and expert panels were convened by the National Science Advisory Board for Biosecurity (NSABB) and National Research Council (NRC). In May 2016, the NSABB published "Recommendations for the Evaluation and Oversight of Proposed Gain-of-Function Research". On 9 January 2017, the HHS published the "Recommended Policy Guidance for Departmental Development of Review Mechanisms for Potential Pandemic Pathogen Care and Oversight" (P3CO). This report sets out how "pandemic potential pathogens" should be regulated, funded, stored, and researched to minimize threats to public health and safety.
On 19 December 2017, the NIH lifted the moratorium because gain-of-function research was deemed "important in helping us identify, understand, and develop strategies and effective countermeasures against rapidly evolving pathogens that pose a threat to public health."
== COVID-19 pandemic ==
During the COVID-19 pandemic a number of conspiracy theories emerged about the origin of the SARS-CoV-2 virus and links to gain-of-function research. In January 2021, University of Saskatchewan virologist Angela Rasmussen wrote that one version of the information invoked previous gain-of-function work on coronaviruses to promulgate the idea that the virus was of laboratory origin. Rasmussen stated that this was unlikely, due to the intense scrutiny and government oversight to which GoFR is subject, and it is improbable that research on hard-to-obtain coronaviruses could occur under the radar.
In a congressional hearing on May 11, 2021, about Anthony Fauci's role as the Chief Medical Advisor to the United States Office of the President, senator Rand Paul stated that "the U.S. has been collaborating with Shi Zhengli of the Wuhan Virology Institute, sharing discoveries about how to create super viruses. This gain-of-function research has been funded by the NIH." Fauci responded "with all due respect, you are entirely and completely incorrect...the NIH has not ever and does not now fund gain-of-function research [conducted at] the Wuhan Institute of Virology." The Washington Post fact-checking team later rated Paul's statements as containing "significant omissions and/or exaggerations". NIH funding to the EcoHealth Alliance and later sub-contracted to the Wuhan Institute of Virology was not to support gain-of-function experiments, but instead to enable the collection of bat samples in the wild. EcoHealth Alliance spokesperson Robert Kessler has also categorically denied the accusation.
The Washington Post also quoted Rutgers University biosecurity expert Richard Ebright's dissenting opinion about Fauci's testimony, demonstrating that there is disagreement about what qualifies as "gain of function" research. Ebright asserted that experiments conducted under the EcoHealth grant "met the definition for gain-of-function research of concern under the 2014 Pause." MIT molecular biologist Alina Chan has argued that these experiments would not have been affected by the 2014 moratorium, because the experiments involved "naturally-occurring viruses" adding that the moratorium had "no teeth".
Several scientists have criticized the US government's GoFR regulations as having serious shortcomings (especially with regard to the NIH's funding of the EcoHealth Alliance grant proposal). Ebright has remarked that the process is not applied to all the experiments covered by the government's policies, while virologists David Relman and Angela Rasmussen have cited a worrying lack of transparency from oversight panels.
== See also ==
Biotechnology risk
Directed evolution
Dual-use technology
Global Virome Project
== References ==
== Further reading ==
European Academies' Science Advisory Council: Gain of function: experimental applications relating to potentially pandemic pathogens (Report)
Lowen, Anice; Lakdawala, Seema (8 May 2023). "Gain-of-function research is more than just tweaking risky viruses – it's a routine and essential tool in all biology research". The Conversation. | Wikipedia/Gain-of-function_research |
PLOS (for Public Library of Science; PLoS until 2012 ) is a nonprofit publisher of open-access journals in science, technology, and medicine and other scientific literature, under an open-content license. It was founded in 2000 and launched its first journal, PLOS Biology, in October 2003.
As of 2024, PLOS publishes 14 academic journals, including 7 journals indexed within the Science Citation Index Expanded, and consequently 7 journals ranked with an impact factor.
PLOS journals are included in the Directory of Open Access Journals (DOAJ). PLOS is also a member of the Open Access Scholarly Publishers Association (OASPA), a participating publisher and supporter of the Initiative for Open Citations, and a member of the Committee on Publication Ethics (COPE).
== History ==
The Public Library of Science began in 2000 with an online petition initiative by Nobel Prize winner Harold Varmus, formerly director of the National Institutes of Health and at that time director of Memorial Sloan–Kettering Cancer Center; Patrick O. Brown, a biochemist at Stanford University; and Michael Eisen, a computational biologist at the University of California, Berkeley, and the Lawrence Berkeley National Laboratory. The petition called for all scientists to pledge that, from September 2001, they would discontinue submission of articles to journals that did not make the full text of their articles available to all, free and unfettered, either immediately or after a delay of no more than six months. Although tens of thousands signed the petition, most did not act upon its terms; and in August 2001, Brown and Eisen announced that they would start their own nonprofit publishing operation. In December 2002, the Gordon and Betty Moore Foundation awarded PLOS a $9 million grant, which it followed in May 2006 with a $1 million grant to help PLOS achieve financial sustainability and launch new free-access biomedical journals.
The PLOS organizers turned their attention to starting their own journal along the lines of the UK-based BioMed Central, which has been publishing open-access scientific articles in the biological sciences in journals such as Genome Biology since 2000. The PLOS journals are what is described as "open-access content"; all content is published under the Creative Commons "attribution" license. The project states (quoting the Budapest Open Access Initiative) that: "The only constraint on reproduction and distribution, and the only role for copyright in this domain, should be to give authors control over the integrity of their work and the right to be properly acknowledged and cited."
As a publishing company, the Public Library of Science officially launched its operation on 13 October 2003, with the publication of a print and online scientific journal entitled PLOS Biology, and has since launched 11 more journals.
One, PLOS Clinical Trials, has since been merged into PLOS ONE. Following the merger, the company started the PLOS Hub for Clinical Trials to collect journal articles published in any PLOS journal that related to clinical trials; the hub was discontinued in July 2013.
PLOS became a signatory of the SDG Publishers Compact in 2023, and has taken steps to support the achievement of the Sustainable Development Goals (SDGs). These include the introduction of five new open-access journals in 2021 to publish research relevant to the SDGs: PLOS Climate, PLOS Water, PLOS Sustainability and Transformation, PLOS Digital Health, and PLOS Global Public Health.
In 2011, the Public Library of Science became an official financial supporting organization of Healthcare Information For All by 2015, a global initiative that advocates unrestricted access to medical knowledge, sponsoring the first HIFA2015 Webinar in 2012.
In 2012, the organization quit using the stylization "PLoS" to identify itself and began using only "PLOS".
In 2016, PLOS confirmed that its chief executive officer, Elizabeth Marincola, would be leaving for personal and professional reasons at the end of that year. In May 2017, PLOS announced that their new CEO would be Alison Mudditt with effect from June.
In 2021, PLOS announced a policy that required changes in reporting for researchers working in other countries as an attempt to address neo-colonial parachute research practices.
== Financial model ==
To fund the journals, PLOS charges an article processing charge (APC) to be paid by the author or the author's employer or funder. In the United States, institutions such as the National Institutes of Health and the Howard Hughes Medical Institute have pledged that recipients of their grants will be allocated funds to cover such author charges. The Global Participation Initiative (GPI) was instituted in 2012, by which authors in "group-one countries" are not charged a fee and those in "group-two countries" are given a fee reduction. (In all cases, decisions to publish are based solely on editorial criteria.)
PLOS was launched with grants totaling US$13 million from the Gordon and Betty Moore Foundation and the Sandler Family Supporting Foundation. PLOS confirmed in July 2011 that it no longer relies on subsidies from foundations and is covering all of its operational costs. Since then, the PLOS balance sheet has improved from $20,511,000 net assets in 2012–2013 to $36,591,000 in 2014–2015.
== Publications ==
== Other partners ==
In April 2017, PLOS was one of the founding partners in the Initiative for Open Citations.
== Headquarters ==
PLOS has its main headquarters in Suite 225 in the Koshland East Building in Levi's Plaza in San Francisco. Previously, the company had been located at 185 Berry Street. In June 2010, PLOS announced that it was moving to a new location in order to accommodate its rapid growth. The move to the Koshland East Building went into effect on 21 June 2010.
== See also ==
List of open-access journals
arXiv e-print archive
Open Archives Initiative
Open Access Scholarly Publishers Association, of which PLOS is a founding member
== Footnotes ==
== References ==
== External links ==
Official website | Wikipedia/Public_Library_of_Science |
An integral, or intrinsic, membrane protein (IMP) is a type of membrane protein that is permanently attached to the biological membrane. All transmembrane proteins can be classified as IMPs, but not all IMPs are transmembrane proteins. IMPs comprise a significant fraction of the proteins encoded in an organism's genome. Proteins that cross the membrane are surrounded by annular lipids, which are defined as lipids that are in direct contact with a membrane protein. Such proteins can only be separated from the membranes by using detergents, nonpolar solvents, or sometimes denaturing agents.
Proteins that adhere only temporarily to cellular membranes are known as peripheral membrane proteins. These proteins can either associate with integral membrane proteins, or independently insert in the lipid bilayer in several ways.
== Structure ==
Three-dimensional structures of ~160 different integral membrane proteins have been determined at atomic resolution by X-ray crystallography or nuclear magnetic resonance spectroscopy. They are challenging subjects for study owing to the difficulties associated with extraction and crystallization. In addition, structures of many water-soluble protein domains of IMPs are available in the Protein Data Bank. Their membrane-anchoring α-helices have been removed to facilitate the extraction and crystallization. Search integral membrane proteins in the PDB (based on gene ontology classification)
IMPs can be divided into two groups:
Integral polytopic proteins (Transmembrane proteins)
Integral monotopic proteins
=== Integral polytopic protein ===
The most common type of IMP is the transmembrane protein, which spans the entire biological membrane. Single-pass membrane proteins cross the membrane only once, while multi-pass membrane proteins weave in and out, crossing the membrane several times. Single pass membrane proteins can be categorized as Type I, which are positioned such that their carboxyl-terminus is towards the cytosol, or Type II, which have their amino-terminus towards the cytosol. Type III proteins have multiple transmembrane domains in a single polypeptide, while type IV consists of several different polypeptides assembled together in a channel through the membrane. Type V proteins are anchored to the lipid bilayer through covalently linked lipids. Finally Type VI proteins have both transmembrane domains and lipid anchors.
=== Integral monotopic proteins ===
Integral monotopic proteins are permanently attached to the cell membrane from one side.
Three-dimensional structures of the following integral monotopic proteins have been determined:
prostaglandin H2 syntheses 1 and 2 (cyclooxygenases)
lanosterol synthase and squalene-hopene cyclase
microsomal prostaglandin E synthase
carnitine O-palmitoyltransferase 2
Phosphoglycosyl transferase C
There are also structures of integral monotopic domains of transmembrane proteins:
monoamine oxidases A and B
fatty acid amide hydrolase
mammalian cytochrome P450 oxidases
corticosteroid 11-beta-dehydrogenases
=== Extraction ===
Many challenges facing the study of integral membrane proteins are attributed to the extraction of those proteins from the phospholipid bilayer. Since integral proteins span the width of the phospholipid bilayer, their extraction involves disrupting the phospholipids surrounding them, without causing any damage that would interrupt the function or structure of the proteins. Several successful methods are available for performing the extraction including the uses of "detergents, low ionic salt (salting out), shearing force, and rapid pressure change".
=== Determination of protein structure ===
The Protein Structure Initiative (PSI), funded by the U.S. National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health (NIH), has among its aim to determine three-dimensional protein structures and to develop techniques for use in structural biology, including for membrane proteins. Homology modeling can be used to construct an atomic-resolution model of the "target" integral protein from its amino acid sequence and an experimental three-dimensional structure of a related homologous protein. This procedure has been extensively used for ligand-G protein–coupled receptors (GPCR) and their complexes.
== Function ==
IMPs include transporters, linkers, channels, receptors, enzymes, structural membrane-anchoring domains, proteins involved in accumulation and transduction of energy, and proteins responsible for cell adhesion. Classification of transporters can be found in Transporter Classification Database.
As an example of the relationship between the IMP (in this case the bacterial phototrapping pigment, bacteriorhodopsin) and the membrane formed by the phospholipid bilayer is illustrated below. In this case the integral membrane protein spans the phospholipid bilayer seven times. The part of the protein that is embedded in the hydrophobic regions of the bilayer are alpha helical and composed of predominantly hydrophobic amino acids. The C terminal end of the protein is in the cytosol while the N terminal region is in the outside of the cell. A membrane that contains this particular protein is able to function in photosynthesis.
== Examples ==
Examples of integral membrane proteins:
Insulin receptor
Some types of cell adhesion proteins or cell adhesion molecules (CAMs) such as integrins, cadherins, NCAMs, or selectins
Some types of receptor proteins
Glycophorin
Rhodopsin
Band 3
CD36
Glucose Permease
Ion channels and Gates
Gap junction Proteins
G protein coupled receptors (e.g., Beta-adrenergic receptor)
Seipin
Photosystem I
== See also ==
Membrane protein
Transmembrane protein
Peripheral membrane protein
Annular lipid shell
Hydrophilicity plot
Inner nuclear membrane protein
== References == | Wikipedia/Integral_membrane_protein |
The Bateson–Dobzhansky–Muller model, also known as Dobzhansky–Muller model, is a model of the evolution of genetic incompatibility, important in understanding the evolution of reproductive isolation during speciation and the role of natural selection in bringing it about. The theory was first described by William Bateson in 1909, then independently described by Theodosius Dobzhansky in 1934, and later elaborated in different forms by Herman Muller, H. Allen Orr and Sergey Gavrilets.
The Dobzhansky–Muller model describes the negative epistatic interactions that occur between different alleles (versions) of different genes with a different evolutionary history. These genetic incompatibilities can occur when populations are hybridising. When two populations diverge from a common ancestor and become isolated from each other, thus meaning there is no interbreeding between the two, mutations can accumulate in both populations. These changes represent evolutionary change in the populations. When the populations are reintroduced to each other, these diverged genes can interact with each other in the hybridising species.
For example, an ancestral species has the alleles a and b fixed in its population, resulting in all individuals having the aabb genotype. When two descendant populations are separated from each other and each undergo several mutations the allele A can occur in one population while the allele B occurs in the second population. When the two populations start hybridising the genotypes AAbb and aaBB hybridise with each other resulting in AaBb (figure 1). Interactions between A and B are introduced which have never occurred before. These two alleles can turn out to be incompatible, which are the Dobzhansky–Muller incompatibilities. The model states that genetic incompatibility is most likely evolved by alternative fixation of two or more loci instead of just one, so that when hybridisation occurs, it is the first time for some of the alleles to co-occur in the same individual.
The Dobzhansky–Muller incompatibilities can result from purely random, neutral or non-selected differences between the populations. They can also be driven by natural selection in at least two ways. When two populations diverge from each other and encounter new - and different - environments they may adapt to these environments. These adaptations can result in hybrid sterility as a side effect. The genes that have arisen to adapt to different ecological surroundings can thus cause hybrid incompatibilities. A second way is when the two diverging populations adapt to a same or similar environment but they do that in a genetically different way. This can result in the populations having different genotypes, that can cause Dobzhansky–Muller incompatibilities.
Genes that are incompatible according to the Dobzhansky–Muller model require three criteria. 1. The gene reduces the fitness of the hybrid, 2. The gene has functionally diverged in each of the hybridising species and, 3. The hybrid incompatibility is only present in combination with a partner gene. Whether the genes are actually incompatible is also dependent on whether the genes are dominant or recessive. Incompatibility will only occur if both alleles are expressed and not if one is recessive.
The genetic changes that are accumulated when populations diverge from a common ancestor will not severely decrease viability or fertility because natural selection influences these strongly deleterious alleles. However, natural selection cannot act when alleles have never occurred together, as they would in the genome of a hybrid. Therefore, it is possible that when these alleles interact, these alleles prove to be incompatible. An incompatible gene prevents the populations from successfully hybridising. These Dobzhansky–Muller incompatibilities can therefore also increase the chance of speciation.
Certain patterns in the Dobzhansky–Muller incompatibilities can provide information of modes of divergence. For instance, if divergence is due to different selection pressures, thus causing natural selection to act, or to random genetic drift. Therefore, Dobzhansky–Muller incompatibilities can also provide information on the time and type of divergence which can help in phylogenetic studies.
== References == | Wikipedia/Bateson–Dobzhansky–Muller_model |
The scholarly method or scholarship is the body of principles and practices used by scholars and academics to make their claims about their subjects of expertise as valid and trustworthy as possible, and to make them known to the scholarly public. It comprises the methods that systemically advance the teaching, research, and practice of a scholarly or academic field of study through rigorous inquiry. Scholarship is creative, can be documented, can be replicated or elaborated, and can be and is peer reviewed through various methods. The scholarly method includes the subcategories of the scientific method, with which scientists bolster their claims, and the historical method, with which historians verify their claims.
== Methods ==
The historical method comprises the techniques and guidelines by which historians research primary sources and other evidence, and then write history. The question of the nature, and indeed the possibility, of sound historical method is raised in the philosophy of history, as a question of epistemology. History guidelines commonly used by historians in their work require external criticism, internal criticism, and synthesis.
The empirical method is generally taken to mean the collection of data on which to base a hypothesis or derive a conclusion in science. It is part of the scientific method, but is often mistakenly assumed to be synonymous with other methods. The empirical method is not sharply defined and is often contrasted with the precision of experiments, where data emerges from the systematic manipulation of variables. The experimental method investigates causal relationships among variables. An experiment is a cornerstone of the empirical approach to acquiring data about the world and is used in both natural sciences and social sciences. An experiment can be used to help solve practical problems and to support or negate theoretical assumptions.
The scientific method refers to a body of techniques for investigating phenomena, acquiring new knowledge, or correcting and integrating previous knowledge. To be termed scientific, a method of inquiry must be based on gathering observable, empirical and measurable evidence subject to specific principles of reasoning. A scientific method consists of the collection of data through observation and experimentation, and the formulation and testing of hypotheses.
== See also ==
== References == | Wikipedia/Scholarly_method |
In geography, regions, otherwise referred to as areas, zones, lands or territories, are portions of the Earth's surface that are broadly divided by physical characteristics (physical geography), human impact characteristics (human geography), and the interaction of humanity and the environment (environmental geography). Geographic regions and sub-regions are mostly described by their imprecisely defined, and sometimes transitory boundaries, except in human geography, where jurisdiction areas such as national borders are defined in law. More confined or well bounded portions are called locations or places.
Apart from the global continental regions, there are also hydrospheric and atmospheric regions that cover the oceans, and discrete climates above the land and water masses of the planet. The land and water global regions are divided into subregions geographically bounded by large geological features that influence large-scale ecologies, such as plains and features.
As a way of describing spatial areas, the concept of regions is important and widely used among the many branches of geography, each of which can describe areas in regional terms. For example, ecoregion is a term used in environmental geography, cultural region in cultural geography, bioregion in biogeography, and so on. The field of geography that studies regions themselves is called regional geography. Regions are an area or division, especially part of a country or the world having definable characteristics but not always fixed boundaries.
In the fields of physical geography, ecology, biogeography, zoogeography, and environmental geography, regions tend to be based on natural features such as ecosystems or biotopes, biomes, drainage basins, natural regions, mountain ranges, soil types. Where human geography is concerned, the regions and subregions are described by the discipline of ethnography.
== Globalization ==
Global regions are distinguishable from space, and are therefore clearly distinguished by the two basic terrestrial environments, land and water. However, they have been generally recognized as such much earlier by terrestrial cartography because of their impact on human geography. They are divided into the largest of land regions, known as continents and the largest of water regions known as oceans. There are also significant regions that do not belong to either classification, such as archipelago regions that are littoral regions, or earthquake regions that are defined in geology.
=== Continental regions ===
Continental regions are usually based on broad experiences in human history and attempt to reduce very large areas to more manageable regionalization for the purpose of the study. As such they are conceptual constructs, usually lacking distinct boundaries. The oceanic division into maritime regions is used in conjunction with the relationship to the central area of the continent, using directions of the compass.
Some continental regions are defined by the major continental feature of their identity, such as the Amazon basin, or the Sahara, which both occupy a significant percentage of their respective continental land area.
To a large extent, major continental regions are mental constructs created by considering an efficient way to define large areas of the continents. For the most part, the images of the world are derived as much from academic studies, from all types of media, or from personal experience of global exploration. They are a matter of collective human knowledge of their own planet and are attempts to better understand their environments.
=== Regional geography ===
Regional geography is a branch of geography that studies regions of all sizes across the Earth. It has a prevailing descriptive character. The main aim is to understand or define the uniqueness or character of a particular region, which consists of natural as well as human elements. Attention is paid also to regionalization, which covers the proper techniques of space delimitation into regions.
Regional geography is also considered as a certain approach to study in geographical sciences (similar to quantitative or critical geographies; for more information, see history of geography).
== Human geography ==
Human geography is a branch of geography that focuses on the study of patterns and processes that shape human interaction with various discrete environments. It encompasses human, political, cultural, social, and economic aspects among others that are often clearly delineated. While the major focus of human geography is not the physical landscape of the Earth (see physical geography), it is hardly possible to discuss human geography without referring to the physical landscape on which human activities are being played out, and environmental geography is emerging as a link between the two. Regions of human geography can be divided into many broad categories:
=== Historical regions ===
The field of historical geography involves the study of human history as it relates to places and regions, or the study of how places and regions have changed over time.
D. W. Meinig, a historical geographer of America, describes many historical regions in his book The Shaping of America: A Geographical Perspective on 500 Years of History. For example, in identifying European "source regions" in early American colonization efforts, he defines and describes the Northwest European Atlantic Protestant Region, which includes sub-regions such as the "Western Channel Community", which itself is made of sub-regions such as the English West Country of Cornwall, Devon, Somerset, and Dorset.
In describing historic regions of America, Meinig writes of "The Great Fishery" off the coast of Newfoundland and New England, an oceanic region that includes the Grand Banks. He rejects regions traditionally used in describing American history, like New France, "West Indies", the Middle Colonies, and the individual colonies themselves (Province of Maryland, for example). Instead he writes of "discrete colonization areas", which may be named after colonies but rarely adhere strictly to political boundaries. Among other historic regions of this type, he writes about "Greater New England" and its major sub-regions of "Plymouth", "New Haven shores" (including parts of Long Island), "Rhode Island" (or "Narragansett Bay"), "the Piscataqua", "Massachusetts Bay", "Connecticut Valley", and to a lesser degree, regions in the sphere of influence of Greater New England, "Acadia" (Nova Scotia), "Newfoundland and The Fishery/The Banks".
Other examples of historical regions are Iroquoia, Ohio Country, Illinois Country, and Rupert's Land.
In Russia, historical regions include Siberia and the Russian North, as well as the Ural Mountains. These regions had an identity that developed from the early modern period and led to Siberian regionalism.
=== Tourism region ===
A tourism region is a geographical region that has been designated by a governmental organization or tourism bureau as having common cultural or environmental characteristics. These regions are often named after a geographical, former, or current administrative region or may have a name created for tourism purposes. The names often evoke certain positive qualities of the area and suggest a coherent tourism experience to visitors. Countries, states, provinces, and other administrative regions are often carved up into tourism regions to facilitate attracting visitors.
Some of the more famous tourism regions based on historical or current administrative regions include Tuscany in Italy and Yucatán in Mexico. Famous examples of regions created by a government or tourism bureau include the United Kingdom's Lake District and California's Wine Country.
great plains region
=== Natural resource regions ===
Natural resources often occur in distinct regions. Natural resource regions can be a topic of physical geography or environmental geography, but also have a strong element of human geography and economic geography. A coal region, for example, is a physical or geomorphological region, but its development and exploitation can make it into an economic and a cultural region. Examples of natural resource regions are the Rumaila Field, the oil field that lies along the border or Iraq and Kuwait and played a role in the Gulf War; the Coal Region of Pennsylvania, which is a historical region as well as a cultural, physical, and natural resource region; the South Wales Coalfield, which like Pennsylvania's coal region is a historical, cultural, and natural region; the Kuznetsk Basin, a similarly important coal mining region in Russia; Kryvbas, the economic and iron ore mining region of Ukraine; and the James Bay Project, a large region of Quebec where one of the largest hydroelectric systems in the world has been developed.
=== Religious regions ===
Sometimes a region associated with a religion is given a name, like Christendom, a term with medieval and renaissance connotations of Christianity as a sort of social and political polity. The term Muslim world is sometimes used to refer to the region of the world where Islam is dominant. These broad terms are somewhat vague when used to describe regions.
Within some religions there are clearly defined regions. The Roman Catholic Church, the Church of England, the Eastern Orthodox Church, and others, define ecclesiastical regions with names such as diocese, eparchy, ecclesiastical provinces, and parish.
For example, the United States is divided into 32 Roman Catholic ecclesiastical provinces. The Lutheran Church–Missouri Synod is organized into 33 geographic districts, which are subdivided into circuits (the Atlantic District (LCMS), for example). The Church of Jesus Christ of Latter-day Saints uses regions similar to dioceses and parishes, but uses terms like ward and stake.
=== Political regions ===
In the field of political geography, regions tend to be based on political units such as sovereign states; subnational units such as administrative regions, provinces, states (in the United States), counties, townships, territories, etc.; and multinational groupings, including formally defined units such as the European Union, the Association of Southeast Asian Nations, and NATO, as well as informally defined regions such as the Third World, Western Europe, and the Middle East.
=== Administrative regions ===
The word "region" is taken from the Latin regio (derived from regere, 'to rule'), and a number of countries have borrowed the term as the formal name for a type of subnational entity (e.g., the región, used in Chile). In English, the word is also used as the conventional translation for equivalent terms in other languages (e.g., the область (oblast), used in Russia alongside a broader term регион).
The following countries use the term "region" (or its cognate) as the name of a type of subnational administrative unit:
Belgium (in French, région; in German, Region; the Dutch term gewest is often mistakenly translated as "regio")
Chile (región)
Côte d'Ivoire (région)
Denmark (effective from 2007)
Eritrea
France (région)
Ghana
Guinea (région)
Guinea-Bissau (região)
Guyana
Hungary (régió)
Italy (regione)
Madagascar (région)
Mali (région)
Malta (reġjun)
Namibia
New Zealand
Peru (región)
Portugal (região)
Philippines (rehiyon)
Senegal (région)
Tanzania
Thailand
Togo (région)
Trinidad and Tobago (Regional Corporation)
The Canadian province of Québec also uses the "administrative region" (région administrative).
Regions of England (not the United Kingdom as a whole) used to be administrative units until 2011. Since then they're only used for statistical purposes.
Scotland had local government regions from 1975 to 1996.
In Spain the official name of the autonomous community of Murcia is Región de Murcia. Also, some single-province autonomous communities such as Madrid use the term región interchangeably with comunidad autónoma.
Two län (counties) in Sweden are officially called 'regions': Skåne and Västra Götaland, and there is currently a controversial proposal to divide the rest of Sweden into large regions, replacing the current counties.
The government of the Philippines uses the term "region" (in Filipino, rehiyon) when it is necessary to group provinces, the primary administrative subdivision of the country. This is also the case in Brazil, which groups its primary administrative divisions (estados; "states") into grandes regiões (greater regions) for statistical purposes, while Russia uses экономические районы (economic regions) in a similar way, as does Romania and Venezuela.
The government of Singapore makes use of the term "region" for its own administrative purposes.
The following countries use an administrative subdivision conventionally referred to as a region in English:
Bulgaria, which uses the област (oblast)
Greece, which uses the Περιφέρεια (periferia)
Russia, which uses the область (oblast'), and for some regions the край (krai)
Ukraine, which uses the область (oblast')
Slovakia (kraj)
China has five 自治区 (zìzhìqū) and two 特別行政區 (or 特别行政区; tèbiéxíngzhèngqū), which are translated as "autonomous region" and "special administrative region", respectively.
==== Local administrative regions ====
There are many relatively small regions based on local government agencies such as districts, agencies, or regions. In general, they are all regions in the general sense of being bounded spatial units. Examples include electoral districts such as Washington's 6th congressional district and Tennessee's 1st congressional district; school districts such as Granite School District and Los Angeles Unified School District; economic districts such as the Reedy Creek Improvement District; metropolitan areas such as the Seattle metropolitan area, and metropolitan districts such as the Metropolitan Water Reclamation District of Greater Chicago, the Las Vegas-Clark County Library District, the Metropolitan Police Service of Greater London, as well as other local districts like the York Rural Sanitary District, the Delaware River Port Authority, the Nassau County Soil and Water Conservation District, and C-TRAN.
=== Traditional or informal regions ===
The traditional territorial divisions of some countries are also commonly rendered in English as "regions". These informal divisions do not form the basis of the modern administrative divisions of these countries, but still define and delimit local regional identity and sense of belonging. Examples are:
England
Finland
Japan
Korea
Norway (landsdeler)
Romania
Slovakia
United States
=== Functional regions ===
Functional regions are usually understood to be the areas organised by the horizontal functional relations (flows, interactions) that are maximised within a region and minimised across its borders so that the principles of internal cohesiveness and external separation regarding spatial interactions are met (see, for instance, Farmer and Fotheringham, 2011; Klapka, Halas, 2016; Smart, 1974). A functional region is not an abstract spatial concept, but to a certain extent it can be regarded as a reflection of the spatial behaviour of individuals in a geographic space.
The functional region is conceived as a general concept while its inner structure, inner spatial flows, and interactions need not necessarily show any regular pattern, only selfcontainment. The concept of self-containment remains the only crucial defining characteristic of a functional region. Nodal regions, functional urban regions, daily urban systems, local labour-market areas (LLMAs), or travel-to-work areas (TTWAs) are considered to be special instances of a general functional region that need to fulfil some specific conditions regarding, for instance, the character of the region-organising interaction or the presence of urban cores, (Halas et al., 2015).
=== Military regions ===
In military usage, a region is shorthand for the name of a military formation larger than an Army Group and smaller than a Theater. The full name of the military formation is Army Region. The size of an Army Region can vary widely but is generally somewhere between about 1 million and 3 million soldiers. Two or more Army Regions could make up a Theater. An Army Region is typically commanded by a full General (US four stars), a Field Marshal or General of the Army (US five stars), or Generalissimo (Soviet Union); and in the US Armed Forces an Admiral (typically four stars) may also command a region. Due to the large size of this formation, its use is rarely employed. Some of the very few examples of an Army Region are each of the Eastern, Western, and southern (mostly in Italy) fronts in Europe during World War II. The military map unit symbol for this echelon of formation (see Military organization and APP-6A) is identified with six Xs.
=== Media geography ===
Media geography is a spatio-temporal understanding, brought through different gadgets of media, nowadays, media became inevitable at different proportions and everyone supposed to consumed at different gravity. The spatial attributes are studied with the help of media outputs in shape of images which are contested in nature and pattern as well where politics is inseparable. Media geography is giving spatial understanding of mediated image.
== See also ==
Autonomous region
Committee of the Regions
Continent
Continental fragment
Euroregion
Field (geography)
Latin names of regions
Military district
Regional district
Regionalism (disambiguation)
Regional municipality
Subcontinent
Submerged continents
Subregion
Supercontinent
United Nations geoscheme
== Notes ==
== References ==
Bailey, Robert G. (1996) Ecosystem Geography. New York: Springer-Verlag. ISBN 0-387-94586-5
Meinig, D.W. (1986). The Shaping of America: A Geographical Perspective on 500 Years of History, Volume 1: Atlantic America, 1492-1800. New Haven: Yale University Press. ISBN 0-300-03548-9
Moinuddin Shekh. (2017) " Mediascape and the State: A Geographical Interpretation of Image Politics in Uttar Pradesh, India. Netherland, Springer.
Smith-Peter, Susan (2018) Imagining Russian Regions: Subnational Identity and Civil Society in Nineteenth-Century Russia. Leiden: Brill, 2017. ISBN 9789004353497
== External links ==
Map and descriptions of hydrologic unit regions of the United States
Federal Standards for Delineation of Hydrologic Unit Boundaries
Physiographic regions of the United States | Wikipedia/Geographical_area |
A transport protein (variously referred to as a transmembrane pump, transporter, escort protein, acid transport protein, cation transport protein, or anion transport protein) is a protein that serves the function of moving other materials within an organism. Transport proteins are vital to the growth and life of all living things. There are several different kinds of transport proteins.
Carrier proteins are proteins involved in the movement of ions, small molecules, or macromolecules, such as another protein, across a biological membrane. Carrier proteins are integral membrane proteins; that is, they exist within and span the membrane across which they transport substances.
The proteins may assist in the movement of substances by facilitated diffusion (i.e., passive transport) or active transport. These mechanisms of movement are known as carrier-mediated transport. Each carrier protein is designed to recognize only one substance or one group of very similar substances. Research suggests that potassium, calcium and sodium channels can function as oxygen sensors in mammals and plants, and has correlated defects in specific carrier proteins with specific diseases.
A membrane transport protein (or simply transporter) is a membrane protein that acts as such a carrier.
A vesicular transport protein is a transmembrane or membrane associated protein. It regulates or facilitates the movement by vesicles of the contents of the cell.
== See also ==
Solute carrier family
Neurotransmitter transporter
== References == | Wikipedia/Transport_protein |
Humboldtian science refers to a movement in science in the 19th century closely connected to the work and writings of German scientist, naturalist, and explorer Alexander von Humboldt. It maintained a certain ethics of precision and observation, which combined scientific field work with the sensitivity and aesthetic ideals of the age of Romanticism. Like Romanticism in science, it was rather popular in the 19th century. The term was coined by Susan Faye Cannon in 1978.
The example of Humboldt's life and his writings allowed him to reach out beyond the academic community with his natural history and address a wider audience with popular science aspects. It has supplanted the older Baconian method, related as well to a single person, Francis Bacon.
== Brief biography ==
Humboldt was born in Berlin in 1769 and worked as a Prussian mining official in the 1790s until 1797 when he quit and began collecting scientific knowledge and equipment. His extensive wealth aided his infatuation with the spirit of Romanticism; he amassed an extensive collection of scientific instruments and tools as well as a sizeable library. In 1799 Humboldt, under the protection of King Charles IV of Spain, left for South America and New Spain, toting all of his tools and books. The purpose of the voyage was steeped in Romanticism; Humboldt intended to investigate how the forces of nature interact with one another and find out about the unity of nature. Humboldt returned to Europe in 1804 and was acclaimed as a public hero. The details and findings of Humboldt's journey were published in his Personal Narrative of Travels to the Equatorial Regions of the New Continent (30 volumes). This Personal Narrative was taken by Charles Darwin on his famous voyage on H.M.S Beagle. Humboldt spent the rest of his life mainly in Europe, although he did embark on a short expedition to Siberia and the Russian steppes in 1829. Humboldt's last works were contained in his book, Kosmos: Entwurf einer physischen Weltbeschreibung ("Cosmos. Sketch for a Physical Description of the Universe"). The book mainly described the development of a life-force from the cosmos, but also included the formation of stars from nebular clouds as well as the geography of planets. Alexander von Humboldt died in 1859, while working on the fifth volume of Kosmos.
Through his travels to South America and his observational records in An Essay on the Geography of Plants as well as Kosmos, an important trend emerged through his techniques of observation, scientific instruments used and unique perspective on nature. Humboldt's novel style has been defined as Humboldtian Science. Humboldt had the ability to combine the study of empirical data with a holistic view of nature and its aesthetically pleasing characteristics, which is now held to be the true definition of the study of vegetation and plant geography. Humboldtian science is one of the first techniques for studying both organic and inorganic branches of science. Examining the interconnectedness of vegetation and its respective environment is one of the new and important aspects of Humboldt's work, an idea labeled as "terrestrial physics," something that scientists who preceded him, such as Linnaeus, failed to do. Humboldtian science is founded on a principle of "general equilibrium of forces." General equilibrium was the idea that there are infinite forces in nature that are in constant conflict, yet all forces balance each other out.
Humboldt laid the groundwork for future scientific endeavors by establishing the importance of studying organisms and their environment in conjunction .
== Humboldtian science defined ==
Humboldtian science includes both the extensive work of Alexander von Humboldt, as well as many of the works of 19th century scientists. Susan Cannon is attributed with coining the term Humboldtian science. According to Cannon, Humboldtian science is, "the accurate, measured study of widespread but interconnected real phenomena in order to find a definite law and a dynamical cause." Humboldtian science is used now in place of the traditional, "Baconianism," as a more appropriate and less vague term for the themes of 19th century science.
Natural history in the eighteenth century was the "nomination of the visible". Carl Linnaeus was preoccupied with fitting all nature into taxonomy, fixated on only what was visible. Towards the turn of the eighteenth century, Immanuel Kant became interested in understanding where species derived from, and was less concerned with an organism's physical attributes. Next, Johann Reinhold Forster, one of Humboldt's future partners, became interested in the study of vegetation as an essential way of understanding nature and its relationship with human society. Proceeding Forster, Karl Willdenow examined floristic plant geography, the distribution of plants and regionality as a whole. All of these pieces in the history before Humboldt help to shape what is defined as Humboldtian science. Humboldt took into account both the outward appearance and inward meaning of plant species. His attention to natural aesthetics and empirical data and evidence is what set his scientific work apart from ecologists before him. Nicolson so aptly puts it as: "Humboldt effortlessly combined a commitment to empiricism and the experimental elucidation of the laws of nature with an equally strong commitment to holism and to a view of nature which was intended to be aesthetically and spiritually satisfactory". It was through this holistic approach to science and the study of nature that Humboldt was able to find a web of interconnectedness despite a multitude of extensive differences between different species of organisms.
According to Malcolm Nicholson, "Susan Cannon characterized Humboldtian science as synthetic, empirical, quantitative and impossible to fit into any one of our twentieth century disciplinary boundaries." A central element of Humboldtian science was its use of the latest advances in scientific instrumentation to observe and measure physical variables, while attending to all possible sources of error. Humboldtian science revolved around understanding the relationship between accurate measurement, sources of error and mathematical laws. Cannon identifies four distinctive features that marked Humboldtian science out from previous versions of science:
insistence on accuracy for all scientific instruments and observations;
a mental sophistication in which theoretical mechanisms and entities of past science were taken lightly;
a new set of conceptual tools, including isomaps, graphs, and a theory of errors;
the application of accuracy, mental sophistication, and tools not to isolated science in laboratories, but to greatly variable real phenomena.
== Humboldt's "terrestrial physicist" ==
Humboldt was committed to what he called 'terrestrial physics.' Essentially Humboldt's new scientific approach required a new type of scientist: Humboldtian science demanded a transition from the naturalist to the physicist. Humboldt described how his idea of terrestrial physics differs from traditional "descriptive" natural history when he stated, "[traveling naturalists] have neglected to track the great and constant laws of nature manifested in the rapid flux of phenomena…and to trace the reciprocal interaction of the divided physical forces." Humboldt did not consider himself an explorer, but rather a scientific traveler, who accurately measured what explorers had reported inaccurately. According to Humboldt, the goal of the terrestrial physicist was to investigate the confluence and interweaving of all physical forces. An incredibly extensive array of precise instrumentation had to be readily available for Humboldt's terrestrial physicist. The expansive amount of scientific resources that characterized the Humboldtian scientist is best described in the book Science in Culture,
Thus the complete Humboldtian traveller, in order to make satisfactory observations, should be able to cope with everything from the revolution of the satellites of Jupiter to the carelessness of clumsy donkeys.
Just some of such instruments included chronometers, telescopes, sextants, microscopes, magnetic compasses, thermometers, hygrometers, barometers, electrometers, and eudiometers. Furthermore, it was necessary to have multiple makes and models of each specific instrument to compare errors and constancy among each type.
== Humboldt's equilibrium ==
One concept that is central to Humboldtian science is that of a general equilibrium of forces. Humboldt explains: "The general equilibrium which reigns amongst disturbances and apparent turmoil, is the result of infinite number of mechanical forces and chemical attractions balancing each other out." Equilibrium is derived from an infinite number of forces acting simultaneously and varying globally. In other words, the lawfulness of nature, according to Humboldt, is a result of infinity and complexity. Humboldtian science promotes the idea that the more forces that are accurately measured over more of the earth's surface results in a greater understanding of the order of nature.
The voyage to the Americas produced many discoveries and developments that help to illustrate Humboldt's ideas about this equilibrium of forces. Humboldt produced the Tableau physique des Andes ("Physical Profile of the Andes), which aimed at capturing his voyage to the Americas in a single graphic table. Humboldt meant to capture all of the physical forces, from organisms to electricity, in this single table. Among many other complex empirical recordings of elevation-specific data, the table included a detailed biodistribution. This biodistribution mapped the specific distributions of flora and fauna at every elevation level on the mountain.
Humboldt's study of plants provides an example of the movement of Humboldtian science away from traditional science. Humboldt's botany also further illustrates the concept of equilibrium and the Humboldtian ideas of the interrelationship of nature's elements. Although he was concerned with physical features of plants, he was largely focused on the investigation of underlying connections and relations among plant organisms. Humboldt worked for years on developing an understanding of plant distributions and geography. The link between the balancing equilibrium of natural forces and organism distribution is evident when Humboldt states:
As in all other phenomena of the physical universe, so in the distribution of organic beings: amidst the apparent disorder which seems to result from the influence of a multitude of local causes, the unchanging law of nature become evident as soon as one surveys an extensive territory, or uses a mass of facts in which the partial disturbances compensate one another.
The study of vegetation and plant geography arose out of new concerns that emerged with Humboldtian science. These new areas of concern in science included integrative processes, invisible connections, historical development, and natural wholes.
Humboldtian science applied the idea of general equilibrium of forces to the continuities in the history of the generation of the planet. Humboldt saw the history of the earth as a continuous global distribution of such things as heat, vegetation, and rock formations. In order to graphically represent this continuity Humboldt developed isothermal lines. These isothermal lines functioned in the general balancing of forces in that isothermal lines preserved local peculiarities within a general regularity. According to Humboldtian science, nature's order and equilibrium emerged "gradually and progressively from laborious observing, averaging, and mapping over increasingly extended areas."
== Transformation of Humboldtian science ==
Ralph Waldo Emerson once dubbed Humboldt to be "one of those wonders of the world… who appear from time to time, as if to show us the possibilities of the human mind."
When Humboldt first began his studies of organisms and the environment he claimed that he wanted to "reorganize the general connections that link organic beings and to study the great harmonies of Nature". He is often considered one of the world's first genuine ecologists. Humboldt succeeded in developing a comprehensive science that joined the separate branches of natural philosophy under a model of natural order founded on the concept of dynamic equilibrium. Humboldt's work reached far beyond his personal expeditions and discoveries. Figures from all across the globe participated on his work. Some such participants included French naval officers, East India Company physicians, Russian provincial administrators, Spanish military commanders, and German diplomats. As was mentioned previously, Charles Darwin carried a copy of Humboldt's Personal Narrative aboard H.M.S. Beagle. Humboldt's projects, particularly those related to natural philosophy, played a significant role in the influx of European money and travelers to Spanish America in increasing numbers in the early 19th century. Sir Edward Sabine, a British scientist, worked on terrestrial magnetism in a manner that was certainly Humboldtian. Also, British scientist George Gabriel Stokes depended heavily on abstract mathematical measurement to deal with error in a precision instrument, certainly Humboldtian science. Maybe the most prominent figure whose work can be considered representative of Humboldtian science is geologist Charles Lyell. Despite a lack of emphasis on precise measurement in geology at the time, Lyell insisted on precision in a Humboldtian manner.
The promotion and development of terrestrial physics under Humboldtian science produced not only useful maps and statistics, but offered both European and Creole societies tools for essentially 're-imaging' America. The lasting impact of Humboldtian science is described in Cultures of Natural History, "Humboldtian science illuminates the reorganization of knowledge and disciplines in the early nineteenth century that defined the emergence of natural history out of natural philosophy."
== Revision of Humboldtian science as a concept ==
In recent years, historian Andreas Daum has explored the history of Humboldtian science as a concept and suggests a fundamental revision. He points out that William Goetzman was the first to establish it. Daum distinguishes between Humboldt's science as an individual form of knowledge production and Humboldtian science as a generalization (and useful ideal type), which later generations coined. Humboldt's science constituted a set of research practices that often defied the ideal of precision and minute comparative data analysis, which Humboldtian science in Cannon's understanding sees as the foundation of large-scale scientific research. Especially in his early years before leaving Europe to the Americas in 1799, Humboldt's research was impromptu, marked by epistemological and personal insecurities, and embedded in his peripatetic way of living. The constant moving around found its expression in an erratic writing style. The revision of Humboldtian Science and a renewed focus on Humboldt’s science undermine the "Humboldt exceptionalism" (Daum) found especially in popular accounts, such as Andrea Wulf's book on the Invention of Nature. This critical approach places Humboldt and his oeuvre firmly in the turbulent epoch he lived in – instead of portraying Humboldt as a man above his time. A review of Humboldtian science encourages historians to study standards of ‘objectivity’ and the belief in instruments yieding precise results as its guarantee. While Humboldt did advocate using widespread comparative measurements and strove to achieve accuracy in his research, his research practices often missed that ideal.
== See also ==
History of biology
History of ecology
History of geography
History of geology
Romanticism
Romanticism in science
== Notes ==
== References ==
Cannon, Susan Faye. Science in Culture: The Early Victorian Period. Science History Publications. NY. 1978
Andreas W. Daum, "Humboldtian Science and Humboldt’s Science". History of Science, 62 (2024), https://doi.org/10.1177/00732753241252478
Andreas W. Daum, “Social Relations, Shared Practices, and Emotions: Alexander von Humboldt’s Excursion into Literary Classicism and the Challenges to Science around 1800”. The Journal of Modern History, 91 (March 2019), 1-37.
Andreas W. Daum, Alexander von Humboldt: A Concise Biography. Trans. Robert Savage. Princeton, NJ: Princeton University Press, 2024.
Jardine, N; Secord, J.A.; Spary, E.C. Cultures of Natural History. Cambridge University Press. Cambridge, NY. 1996
Nicolson, Malcolm. "Alexander von Humboldt, Humboldtian science, and the origins of the study of vegetation." History of Science, 25:2. June 1987 | Wikipedia/Humboldtian_science |
Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.
Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other. Such measurements are used as a research tool in fields including biology and chemistry.
FRET is analogous to near-field communication, in that the radius of interaction is much smaller than the wavelength of light emitted. In the near-field region, the excited chromophore emits a virtual photon that is instantly absorbed by a receiving chromophore. These virtual photons are undetectable, since their existence violates the conservation of energy and momentum, and hence FRET is known as a radiationless mechanism. Quantum electrodynamical calculations have been used to determine that radiationless FRET and radiative energy transfer are the short- and long-range asymptotes of a single unified mechanism.
== Terminology ==
Förster resonance energy transfer is named after the German scientist Theodor Förster. When both chromophores are fluorescent, the term "fluorescence resonance energy transfer" is often used instead, although the energy is not actually transferred by fluorescence. In order to avoid an erroneous interpretation of the phenomenon that is always a nonradiative transfer of energy (even when occurring between two fluorescent chromophores), the name "Förster resonance energy transfer" is preferred to "fluorescence resonance energy transfer"; however, the latter enjoys common usage in scientific literature. FRET is not restricted to fluorescence and occurs in connection with phosphorescence as well.
== Theoretical basis ==
The FRET efficiency (
E
{\displaystyle E}
) is the quantum yield of the energy-transfer transition, i.e. the probability of energy-transfer event occurring per donor excitation event:
E
=
k
ET
k
f
+
k
ET
+
∑
k
i
,
{\displaystyle E={\frac {k_{\text{ET}}}{k_{f}+k_{\text{ET}}+\sum {k_{i}}}},}
where
k
f
{\displaystyle k_{f}}
the radiative decay rate of the donor,
k
ET
{\displaystyle k_{\text{ET}}}
is the rate of energy transfer, and
k
i
{\displaystyle k_{i}}
the rates of any other de-excitation pathways excluding energy transfers to other acceptors.
The FRET efficiency depends on many physical parameters that can be grouped as: 1) the distance between the donor and the acceptor (typically in the range of 1–10 nm), 2) the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum, and 3) the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.
E
{\displaystyle E}
depends on the donor-to-acceptor separation distance
r
{\displaystyle r}
with an inverse 6th-power law due to the dipole–dipole coupling mechanism:
E
=
1
1
+
(
r
/
R
0
)
6
{\displaystyle E={\frac {1}{1+(r/R_{0})^{6}}}}
with
R
0
{\displaystyle R_{0}}
being the Förster distance of this pair of donor and acceptor, i.e. the distance at which the energy transfer efficiency is 50%. The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation all in SI units:
R
0
6
=
9
log
(
10
)
κ
2
Q
D
J
128
π
5
N
A
n
4
{\displaystyle R_{0}^{6}={\frac {9\,\log(10)\,\kappa ^{2}\,Q_{D}\,J}{128\,\pi ^{5}\,N_{A}\,n^{4}}}}
where
Q
D
{\displaystyle Q_{\text{D}}}
is the fluorescence quantum yield of the donor in the absence of the acceptor,
κ
2
{\displaystyle \kappa ^{2}}
is the dipole orientation factor,
n
{\displaystyle n}
is the refractive index of the medium,
N
A
{\displaystyle N_{\text{A}}}
is the Avogadro constant, and
J
{\displaystyle J}
is the spectral overlap integral calculated as
J
=
∫
f
D
(
λ
)
ϵ
A
(
λ
)
λ
4
d
λ
∫
f
D
(
λ
)
d
λ
=
∫
f
D
¯
(
λ
)
ϵ
A
(
λ
)
λ
4
d
λ
,
{\displaystyle J={\frac {\int f_{\text{D}}(\lambda )\epsilon _{\text{A}}(\lambda )\lambda ^{4}\,d\lambda }{\int f_{\text{D}}(\lambda )\,d\lambda }}=\int {\overline {f_{\text{D}}}}(\lambda )\epsilon _{\text{A}}(\lambda )\lambda ^{4}\,d\lambda ,}
where
f
D
{\displaystyle f_{\text{D}}}
is the donor emission spectrum,
f
D
¯
{\displaystyle {\overline {f_{\text{D}}}}}
is the donor emission spectrum normalized to an area of 1, and
ϵ
A
{\displaystyle \epsilon _{\text{A}}}
is the acceptor molar extinction coefficient, normally obtained from an absorption spectrum. The orientation factor κ is given by
κ
=
μ
^
A
⋅
μ
^
D
−
3
(
μ
^
D
⋅
R
^
)
(
μ
^
A
⋅
R
^
)
,
{\displaystyle \kappa ={\hat {\mu }}_{\text{A}}\cdot {\hat {\mu }}_{\text{D}}-3({\hat {\mu }}_{\text{D}}\cdot {\hat {R}})({\hat {\mu }}_{\text{A}}\cdot {\hat {R}}),}
where
μ
^
i
{\displaystyle {\hat {\mu }}_{i}}
denotes the normalized transition dipole moment of the respective fluorophore, and
R
^
{\displaystyle {\hat {R}}}
denotes the normalized inter-fluorophore displacement.
κ
2
{\displaystyle \kappa ^{2}}
= 2/3 is often assumed. This value is obtained when both dyes are freely rotating and can be considered to be isotropically oriented during the excited-state lifetime. If either dye is fixed or not free to rotate, then
κ
2
{\displaystyle \kappa ^{2}}
= 2/3 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging that
κ
2
{\displaystyle \kappa ^{2}}
= 2/3 does not result in a large error in the estimated energy-transfer distance due to the sixth-power dependence of
R
0
{\displaystyle R_{0}}
on
κ
2
{\displaystyle \kappa ^{2}}
. Even when
κ
2
{\displaystyle \kappa ^{2}}
is quite different from 2/3, the error can be associated with a shift in
R
0
{\displaystyle R_{0}}
, and thus determinations of changes in relative distance for a particular system are still valid. Fluorescent proteins do not reorient on a timescale that is faster than their fluorescence lifetime. In this case 0 ≤
κ
2
{\displaystyle \kappa ^{2}}
≤ 4.
The units of the data are usually not in SI units. Using the original units to calculate the Förster distance is often more convenient. For example, the wavelength is often in unit nm and the extinction coefficient is often in unit
M
−
1
c
m
−
1
{\displaystyle M^{-1}cm^{-1}}
, where
M
{\displaystyle M}
is concentration
m
o
l
/
L
{\displaystyle mol/L}
.
J
{\displaystyle J}
obtained from these units will have unit
M
−
1
c
m
−
1
n
m
4
{\displaystyle M^{-1}cm^{-1}nm^{4}}
. To use unit Å (
10
−
10
m
{\displaystyle 10^{-10}m}
) for the
R
0
{\displaystyle R_{0}}
, the equation is adjusted to
R
0
6
=
8.785
×
10
−
5
κ
2
Q
D
n
4
J
{\displaystyle {R_{0}}^{6}=8.785\times 10^{-5}{\frac {\kappa ^{2}\,Q_{D}}{n^{4}}}J}
(Å
6
{\displaystyle ^{6}}
)
For time-dependent analyses of FRET, the rate of energy transfer (
k
ET
{\displaystyle k_{\text{ET}}}
) can be used directly instead:
k
ET
=
(
R
0
r
)
6
1
τ
D
{\displaystyle k_{\text{ET}}=\left({\frac {R_{0}}{r}}\right)^{6}\,{\frac {1}{\tau _{D}}}}
where
τ
D
{\displaystyle \tau _{D}}
is the donor's fluorescence lifetime in the absence of the acceptor.
The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the donor molecule as follows:
E
=
1
−
τ
D
′
/
τ
D
,
{\displaystyle E=1-\tau '_{\text{D}}/\tau _{\text{D}},}
where
τ
D
′
{\displaystyle \tau _{\text{D}}'}
and
τ
D
{\displaystyle \tau _{\text{D}}}
are the donor fluorescence lifetimes in the presence and absence of an acceptor respectively, or as
E
=
1
−
F
D
′
/
F
D
,
{\displaystyle E=1-F_{\text{D}}'/F_{\text{D}},}
where
F
D
′
{\displaystyle F_{\text{D}}'}
and
F
D
{\displaystyle F_{\text{D}}}
are the donor fluorescence intensities with and without an acceptor respectively.
== Experimental confirmation of the FRET theory ==
The inverse sixth-power distance dependence of Förster resonance energy transfer was experimentally confirmed by Wilchek, Edelhoch and Brand using tryptophyl peptides. Stryer, Haugland and Yguerabide also experimentally demonstrated the theoretical dependence of Förster resonance energy transfer on the overlap integral by using a fused indolosteroid as a donor and a ketone as an acceptor. Calculations on FRET distances of some example dye-pairs can be found here. However, a lot of contradictions of special experiments with the theory was observed under complicated environment when the orientations and quantum yields of the molecules are difficult to estimate.
== Methods to measure FRET efficiency ==
In fluorescence microscopy, fluorescence confocal laser scanning microscopy, as well as in molecular biology, FRET is a useful tool to quantify molecular dynamics in biophysics and biochemistry, such as protein-protein interactions, protein–DNA interactions, DNA-DNA interactions, and protein conformational changes. For monitoring the complex formation between two molecules, one of them is labeled with a donor and the other with an acceptor. The FRET efficiency is measured and used to identify interactions between the labeled complexes. There are several ways of measuring the FRET efficiency by monitoring changes in the fluorescence emitted by the donor or the acceptor.
=== Sensitized emission ===
One method of measuring FRET efficiency is to measure the variation in acceptor emission intensity. When the donor and acceptor are in proximity (1–10 nm) due to the interaction of the two molecules, the acceptor emission will increase because of the intermolecular FRET from the donor to the acceptor. For monitoring protein conformational changes, the target protein is labeled with a donor and an acceptor at two loci. When a twist or bend of the protein brings the change in the distance or relative orientation of the donor and acceptor, FRET change is observed. If a molecular interaction or a protein conformational change is dependent on ligand binding, this FRET technique is applicable to fluorescent indicators for the ligand detection.
=== Photobleaching FRET ===
FRET efficiencies can also be inferred from the photobleaching rates of the donor in the presence and absence of an acceptor. This method can be performed on most fluorescence microscopes; one simply shines the excitation light (of a frequency that will excite the donor but not the acceptor significantly) on specimens with and without the acceptor fluorophore and monitors the donor fluorescence (typically separated from acceptor fluorescence using a bandpass filter) over time. The timescale is that of photobleaching, which is seconds to minutes, with fluorescence in each curve being given by
background
+
constant
⋅
e
−
time
/
τ
pb
,
{\displaystyle {\text{background}}+{\text{constant}}\cdot e^{-{\text{time}}/\tau _{\text{pb}}},}
where
τ
pb
{\displaystyle \tau _{\text{pb}}}
is the photobleaching decay time constant and depends on whether the acceptor is present or not. Since photobleaching consists in the permanent inactivation of excited fluorophores, resonance energy transfer from an excited donor to an acceptor fluorophore prevents the photobleaching of that donor fluorophore, and thus high FRET efficiency leads to a longer photobleaching decay time constant:
E
=
1
−
τ
pb
/
τ
pb
′
,
{\displaystyle E=1-\tau _{\text{pb}}/\tau _{\text{pb}}',}
where
τ
pb
′
{\displaystyle \tau _{\text{pb}}'}
and
τ
pb
{\displaystyle \tau _{\text{pb}}}
are the photobleaching decay time constants of the donor in the presence and in the absence of the acceptor respectively. (Notice that the fraction is the reciprocal of that used for lifetime measurements).
This technique was introduced by Jovin in 1989. Its use of an entire curve of points to extract the time constants can give it accuracy advantages over the other methods. Also, the fact that time measurements are over seconds rather than nanoseconds makes it easier than fluorescence lifetime measurements, and because photobleaching decay rates do not generally depend on donor concentration (unless acceptor saturation is an issue), the careful control of concentrations needed for intensity measurements is not needed. It is, however, important to keep the illumination the same for the with- and without-acceptor measurements, as photobleaching increases markedly with more intense incident light.
=== Lifetime measurements ===
FRET efficiency can also be determined from the change in the fluorescence lifetime of the donor. The lifetime of the donor will decrease in the presence of the acceptor. Lifetime measurements of the FRET-donor are used in fluorescence-lifetime imaging microscopy (FLIM).
=== Single-molecule FRET (smFRET) ===
smFRET is a group of methods using various microscopic techniques to measure a pair of donor and acceptor fluorophores that are excited and detected at the single molecule level. In contrast to "ensemble FRET" or "bulk FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium. Heterogeneity among different molecules can also be observed. This method has been applied in many measurements of biomolecular dynamics such as DNA/RNA/protein folding/unfolding and other conformational changes, and intermolecular dynamics such as reaction, binding, adsorption, and desorption that are particularly useful in chemical sensing, bioassays, and biosensing.
== Fluorophores used for FRET ==
=== CFP-YFP pairs ===
One common pair fluorophores for biological use is a cyan fluorescent protein (CFP) – yellow fluorescent protein (YFP) pair. Both are color variants of green fluorescent protein (GFP). Labeling with organic fluorescent dyes requires purification, chemical modification, and intracellular injection of a host protein. GFP variants can be attached to a host protein by genetic engineering which can be more convenient. Additionally, a fusion of CFP and YFP ("tandem-dimer") linked by a protease cleavage sequence can be used as a cleavage assay.
=== BRET ===
A limitation of FRET performed with fluorophore donors is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise in the results from direct excitation of the acceptor or to photobleaching. To avoid this drawback, bioluminescence resonance energy transfer (or BRET) has been developed. This technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than CFP to produce an initial photon emission compatible with YFP.
BRET has also been implemented using a different luciferase enzyme, engineered from the deep-sea shrimp Oplophorus gracilirostris. This luciferase is smaller (19 kD) and brighter than the more commonly used luciferase from Renilla reniformis, and has been named NanoLuc or NanoKAZ. Promega has developed a patented substrate for NanoLuc called furimazine, though other valuables coelenterazine substrates for NanoLuc have also been published. A split-protein version of NanoLuc developed by Promega has also been used as a BRET donor in experiments measuring protein-protein interactions.
=== Homo-FRET ===
In general, "FRET" refers to situations where the donor and acceptor proteins (or "fluorophores") are of two different types. In many biological situations, however, researchers might need to examine the interactions between two, or more, proteins of the same type—or indeed the same protein with itself, for example if the protein folds or forms part of a polymer chain of proteins or for other questions of quantification in biological cells or in vitro experiments.
Obviously, differences in conventional UV-vis spectra will not be the tool used to detect and measure homogeneous FRET, as both the acceptor and donor emit light with the same wavelengths. However, there is evidence that certain nonlinear spectroscopies may provide signatures of homogeneous FRET that would be invisible in linear spectra. Yet researchers can detect differences in the polarisation between the light which excites the fluorophores and the light which is emitted, in a technique called FRET anisotropy imaging; the level of quantified anisotropy (difference in polarisation between the excitation and emission beams) then becomes an indicative guide to how many FRET events have happened.
In the field of nano-photonics, FRET can be detrimental if it funnels excitonic energy to defect sites, but it is also essential to charge collection in organic and quantum-dot-sensitized solar cells, and various FRET-enabled strategies have been proposed for different opto-electronic devices. It is then essential to understand how isolated nano-emitters behave when they are stacked in a dense layer. Nanoplatelets are especially promising candidates for strong homo-FRET exciton diffusion because of their strong in-plane dipole coupling and low Stokes shift. Fluorescence microscopy study of such single chains demonstrated that energy transfer by FRET between neighbor platelets causes energy to diffuse over a typical 500-nm length (about 80 nano emitters), and the transfer time between platelets is on the order of 1 ps.
=== Others ===
Various compounds beside fluorescent proteins.
== Applications ==
The applications of fluorescence resonance energy transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple in many biological and biophysical fields. FRET can be used as a spectroscopic ruler to measure distance and detect molecular interactions in a number of systems and has applications in biology and biochemistry.
=== Proteins ===
FRET is often used to detect and track interactions between proteins. Additionally, FRET can be used to measure distances between domains in a single protein by tagging different regions of the protein with fluorophores and measuring emission to determine distance. This provides information about protein conformation, including secondary structures and protein folding. This extends to tracking functional changes in protein structure, such as conformational changes associated with myosin activity. Applied in vivo, FRET has been used to detect the location and interactions of cellular structures including integrins and membrane proteins.
=== Membranes ===
FRET can be used to observe membrane fluidity, movement and dispersal of membrane proteins, membrane lipid-protein and protein-protein interactions, and successful mixing of different membranes. FRET is also used to study formation and properties of membrane domains and lipid rafts in cell membranes and to determine surface density in membranes.
=== Chemosensor ===
FRET-based probes can detect the presence of various molecules: the probe's structure is affected by small molecule binding or activity, which can turn the FRET system on or off. This is often used to detect anions, cations, small uncharged molecules, and some larger biomacromolecules as well. Similarly, FRET systems have been designed to detect changes in the cellular environment due to such factors as pH, hypoxia, or mitochondrial membrane potential.
=== Signaling pathways ===
Another use for FRET is in the study of metabolic or signaling pathways. For example, FRET and BRET have been used in various experiments to characterize G-protein coupled receptor activation and consequent signaling mechanisms. Other examples include the use of FRET to analyze such diverse processes as bacterial chemotaxis and caspase activity in apoptosis.
=== Proteins and nucleotides folding kinetics ===
Proteins, DNAs, RNAs, and other polymer folding dynamics have been measured using FRET. Usually, these systems are under equilibrium whose kinetics is hidden. However, they can be measured by measuring single-molecule FRET with proper placement of the acceptor and donor dyes on the molecules. See single-molecule FRET for a more detailed description.
=== Other applications ===
In addition to common uses previously mentioned, FRET and BRET are also effective in the study of biochemical reaction kinetics. FRET is increasingly used for monitoring pH dependent assembly and disassembly and is valuable in the analysis of nucleic acids encapsulation. This technique can be used to determine factors affecting various types of nanoparticle formation as well as the mechanisms and effects of nanomedicines.
== Other methods ==
A different, but related, mechanism is Dexter electron transfer.
An alternative method to detecting protein–protein proximity is the bimolecular fluorescence complementation (BiFC), where two parts of a fluorescent protein are each fused to other proteins. When these two parts meet, they form a fluorophore on a timescale of minutes or hours.
== See also ==
Dexter electron transfer
Förster coupling
Surface energy transfer
Time-resolved fluorescence energy transfer
== References ==
== External links ==
FRET effect in a thin film on YouTube
FRET Imaging (Tutorial of Becker & Hickl, website) | Wikipedia/Förster_resonance_energy_transfer |
In biology, a substrate is the surface on which an organism (such as a plant, fungus, or animal) lives. A substrate can include biotic or abiotic materials and animals. For example, encrusting algae that lives on a rock (its substrate) can be itself a substrate for an animal that lives on top of the algae. Inert substrates are used as growing support materials in the hydroponic cultivation of plants. In biology substrates are often activated by the nanoscopic process of substrate presentation.
== In agriculture and horticulture ==
Cellulose substrate
Expanded clay aggregate (LECA)
Rock wool
Potting soil
Soil
== In animal biotechnology ==
=== Requirements for animal cell and tissue culture ===
Requirements for animal cell and tissue culture are the same as described for plant cell, tissue and organ culture (In Vitro Culture Techniques: The Biotechnological Principles). Desirable requirements are (i) air conditioning of a room, (ii) hot room with temperature recorder, (iii) microscope room for carrying out microscopic work where different types of microscopes should be installed, (iv) dark room, (v) service room, (vi) sterilization room for sterilization of glassware and culture media, and (vii) preparation room for media preparation, etc. In addition the storage areas should be such where following should be kept properly : (i) liquids-ambient (4–20 °C), (ii) glassware-shelving, (iii) plastics-shelving, (iv) small items-drawers, (v) specialized equipments-cupboard, slow turnover, (vi) chemicals-sidled containers.
=== For cell growth ===
There are many types of vertebrate cells that require support for their growth in vitro otherwise they will not grow properly. Such cells are called anchorage-dependent cells. Therefore, many substrates which may be adhesive (e.g. plastic, glass, palladium, metallic surfaces, etc.) or non-adhesive (e.g. agar, agarose, etc.) types may be used as discussed below:
Plastic as a substrate. Disposable plastics are cheaper substrate as they are commonly made up of polystyrene. After use they should be disposed of properly. Before use they are treated with gamma radiation or electric arc simply to develop charges on the surface of substrate. After cell growth its rate of proliferation should be measured. In addition, the other plastic materials used as substrate are teflon or polytetrafluoroethylene (PTFE), thermamox (TPX), polyvinylchloride (PVC), polycarbonate, etc. Monolayer of cell must be grown. Moreover, plastic beads of polystyrene, sephadex and polyacrylamide are also available for cell growth in suspension culture.
Glass as a substrate. Glass is an important substrate used in laboratory in several forms such as test tubes, slides, coverslips, pipettes, flasks, rods, bottles, Petri dishes, several apparatus, etc. These are sterilized by using chemicals, radiations, dry heat (in oven) and moist heat (in autoclave).
Palladium as a substrate. For the first time palladium deposited on agarose was used as a substrate for growth of fibroblast and glia.
== See also ==
Substrate (aquatic environment) for the specific substrate in aquatic habitats
== References ==
== External links ==
"Micro-vegetable growing" using abiotic substrates at home | Wikipedia/Substrate_(biology) |
Red fluorescent protein (RFP) is a protein which acts as a fluorophore, fluorescing red-orange when excited. The original variant occurs naturally in the coral genus Discosoma, and is named DsRed. Several new variants have been developed using directed mutagenesis which fluoresce orange, red, and far-red.
== Characteristics and Properties ==
Like GFP and other fluorescent proteins, RFP is a barrel-shaped protein made primarily out of β-sheet motifs; this type of protein fold is commonly known as a β-barrel.
The mass of RFP is approximately 25.9 kDa. Its excitation maximum is 558 nm, and its emission maximum is 583 nm.
== Applications ==
RFP is frequently used in molecular biology research as a fluorescent marker, for a variety of purposes. DsRed has been shown to be more suitable for optical imaging approaches than EGFP.
Issues with fluorescent proteins include the length of time between protein synthesis and expression of fluorescence. DsRed has a maturation time of around 24 hours, which renders it unsuited for experiments that take place over a shorter time frame. Additionally, DsRed exists in a tetrameric form, which can affect the function of proteins to which it is attached. Genetic engineering has improved the utility of RFP by increasing the speed of fluorescence development and creating monomeric variants. Improved variants of RFP include the mFruits variants (mCherry, mOrange, mRaspberry), mKO, TagRFP, mKate, mRuby, FusionRed, mScarlet and DsRed-Express.
== Other Fluorescent Proteins ==
The first fluorescent protein to be discovered, green fluorescent protein (GFP), has been adapted to identify and develop fluorescent markers in other colors. Variants such as yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) were discovered in Anthozoa.
== See Also ==
Cyan fluorescent protein (CFP)
Green fluorescent protein (GFP)
Yellow fluorescent protein (YFP)
== References ==
== External links ==
DsRed on FPBase | Wikipedia/Red_fluorescent_protein |
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags.
Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique. Affinity tags include chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag and glutathione-S-transferase (GST). The poly(His) tag is a widely used protein tag, which binds to matrices bearing immobilized metal ions.
Solubilization tags are used, especially for recombinant proteins expressed in species such as E. coli, to assist in the proper folding in proteins and keep them from aggregating in inclusion bodies. These tags include thioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role as a solubilization agent, such as MBP and GST.
Chromatography tags are used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. Often, these consist of polyanionic amino acids, such as FLAG-tag or polyglutamate tag.
Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include ALFA-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag and NE-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification.
Fluorescence tags are used to give visual readout on a protein. Green fluorescent protein (GFP) and its variants are the most commonly used fluorescence tags. More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colorless if not).
Protein tags may allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as coupling to other proteins through SpyCatcher or reaction with FlAsH-EDT2 for fluorescence imaging). Often tags are combined, in order to connect proteins to multiple other components. However, with the addition of each tag comes the risk that the native function of the protein may be compromised by interactions with the tag. Therefore, after purification, tags are sometimes removed by specific proteolysis (e.g. by TEV protease, Thrombin, Factor Xa or Enteropeptidase) or intein splicing.
== List of protein tags ==
(See Proteinogenic amino acid#Chemical properties for the A-Z amino-acid codes)
=== Peptide tags ===
ALFA-tag, a de novo designed helical peptide tag (SRLEEELRRRLTE) for biochemical and microscopy applications. The tag is recognized by a repertoire of single-domain antibodies
AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE)
C-tag, a peptide that binds to a single-domain camelid antibody developed through phage display (EPEA)
Calmodulin-tag, a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL)
iCapTag™ (intein Capture Tag), a self-removing peptide-based tag (MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIAHN). The iCapTag™ is controlled by pH change (typically pH 8.5 to pH 6.2). Therefore, this technology can be adapted to a wide range of buffers adjusted to the target pH values of 8.5 and 6.2. The expected purity of target proteins or peptides is between 95-99%. The iCapTag™ contains patented component derived from Nostoc punctiforme (Npu) intein. This tag is used for protein purification of recombinant proteins and its fragments. It can be used in research labs and it is intended for large-scale purification during downstream manufacturing process as well. The iCapTag™-target protein complex can be expressed in a wide range of expression hosts (e.g. CHO and E.coli cells). It is not intended for fully expressed mAbs
polyglutamate tag, a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE)
polyarginine tag, a peptide binding efficiently to cation-exchange resin (from 5 to 9 consecutive R)
E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR)
FLAG-tag, a peptide recognized by an antibody (DYKDDDDK)
HA-tag, a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA)
His-tag, 5-10 histidines bound by a nickel or cobalt chelate (HHHHHH)
Gly-His-tags are N-terminal His-Tag variants (e.g. GHHHH, or GHHHHHH, or GSSHHHHHH) that still bind to immobilised metal cations but can also be activated via azidogluconoylation to enable click-chemistry applications
Myc-tag, a peptide derived from c-myc recognized by an antibody (EQKLISEEDL)
NE-tag, an 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES) recognized by a monoclonal IgG1 antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins
Rho1D4-tag, refers to the last 9 amino acids of the intracellular C-terminus of bovine rhodopsin (TETSQVAPA). It is a very specific tag that can be used for purification of membrane proteins.
S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS)
SBP-tag, a peptide which binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)
Softag 1, for mammalian expression (SLAELLNAGLGGS)
Softag 3, for prokaryotic expression (TQDPSRVG)
Spot-tag, a peptide recognized by a nanobody (PDRVRAVSHWSS) for immunoprecipitation, affinity purification, immunofluorescence and super resolution microscopy
Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK)
T7-tag, an epitope tag derived from the T7 major capsid protein of the T7 gene (MASMTGGQQMG). Used in different immunoassays as well as affinity purification Mainly used
TC tag, a tetracysteine tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC)
Ty tag (EVHTNQDPLD)
V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST)
VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK)
Xpress tag (DLYDDDDK), a peptide recognized by an antibody
=== Covalent peptide tags ===
Isopeptag, a peptide which binds covalently to pilin-C protein (TDKDMTITFTNKKDAE)
SpyTag, a peptide which binds covalently to SpyCatcher protein (AHIVMVDAYKPTK)
SnoopTag, a peptide which binds covalently to SnoopCatcher protein (KLGDIEFIKVNK). A second generation, SnoopTagJr, was also developed to bind to either SnoopCatcher or DogTag (mediated by SnoopLigase) (KLGSIEFIKVNK)
DogTag, a peptide which covalently binds to DogCatcher (DIPATYEFTDGKHYITNEPIPPK), and can also covalently bind to SnoopTagJr, mediated by SnoopLigase
SdyTag, a peptide which binds covalently to SdyCatcher protein (DPIVMIDNDKPIT). SdyTag/SdyCatcher has a kinetic-dependent cross-reactivity with SpyTag/SpyCatcher.
=== Protein tags ===
BCCP (Biotin Carboxyl Carrier Protein), a protein domain biotinylated by BirA enabling recognition by streptavidin
BromoTag, a "bump-and-hole" mutated version of the second bromodomain of Brd4, Brd4-BD2 L387A, that can be highly selectively bound by tag-specific PROTAC degrader AGB1 to form a ternary complex between the "BromoTagged" protein and the E3 ligase VHL, leading to ubiquitination of the tagged protein and its subsequent rapid and effective proteasomal degradation in cells.
FAST (Fluorescence-Activating and absorption-Shifting Tag), a mutated photoactive yellow protein (PYP) that reversibly binds cognate fluorogenic ligands
CL7-tag, an engineered variant of Colicin E7 that has a strong binding affinity and specificity for immobilized Immunity Protein 7 (Im7).
Glutathione-S-transferase-tag, a protein which binds to immobilized glutathione
Green fluorescent protein-tag, a protein which is spontaneously fluorescent and can be bound by nanobodies
HaloTag, a mutated bacterial haloalkane dehalogenase that covalently attaches to haloalkane substrates
SNAP-tag, a mutated eukaryotic DNA methyltransferase that covalently attaches to benzylguanine derivatives
CLIP-tag, a mutated eukaryotic DNA methyltransferase that covalently attaches to benzylcytosine derivatives
HUH-tag, a sequence-specific single-stranded DNA binding protein that covalently binds to its target sequence
Maltose binding protein-tag, a protein which binds to amylose agarose
Nus-tag
Thioredoxin-tag
Fc-tag, derived from immunoglobulin Fc domain, allow dimerization and solubilization. Can be used for purification on Protein-A Sepharose
Designed Intrinsically Disordered tags containing disorder promoting amino acids (P,E,S,T,A,Q,G,..)
Carbohydrate Recognition Domain or CRDSAT-tag, a protein which binds to lactose agarose or Sepharose
=== Others ===
HiBiT-tag
was developed by Scientists at Promega. It is an 11-amino-acid peptide tag, and it can be fused to the N- or C-terminus or internal locations of proteins. Its small size leads to a rapid knock-in of this tag with other proteins through CRISPR/Cas9 technology.
== Applications ==
Affinity purification
Protein array
TimeSTAMP protein labelling
Western blotting
== References == | Wikipedia/Protein_tag |
A FMN-binding fluorescent protein (FbFP), also known as a LOV-based fluorescent protein, is a small, oxygen-independent fluorescent protein that binds flavin mononucleotide (FMN) as a chromophore.
They were developed from blue-light receptors (so called LOV-domains) found in plants and various bacteria. They complement the GFP-derivatives and –homologues and are particularly characterized by their independence of molecular oxygen and their small size. FbFPs absorb blue light and emit light in the cyan-green spectral range.
== Development ==
LOV-domains are a sub-class of PAS domains and were first identified in plants as part of Phototropin, which plays an essential role in the plant's growth towards light. They noncovalently bind Flavin mononucleotide (FMN) as cofactor. Due to the bound FMN LOV-domains exhibit an intrinsic fluorescence, which is however very weak. Upon illumination with blue light, LOV-domains undergo a photocyle, during which a covalent bond is formed between a conserved cysteine-residue and the FMN. This causes a conformational change in the protein that is necessary for signal propagation and also leads to the loss of fluorescence. The covalent bond is energetically unfavorable and is cleaved with a protein specific time constant ranging from seconds to hours.
In order to make better use of the fluorescence properties of these proteins, the natural photocycle of these LOV-domains was abolished by exchanging the conserved cysteine against an alanine on a genetic level.
Thus, upon blue light irradiation, the protein remains in the fluorescent state and also exhibits a brighter fluorescence.
The first FbFPs that were generated in this fashion were subsequently further optimized using different methods of mutagenesis. Especially the brightness but also the photostability of the proteins were enhanced and their spectral characteristics altered.
== Spectral characteristics ==
Typically FbFPs have an excitation maximum at a wavelength of approximately 450 nm (blue light) and a second distinct excitation peak around 370 nm (UV-A light). The main emission peak is at approx. 495 nm, with a shoulder around 520 nm. One variant of Pp2FbFP (Q116V) exhibits a 10 nm blue shift in both the excitation and emission spectra. Rationally designed variants of iLOV and CagFbFP exhibit 6 and 7 nm red shifts, respectively.
== Photophysical properties ==
The photophysical properties of the FbFPs are determined by the chromophore itself and its chemical surrounding in the protein. The extinction coefficient (ε) is around 14.200 M−1cm−1 at 450 nm for all described FbFPs, which is slightly higher than that of free FMN (ε = 12.200 M−1cm−1 ). The Fluorescence-Quantum yield (Φ) varies significantly between different FbFPs and ranges from 0.2 (phiLOV2.1) to 0.44 (EcFbFP and iLOV). This represents an almost twofold increase compared to free FMN (Φ = 0.25).
The difference to free FMN is even more significant in the case of the photostabaility, the proteins resistance to bleach out during prolonged and intense irradiation with blue light. Based on the bleaching-halftime (the times it takes to reduce the initial fluorescence intensity to 50% upon illumination) the genetically engineered variant phiLOV2.1 is approximately 40x as stable as free FMN. This stabilizing effect can be observed for almost all FbFPs, although it is usually in the range of 5x - 10x.
The average fluorescence lifetime of FbFPs is in the range of 3.17 (Pp2FbFP) and 5.7 ns (e.g. EcFbFP). They are thereby much longer than the ones of GFP derivatives, which are usually between 1,5 and 3 ns.
FbFPs are therefore well suited as donor domains in Förster resonance energy transfer (FRET) systems in conjunction with GFP derivatives like YFP as acceptor domains.
== Advantages and disadvantages ==
The main advantage of FbFPs over GFP is their independence of molecular oxygen. Since all GFP derivatives and homologues require molecular oxygen for the maturation of their chromophore, these fluorescent proteins are of limited use under anaerobic or hypoxic conditions.
Since FbFPs bind FMN as chromophore, which is synthesized independently of molecular oxygen, their fluorescence signal does not differ between aerobic and anaerobic conditions.
Another advantage is the small size of FbFPs, which is typically between 100 and 150 amino acids. This is about half the size of GFP (238 amino acids). It could for example be shown that this renders them superior tags for monitoring tobacco mosaic virus infections in tobacco leaves.
Due to their extraordinary long average fluorescence lifetime of up to 5.7 ns they are also very well suited for the use as donor domains in FRET systems in conjunction with e.g. YFP (see photophysical properties). A fusion of EcFbFP and YFP was e.g. used to develop the first genetically encoded fluorescence biosensor for oxygen (FluBO)
The main disadvantage compared to GFP variants is their lower brightness (the product of ε and Φ). The commonly used EGFP (ε = 55,000 M−1cm−1; Φ = 0.60 ) for example is approximately five times as bright as EcFbFP.
Another disadvantage of the FbFPs is the lack of color variants to tag and distinguish multiple proteins in a single cell or tissue. The largest spectral shift reported for FbFPs so far is 10 nm. Although this variant (Pp2FbFP Q116V) can be visually distinguished from the others with the human eye, the spectral differences are too small for fluorescence microscopy filters.
== References == | Wikipedia/FMN-binding_fluorescent_proteins |
EosFP is a photoactivatable green to red fluorescent protein. Its green fluorescence (516 nm) switches to red (581 nm) upon UV irradiation of ~390 nm (violet/blue light) due to a photo-induced modification resulting from a break in the peptide backbone near the chromophore. Eos was first discovered as a tetrameric protein in the stony coral Lobophyllia hemprichii. Like other fluorescent proteins, Eos allows for applications such as the tracking of fusion proteins, multicolour labelling and tracking of cell movement. Several variants of Eos have been engineered for use in specific study systems including mEos2, mEos4 and CaMPARI.
== History ==
EosFP was first discovered in 2005 during a large scale screen for PAFPs (photoactivatable fluorescent proteins) within the stony coral Lobophyllia hemprichii. It has since been successfully cloned in Escherichia coli and fusion constructs have been developed for use in human cells. Eos was named after the Greek goddess of dawn.
Unlike the tetrameric fluorescent proteins derived from anthozoan coral, which can interfere with normal cellular function due to interactions between protein subunits, EosFP has been broken up into dimeric and monomeric variants through the introduction of single point mutations. These variants have been successful in the tracking of cellular components without disturbing function in the host cell and maintain the same photophysical properties as wild-type Eos.
Since their discovery, monomeric Eos probes (mEos) have been shown to localize in the cytosol, plasma membrane, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, golgi bodies, peroxisomes, mitochondria, invaginations, filamentous actin and cortical microtubules. mEos fusion proteins allow for differential colour labelling in single cells, or groups of cells in developing organs. They can also be used for the understanding of spatial/ temporal interactions between organelles and vesicles. The two fluorescent forms of mEosFP (green and red) are compatible with CFP, GFP, YFP and RFP for multicolour labelling.
== Function ==
EosFP emits a strong green fluorescence (516 nm) that changes irreversibly to red (581 nm) when irradiated with UV-light of 390 nm. This modification occurs due to a break in the peptide backbone next to the chromophore. This mechanism allows for localized tagging of the protein and makes EosFP an appropriate tool for tracking protein movement within living cells. Formation of the red chromophore involves cleaving the peptide backbone but includes almost no other changes in the protein structure.
According to single-molecule fluorescence spectroscopy, EosFP is tetrameric, and exhibits strong Forster resonance coupling within individual fluorophores. Like other fluorescent proteins, Eos can be used to report diverse signals in cells, tissues and organs without disturbing complex biological machinery. While the use of fluorescent proteins was once limited to the green fluorescent protein (GFP), in recent years many other fluorescent proteins have been cloned. Unlike GFPs, which are derived from the luminescent jellyfish Aequorea victoria, fluorescent proteins derived from anthozoa, including Eos, emit fluorescence in the red spectral range. The novel property of photoinduced green-to-red conversion in Eos is useful because it allows for localized tracking of proteins in living cells. EosFP is unique because it has a large separation in the wavelengths it can emit which allows for easy identification of peak colours. All green-to-red photoinducible fluorescent proteins, including Eos, contain a chromophoric unit derived from the tripeptide his-tyr-gly. This green-to-red conversion is completed by light rather than chemical oxidation such as in other FPs.
== Structure and Absorbance Properties ==
=== Primary structure ===
EosFP consists of 226 amino acids. It has a molecular mass of 25.8 kDa and its pI is 6.9. Eos has 84% identical residues to Kaede, a fluorescent protein that originated in a different scleractinian coral Trachyphyllia geoffroyi, but can also be irreversibly converted from a green to red emitting form using UV light. Excluding residues Phe-61 and His-62, the chromophore environment and chromophore itself are unaffected by photochemical modification. Wild-type EosFP has a tetrameric arrangement of subunits where each subunit has the same β-can structure as GFP. This structure includes an 11-stranded barrel and, down the central axis, the fluorophore-containing helix.
=== Structure of Green EosFP ===
In its anionic form, the green chromophore has an absorption maxima at 506 nm and an emission maxima at 516 nm. It is formed autocatalytically from amino acids His-62, Tyr-63 and Gly-64. Immediately surrounding the chromophore there is a cluster of charged or polar amino acids as well as structural water molecules. Above the plane of the chromophore, there is a network of hydrogen bond interactions between Glu-144, His-194, Glu-212 and Gln-38. Arg-66 and Arg-91 participate in hydrogen bonding with the carbonyl oxygen of green Eos's imidazolinone moiety. The His-62 side chain lies in an unpolar environment. Conversion from the green to red form depends on the presence of a histidine in the first position of the tripeptide HYG that forms the chromophore. When this histidine residue is substituted with M, S, T or L, Eos only emits bright green light and no longer acts as a photoconvertible fluorescent protein.
=== Structure of Red EosFP ===
The red chromophore, which is generated by cleavage of the peptide backbone, has an absorption maxima at 571 nm and an emission maxima at 581 nm, in its anionic form. The break in the peptide backbone that leads to this chromophore is between His-62 Nα and Cα. The observed red fluorescence occurs due to an extension of the chromophore's π-conjugation where the His-62 imidazole ring connects to the imidazolinone. The hydrogen bond patterns of the red and green chromophores are almost identical.
== Photochemical conversion ==
Photochemical conversion occurs due to interactions between the chromophoric unit and residues in its vicinity. Glu-212 functions as a base that removes a proton from His-62 aiding in the cleavage of the His-62-Nα-Cα bond. Replacing Glu-212 with glutamine prevents photoconversion. At low pH, the yield of Eos involved in photoconversion is greatly increased as the fraction of molecules in the protonated form increases. The action spectrum for photoconversion is closely related to the action spectrum for Eos's protonated form. These observations suggest that the neutral form of the green chromophore, including a protonated Tyr-63 side chain, is the gateway structure for photoconversion. Proton ejection from the Tyr-63 phenyl side chain is an important event in the conversion mechanism where a proton is transferred from the His-62 imidazole, which is hydrogen-bonded to the Phe-61 carbonyl. The extra proton causes His-62 to donate a proton to the Phe-61 carbonyl forming a leaving group out of the peptide bond between His and Phe in the elimination reaction. The His-62 side chain is protonated during photoexcitation and assists the reaction by donating a proton to the Phe-61 carbonyl in the leaving group. After the backbone is cleaved, the hydrogen bond between His-62 and Phe-61 is reformed. When His-62 is replaced with other amino acids, EosFP loses its ability to photoconvert, providing evidence that His-62 is a necessary component of the photoconversion mechanism. The internal charge distribution of the green chromophore is altered during photo excitation to assist in the elimination reaction.
== Spectroscopy ==
Both the fluorescence excitation and emission spectrums of wild-type EosFP are shifted ~65 nm to the right upon excitation toward the red end of the spectrum. This spectral change is caused by an extension of the chromophore accompanied by a break in the peptide backbone between Phe-61 and His-62 in an irreversible mechanism. The presence of a crisp isosbestic point at 432 nm also suggests an interconversion between two species. An absorption peak at 280 nm is visible due to aromatic amino acids which transfer their excitation energy to the green chromophore. The quantum yield of the green-emitting form of Eos is 0.7. In the red shifted species, there are pronounced vibronic sidebands separate from the main peak at 533 nm and 629 nm in the excitation spectrum and emission spectrum respectively. There is another peak in the red excitation spectrum at 502 nm likely due to FRET excitation of the red fluorophore. The quantum yield of the red-emitting form is 0.55.
EosFPs variants show almost no difference in spectroscopic properties, therefore, it is likely that the structural modifications which arise from separation of interfaces have little to no effect on the structure of the fluorophore-binding site.
== Applications ==
=== Tracking of Fusion Proteins ===
Many different fusion proteins have been created using EosFP and its engineered variants. These fusion proteins allow for the tracking of proteins within living cells while retaining complex biological functions like protein-protein interactions and protein-DNA interactions. Eos fusion constructs include those with recombination signal-binding protein (RBP) and cytokeratin. Studies have shown that it is favourable to attach the protein of interest to the N-terminal side of the EosFP label. These fusion constructs have been used to visualize nuclear translocation with androgen receptors, dynamics of the cytoskeleton with actin and vinculin and intranuclear protein movement with RBP.
=== Multicolour Labelling ===
Since EosFP can be used in fusion constructs while maintaining functionality of the protein of interest, it is a popular choice for multi-colour labelling studies. In a dual-colour labelling experiment to map the stages of mitosis, HEK293 cells were first stably transfected with tubulin-binding protein cDNA fused to EGFP for visualization of the spindle apparatus. Then, transient transfection of recombination signal-binding protein (RBP) fused to d2EosFP was used to visualize the beginning of mitosis. Photoconversion was completed by fluorescent microscopy and highlighted the separation between two sets of chromosomes during anaphase, telophase and cytokinesis.
=== Tracking of Cell Movement in Developmental Biology ===
EosFP has been used to track cell movements during embryonic development of Xenopus laevis. At the two-cell/ early gastrula stage, capped mRNA coding for a dimeric EosFP (d2EosFP) was injected into cells and locally photoconverted using fluorescence microscopy. These fluorescent embryos demonstrated the dynamics of cell movement during neurulation. EosFP was found in part of the notochord which shows the possibility of EosFP to be used in fate-mapping experiments.
== Engineered variants ==
=== mEos4 ===
Many new monomeric versions of EosFP have been developed that offer advantages over wild type EosFP. Developed by a team at the Janelia Farm Research Campus at Howard Hughes Medical Institute, mEos4 has higher photostability and longer imaging abilities than EosFP. It is also highly resistant to chemical fixatives such as PFA, gluteraldehyde and OsO4 which are used to preserve samples. mEos4 is effective at higher temperatures than EosFP, phot-converts at an increased rate and has a higher emission amplitude in both green and red fluorescent states. Applications for the mEos4 protein include photoactivation localization microscopy (PALM), correlative light/ electron microscopy (CLEM), protein activity indication and activity integration (post-hoc imaging for protein activity over time).
=== mEos2 ===
mEosFP is another monomeric Eos variant that folds effectively at 37 degrees Celsius. Where tdEos (tandem dimer) cannot fuse to targets such as histones, tubulin, intermediate filaments and gap junctions, and mEos (monomeric) which can only be used successfully at 30 degrees Celsius, mEos2 is an engineered variant that can fold effectively at 37 degrees Celsius and successfully label targets intolerant to fusion from other fluorescent protein dimers . mEos2 shows almost identical spectral properties, brightness, pKa, photoconversion, contrast and maturation properties to WT Eos. The localization precision of mEos2 is twice as great as other monomeric fluorescent proteins.
=== CaMPARI ===
Also at the Janelia Research Campus, a new fluorescent molecules known as CaMPARI (calcium-modulated photoactivatable ratiometric integrator) was developed using EosFP. The permanent green to red conversion signal was coupled with a calcium-sensitive protein, calmodulin, so that color change in the fusion construct depended on the release of calcium accompanied by neural activity. CaMPARI is able to permanently mark neurons that are active at an any time and can also be targeted to synapses. This visualization is possible across a wide amount of brain tissue as opposed to the limited view available with using a microscope. It also allows for the visualization of neural activity during complicated behaviors as the organism under study is allowed to move freely, rather than under a microscope. It also allows for the observation of neurons during specific behavior periods. CaMPARI has, thus far, been used to label active neural circuits in mice, zebrafish and fruit flies.
== References == | Wikipedia/Eos_(protein) |
A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector.
The sensitive biological element, e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc., is a biologically derived material or biomimetic component that interacts with, binds with, or recognizes the analyte under study. The biologically sensitive elements can also be created by biological engineering.
The transducer or the detector element, which transforms one signal into another one, works in a physicochemical way: optical, piezoelectric, electrochemical,
electrochemiluminescence etc., resulting from the interaction of the analyte with the biological element, to easily measure and quantify.
The biosensor reader device connects with the associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. This sometimes accounts for the most expensive part of the sensor device, however it is possible to generate a user friendly display that includes transducer and sensitive element (holographic sensor). The readers are usually custom-designed and manufactured to suit the different working principles of biosensors.
== Biosensor system ==
A biosensor typically consists of a bio-receptor (enzyme/antibody/cell/nucleic acid/aptamer), transducer component (semi-conducting material/nanomaterial), and electronic system which includes a signal amplifier, processor & display. Transducers and electronics can be combined, e.g., in CMOS-based microsensor systems. The recognition component, often called a bioreceptor, uses biomolecules from organisms or receptors modeled after biological systems to interact with the analyte of interest. This interaction is measured by the biotransducer which outputs a measurable signal proportional to the presence of the target analyte in the sample. The general aim of the design of a biosensor is to enable quick, convenient testing at the point of concern or care where the sample was procured.
== Bioreceptors ==
In a biosensor, the bioreceptor is designed to interact with the specific analyte of interest to produce an effect measurable by the transducer. High selectivity for the analyte among a matrix of other chemical or biological components is a key requirement of the bioreceptor. While the type of biomolecule used can vary widely, biosensors can be classified according to common types of bioreceptor interactions involving: antibody/antigen, enzymes/ligands, nucleic acids/DNA, cellular structures/cells, or biomimetic materials.
=== Antibody/antigen interactions ===
An immunosensor utilizes the very specific binding affinity of antibodies for a specific compound or antigen. The specific nature of the antibody-antigen interaction is analogous to a lock and key fit in that the antigen will only bind to the antibody if it has the correct conformation. Binding events result in a physicochemical change that in combination with a tracer, such as fluorescent molecules, enzymes, or radioisotopes, can generate a signal. There are limitations with using antibodies in sensors: 1. The antibody binding capacity is strongly dependent on assay conditions (e.g. pH and temperature), and 2. the antibody-antigen interaction is generally robust, however, binding can be disrupted by chaotropic reagents, organic solvents, or even ultrasonic radiation.
Antibody-antigen interactions can also be used for serological testing, or the detection of circulating antibodies in response to a specific disease. Importantly, serology tests have become an important part of the global response to the COVID-19 pandemic.
=== Artificial binding proteins ===
The use of antibodies as the bio-recognition component of biosensors has several drawbacks. They have high molecular weights and limited stability, contain essential disulfide bonds and are expensive to produce. In one approach to overcome these limitations, recombinant binding fragments (Fab, Fv or scFv) or domains (VH, VHH) of antibodies have been engineered. In another approach, small protein scaffolds with favorable biophysical properties have been engineered to generate artificial families of Antigen Binding Proteins (AgBP), capable of specific binding to different target proteins while retaining the favorable properties of the parent molecule. The elements of the family that specifically bind to a given target antigen, are often selected in vitro by display techniques: phage display, ribosome display, yeast display or mRNA display. The artificial binding proteins are much smaller than antibodies (usually less than 100 amino-acid residues), have a strong stability, lack disulfide bonds and can be expressed in high yield in reducing cellular environments like the bacterial cytoplasm, contrary to antibodies and their derivatives. They are thus especially suitable to create biosensors.
=== Enzymatic interactions ===
The specific binding capabilities and catalytic activity of enzymes make them popular bioreceptors. Analyte recognition is enabled through several possible mechanisms: 1) the enzyme converting the analyte into a product that is sensor-detectable, 2) detecting enzyme inhibition or activation by the analyte, or 3) monitoring modification of enzyme properties resulting from interaction with the analyte. The main reasons for the common use of enzymes in biosensors are: 1) ability to catalyze a large number of reactions; 2) potential to detect a group of analytes (substrates, products, inhibitors, and modulators of the catalytic activity); and 3) suitability with several different transduction methods for detecting the analyte. Notably, since enzymes are not consumed in reactions, the biosensor can easily be used continuously. The catalytic activity of enzymes also allows lower limits of detection compared to common binding techniques. However, the sensor's lifetime is limited by the stability of the enzyme.
=== Affinity binding receptors ===
Antibodies have a high binding constant in excess of 10^8 L/mol, which stands for a nearly irreversible association once the antigen-antibody couple has formed. For certain analyte molecules like glucose affinity binding proteins exist that bind their ligand with a high specificity like an antibody, but with a much smaller binding constant on the order of 10^2 to 10^4 L/mol. The association between analyte and receptor then is of reversible nature and next to the couple between both also their free molecules occur in a measurable concentration. In case of glucose, for instance, concanavalin A may function as affinity receptor exhibiting a binding constant of 4x10^2 L/mol. The use of affinity binding receptors for purposes of biosensing has been proposed by Schultz and Sims in 1979 and was subsequently configured into a fluorescent assay for measuring glucose in the relevant physiological range between 4.4 and 6.1 mmol/L. The sensor principle has the advantage that it does not consume the analyte in a chemical reaction as occurs in enzymatic assays.
=== Nucleic acid interactions ===
Biosensors employing nucleic acid based receptors can be either based on complementary base pairing interactions referred to as genosensors or specific nucleic acid based antibody mimics (aptamers) as aptasensors. In the former, the recognition process is based on the principle of complementary base pairing, adenine:thymine and cytosine:guanine in DNA. If the target nucleic acid sequence is known, complementary sequences can be synthesized, labeled, and then immobilized on the sensor. The hybridization event can be optically detected and presence of target DNA/RNA ascertained. In the latter, aptamers generated against the target recognise it via interplay of specific non-covalent interactions and induced fitting. These aptamers can be labelled with a fluorophore/metal nanoparticles easily for optical detection or may be employed for label-free electrochemical or cantilever based detection platforms for a wide range of target molecules or complex targets like cells and viruses. Additionally, aptamers can be combined with nucleic acid enzymes, such as RNA-cleaving DNAzymes, providing both target recognition and signal generation in a single molecule, which shows potential applications in the development of multiplex biosensors.
=== Epigenetics ===
It has been proposed that properly optimized integrated optical resonators can be exploited for detecting epigenetic modifications (e.g. DNA methylation, histone post-translational modifications) in body fluids from patients affected by cancer or other diseases. Photonic biosensors with ultra-sensitivity are nowadays being developed at a research level to easily detect cancerous cells within the patient's urine. Different research projects aim to develop new portable devices that use cheap, environmentally friendly, disposable cartridges that require only simple handling with no need of further processing, washing, or manipulation by expert technicians.
=== Organelles ===
Organelles form separate compartments inside cells and usually perform functions independently. Different kinds of organelles have various metabolic pathways and contain enzymes to fulfill its function. Commonly used organelles include lysosome, chloroplast and mitochondria. The spatial-temporal distribution pattern of calcium is closely related to ubiquitous signaling pathway. Mitochondria actively participate in the metabolism of calcium ions to control the function and also modulate the calcium related signaling pathways. Experiments have proved that mitochondria have the ability to respond to high calcium concentrations generated in their proximity by opening the calcium channels. In this way, mitochondria can be used to detect the calcium concentration in medium and the detection is very sensitive due to high spatial resolution. Another application of mitochondria is used for detection of water pollution. Detergent compounds' toxicity will damage the cell and subcellular structure including mitochondria. The detergents will cause a swelling effect which could be measured by an absorbance change. Experiment data shows the change rate is proportional to the detergent concentration, providing a high standard for detection accuracy.
=== Cells ===
Cells are often used in bioreceptors because they are sensitive to surrounding environment and they can respond to all kinds of stimulants. Cells tend to attach to the surface so they can be easily immobilized. Compared to organelles they remain active for longer period and the reproducibility makes them reusable. They are commonly used to detect global parameter like stress condition, toxicity and organic derivatives. They can also be used to monitor the treatment effect of drugs. One application is to use cells to determine herbicides which are main aquatic contaminant. Microalgae are entrapped on a quartz microfiber and the chlorophyll fluorescence modified by herbicides is collected at the tip of an optical fiber bundle and transmitted to a fluorimeter. The algae are continuously cultured to get optimized measurement. Results show that detection limit of certain herbicide can reach sub-ppb concentration level. Some cells can also be used to monitor the microbial corrosion. Pseudomonas sp. is isolated from corroded material surface and immobilized on acetylcellulose membrane. The respiration activity is determined by measuring oxygen consumption. There is linear relationship between the current generated and the concentration of sulfuric acid. The response time is related to the loading of cells and surrounding environments and can be controlled to no more than 5min.
=== Tissue ===
Tissues are used for biosensor for the abundance of enzymes existing. Advantages of tissues as biosensors include the following:
easier to immobilize compared to cells and organelles
the higher activity and stability from maintaining enzymes in the natural environment
the availability and low price
the avoidance of tedious work of extraction, centrifuge, and purification of enzymes
necessary cofactors for an enzyme to function exists
the diversity providing a wide range of choices concerning different objectives.
There also exist some disadvantages of tissues, like the lack of specificity due to the interference of other enzymes and longer response time due to the transport barrier.
=== Microbial biosensors ===
Microbial biosensors exploit the response of bacteria to a given substance. For example, arsenic can be detected using the ars operon found in several bacterial taxon.
== Surface attachment of the biological elements ==
An important part of a biosensor is to attach the biological elements (small molecules/protein/cells) to the surface of the sensor (be it metal, polymer, or glass). The simplest way is to functionalize the surface in order to coat it with the biological elements. This can be done by polylysine, aminosilane, epoxysilane, or nitrocellulose in the case of silicon chips/silica glass. Subsequently, the bound biological agent may also be fixed—for example, by layer by layer deposition of alternatively charged polymer coatings.
Alternatively, three-dimensional lattices (hydrogel/xerogel) can be used to chemically or physically entrap these (whereby chemically entrapped it is meant that the biological element is kept in place by a strong bond, while physically they are kept in place being unable to pass through the pores of the gel matrix). The most commonly used hydrogel is sol-gel, glassy silica generated by polymerization of silicate monomers (added as tetra alkyl orthosilicates, such as TMOS or TEOS) in the presence of the biological elements (along with other stabilizing polymers, such as PEG) in the case of physical entrapment.
Another group of hydrogels, which set under conditions suitable for cells or protein, are acrylate hydrogel, which polymerizes upon radical initiation. One type of radical initiator is a peroxide radical, typically generated by combining a persulfate with TEMED (Polyacrylamide gel are also commonly used for protein electrophoresis), alternatively light can be used in combination with a photoinitiator, such as DMPA (2,2-dimethoxy-2-phenylacetophenone). Smart materials that mimic the biological components of a sensor can also be classified as biosensors using only the active or catalytic site or analogous configurations of a biomolecule.
== Biotransducer ==
Biosensors can be classified by their biotransducer type. The most common types of biotransducers used in biosensors are:
electrochemical biosensors
optical biosensors
electronic biosensors
piezoelectric biosensors
gravimetric biosensors
pyroelectric biosensors
magnetic biosensors
=== Electrochemical ===
Electrochemical biosensors, based on enzymes, work through the enzymatic catalysis of reactions that directly or indirectly produce or consume electrons (such enzymes are rightly called redox enzymes). The sensor design usually consists of three electrodes; a reference electrode, a working electrode, and a counter electrode. The target analyte is involved in the reaction that takes place on the surface of the active working electrode, and the reaction may cause either electron transfer across the double layer (producing a current) or can contribute to the double layer potential (producing a voltage). The current (rate of flow of electrons is now proportional to the analyte concentration) can be measured at a fixed potential or the potential can be measured at zero current (this gives a logarithmic response). Note that potential of the working electrode is space charge sensitive and this is often used. Additionally, the label-free and direct electrical detection of small peptides and proteins is possible by their intrinsic charges using biofunctionalized ion-sensitive field-effect transistors.
Another example, the potentiometric biosensor, (potential produced at zero current) gives a logarithmic response with a high dynamic range. Such biosensors are often made by screen printing the electrode patterns on a plastic substrate, coated with a conducting polymer and then some protein (enzyme or antibody) is attached. They have only two electrodes and are extremely sensitive and robust. They enable the detection of analytes at levels previously only achievable by HPLC and LC/MS and without rigorous sample preparation. All biosensors usually involve minimal sample preparation as the biological sensing component is highly selective for the analyte concerned. The signal is produced by electrochemical and physical changes in the conducting polymer layer due to changes occurring at the surface of the sensor. Such changes can be attributed to ionic strength, pH, hydration and redox reactions, the latter due to the enzyme label turning over a substrate. Field effect transistors, in which the gate region has been modified with an enzyme or antibody, can also detect very low concentrations of various analytes as the binding of the analyte to the gate region of the FET cause a change in the drain-source current.
Impedance spectroscopy based biosensor development has been gaining traction nowadays and many such devices / developments are found in the academia and industry. One such device, based on a 4-electrode electrochemical cell, using a nanoporous alumina membrane, has been shown to detect low concentrations of human alpha thrombin in presence of high background of serum albumin. Also interdigitated electrodes have been used for impedance biosensors.
=== Ion channel switch ===
The use of ion channels has been shown to offer highly sensitive detection
of target biological molecules. By embedding the ion channels in supported or tethered bilayer membranes (t-BLM) attached to a gold electrode, an electrical circuit is created. Capture molecules such as antibodies can be bound to the ion channel so that the binding of the target molecule controls the ion flow through the channel. This results in a measurable change in the electrical conduction which is proportional to the concentration of the target.
An ion channel switch (ICS) biosensor can be created using gramicidin, a dimeric peptide channel, in a tethered bilayer membrane. One peptide of gramicidin, with attached antibody, is mobile and one is fixed. Breaking the dimer stops the ionic current through the membrane. The magnitude of the change in electrical signal is greatly increased by separating the membrane from the metal surface using a hydrophilic spacer.
Quantitative detection of an extensive class of target species, including proteins, bacteria, drug and toxins has been demonstrated using different membrane and capture configurations. The European research project Greensense develops a biosensor to perform quantitative screening of drug-of-abuse such as THC, morphine, and cocaine in saliva and urine.
=== Reagentless fluorescent biosensor ===
A reagentless biosensor can monitor a target analyte in a complex biological mixture without additional reagent. Therefore, it can function continuously if immobilized on a solid support. A fluorescent biosensor reacts to the interaction with its target analyte by a change of its fluorescence properties. A Reagentless Fluorescent biosensor (RF biosensor) can be obtained by integrating a biological receptor, which is directed against the target analyte, and a solvatochromic fluorophore, whose emission properties are sensitive to the nature of its local environment, in a single macromolecule. The fluorophore transduces the recognition event into a measurable optical signal. The use of extrinsic fluorophores, whose emission properties differ widely from those of the intrinsic fluorophores of proteins, tryptophan and tyrosine, enables one to immediately detect and quantify the analyte in complex biological mixtures. The integration of the fluorophore must be done in a site where it is sensitive to the binding of the analyte without perturbing the affinity of the receptor.
Antibodies and artificial families of Antigen Binding Proteins (AgBP) are well suited to provide the recognition module of RF biosensors since they can be directed against any antigen (see the paragraph on bioreceptors). A general approach to integrate a solvatochromic fluorophore in an AgBP when the atomic structure of the complex with its antigen is known, and thus transform it into a RF biosensor, has been described. A residue of the AgBP is identified in the neighborhood of the antigen in their complex. This residue is changed into a cysteine by site-directed mutagenesis. The fluorophore is chemically coupled to the mutant cysteine. When the design is successful, the coupled fluorophore does not prevent the binding of the antigen, this binding shields the fluorophore from the solvent, and it can be detected by a change of fluorescence. This strategy is also valid for antibody fragments.
However, in the absence of specific structural data, other strategies must be applied. Antibodies and artificial families of AgBPs are constituted by a set of hypervariable (or randomized) residue positions, located in a unique sub-region of the protein, and supported by a constant polypeptide scaffold. The residues that form the binding site for a given antigen, are selected among the hypervariable residues. It is possible to transform any AgBP of these families into a RF biosensor, specific of the target antigen, simply by coupling a solvatochromic fluorophore to one of the hypervariable residues that have little or no importance for the interaction with the antigen, after changing this residue into cysteine by mutagenesis. More specifically, the strategy consists in individually changing the residues of the hypervariable positions into cysteine at the genetic level, in chemically coupling a solvatochromic fluorophore with the mutant cysteine, and then in keeping the resulting conjugates that have the highest sensitivity (a parameter that involves both affinity and variation of fluorescence signal). This approach is also valid for families of antibody fragments.
A posteriori studies have shown that the best reagentless fluorescent biosensors are obtained when the fluorophore does not make non-covalent interactions with the surface of the bioreceptor, which would increase the background signal, and when it interacts with a binding pocket at the surface of the target antigen. The RF biosensors that are obtained by the above methods, can function and detect target analytes inside living cells.
=== Magnetic biosensors ===
Magnetic biosensors utilize paramagnetic or supra-paramagnetic particles, or crystals, to detect biological interactions. Examples could be coil-inductance, resistance, or other magnetic properties. It is common to use magnetic nano or microparticles. In the surface of such particles are the bioreceptors, that can be DNA (complementary to a sequence or aptamers) antibodies, or others. The binding of the bioreceptor will affect some of the magnetic particle properties that can be measured by AC susceptometry, a Hall Effect sensor, a giant magnetoresistance device, or others.
=== Others ===
Piezoelectric sensors utilise crystals which undergo an elastic deformation when an electrical potential is applied to them. An alternating potential (A.C.) produces a standing wave in the crystal at a characteristic frequency. This frequency is highly dependent on the elastic properties of the crystal, such that if a crystal is coated with a biological recognition element the binding of a (large) target analyte to a receptor will produce a change in the resonance frequency, which gives a binding signal. In a mode that uses surface acoustic waves (SAW), the sensitivity is greatly increased. This is a specialised application of the quartz crystal microbalance as a biosensor
Electrochemiluminescence (ECL) is nowadays a leading technique in biosensors. Since the excited species are produced with an electrochemical stimulus rather than with a light excitation source, ECL displays improved signal-to-noise ratio compared to photoluminescence, with minimized effects due to light scattering and luminescence background. In particular, coreactant ECL operating in buffered aqueous solution in the region of positive potentials (oxidative-reduction mechanism) definitively boosted ECL for immunoassay, as confirmed by many research applications and, even more, by the presence of important companies which developed commercial hardware for high throughput immunoassays analysis in a market worth billions of dollars each year.
Thermometric biosensors are rare.
== Biosensor MOSFET (BioFET) ==
The MOSFET invented at Bell Labs between 1955 and 1960, Later, Leland C. Clark and Champ Lyons invented the first biosensor in 1962. Biosensor MOSFETs (BioFETs) were later developed, and they have since been widely used to measure physical, chemical, biological and environmental parameters.
The first BioFET was the ion-sensitive field-effect transistor (ISFET), invented by Piet Bergveld for electrochemical and biological applications in 1970. the adsorption FET (ADFET) was patented by P.F. Cox in 1974, and a hydrogen-sensitive MOSFET was demonstrated by I. Lundstrom, M.S. Shivaraman, C.S. Svenson and L. Lundkvist in 1975. The ISFET is a special type of MOSFET with a gate at a certain distance, and where the metal gate is replaced by an ion-sensitive membrane, electrolyte solution and reference electrode. The ISFET is widely used in biomedical applications, such as the detection of DNA hybridization, biomarker detection from blood, antibody detection, glucose measurement, pH sensing, and genetic technology.
By the mid-1980s, other BioFETs had been developed, including the gas sensor FET (GASFET), pressure sensor FET (PRESSFET), chemical field-effect transistor (ChemFET), reference ISFET (REFET), enzyme-modified FET (ENFET) and immunologically modified FET (IMFET). By the early 2000s, BioFETs such as the DNA field-effect transistor (DNAFET), gene-modified FET (GenFET) and cell-potential BioFET (CPFET) had been developed.
== Placement of biosensors ==
The appropriate placement of biosensors depends on their field of application, which may roughly be divided into biotechnology, agriculture, food technology and biomedicine.
In biotechnology, analysis of the chemical composition of cultivation broth can be conducted in-line, on-line, at-line and off-line. As outlined by the US Food and Drug Administration (FDA) the sample is not removed from the process stream for in-line sensors, while it is diverted from the manufacturing process for on-line measurements. For at-line sensors the sample may be removed and analyzed in close proximity to the process stream. An example of the latter is the monitoring of lactose in a dairy processing plant. Off-line biosensors compare to bioanalytical techniques that are not operating in the field, but in the laboratory. These techniques are mainly used in agriculture, food technology and biomedicine.
In medical applications biosensors are generally categorized as in vitro and in vivo systems. An in vitro, biosensor measurement takes place in a test tube, a culture dish, a microtiter plate or elsewhere outside a living organism. The sensor uses a bioreceptor and transducer as outlined above. An example of an in vitro biosensor is an enzyme-conductimetric biosensor for blood glucose monitoring. There is a challenge to create a biosensor that operates by the principle of point-of-care testing, i.e. at the location where the test is needed. Development of wearable biosensors is among such studies. The elimination of lab testing can save time and money. An application of a POCT biosensor can be for the testing of HIV in areas where it is difficult for patients to be tested. A biosensor can be sent directly to the location and a quick and easy test can be used.
An in vivo biosensor is an implantable device that operates inside the body. Of course, biosensor implants have to fulfill the strict regulations on sterilization in order to avoid an initial inflammatory response after implantation. The second concern relates to the long-term biocompatibility, i.e. the unharmful interaction with the body environment during the intended period of use. Another issue that arises is failure. If there is failure, the device must be removed and replaced, causing additional surgery. An example for application of an in vivo biosensor would be the insulin monitoring within the body, which is not available yet.
Most advanced biosensor implants have been developed for the continuous monitoring of glucose. The figure displays a device, for which a Ti casing and a battery as established for cardiovascular implants like pacemakers and defibrillators is used. Its size is determined by the battery as required for a lifetime of one year. Measured glucose data will be transmitted wirelessly out of the body within the MICS 402-405 MHz band as approved for medical implants.
Biosensors can also be integrated into mobile phone systems, making them user-friendly and accessible to a large number of users.
== Applications ==
There are many potential applications of biosensors of various types. The main requirements for a biosensor approach to be valuable in terms of research and commercial applications are the identification of a target molecule, availability of a suitable biological recognition element, and the potential for disposable portable detection systems to be preferred to sensitive laboratory-based techniques in some situations. Some examples are:
glucose monitoring in diabetes patients, other medical health related targets,
environmental applications, e.g. the detection of pesticides, detection and determining of organophosphate, and river water contaminants, such as heavy metal ions,
remote sensing of airborne bacteria, e.g. in counter-bioterrorist activities,
remote sensing of water quality in coastal waters by describing online different aspects of clam ethology (biological rhythms, growth rates, spawning or death records) in groups of abandoned bivalves around the world,
detection of pathogens,
determining levels of toxic substances before and after bioremediation,
routine analytical measurement of folic acid, biotin, vitamin B12 and pantothenic acid as an alternative to microbiological assay,
determination of drug residues in food, such as antibiotics and growth promoters, particularly meat and honey,
drug discovery and evaluation of biological activity of new compounds,
protein engineering in biosensors, and
detection of toxic metabolites such as mycotoxins.
A common example of a commercial biosensor is the blood glucose biosensor, which uses the enzyme glucose oxidase to break blood glucose down. In doing so it first oxidizes glucose and uses two electrons to reduce the FAD (a component of the enzyme) to FADH2. This in turn is oxidized by the electrode in a number of steps. The resulting current is a measure of the concentration of glucose. In this case, the electrode is the transducer and the enzyme is the biologically active component.
A canary in a cage, as used by miners to warn of gas, could be considered a biosensor. Many of today's biosensor applications are similar, in that they use organisms which respond to toxic substances at a much lower concentrations than humans can detect to warn of their presence. Such devices can be used in environmental monitoring, trace gas detection and in water treatment facilities.
=== Glucose monitoring ===
Commercially available glucose monitors rely on amperometric sensing of glucose by means of glucose oxidase, which oxidises glucose producing hydrogen peroxide which is detected by the electrode. To overcome the limitation of amperometric sensors, a flurry of research is present into novel sensing methods, such as fluorescent glucose biosensors.
=== Interferometric reflectance imaging sensor ===
The interferometric reflectance imaging sensor (IRIS) is based on the principles of optical interference and consists of a silicon-silicon oxide substrate, standard optics, and low-powered coherent LEDs. When light is illuminated through a low magnification objective onto the layered silicon-silicon oxide substrate, an interferometric signature is produced. As biomass, which has a similar index of refraction as silicon oxide, accumulates on the substrate surface, a change in the interferometric signature occurs and the change can be correlated to a quantifiable mass. Daaboul et al. used IRIS to yield a label-free sensitivity of approximately 19 ng/mL. Ahn et al. improved the sensitivity of IRIS through a mass tagging technique.
Since initial publication, IRIS has been adapted to perform various functions. First, IRIS integrated a fluorescence imaging capability into the interferometric imaging instrument as a potential way to address fluorescence protein microarray variability. Briefly, the variation in fluorescence microarrays mainly derives from inconsistent protein immobilization on surfaces and may cause misdiagnoses in allergy microarrays. To correct for any variation in protein immobilization, data acquired in the fluorescence modality is then normalized by the data acquired in the label-free modality. IRIS has also been adapted to perform single nanoparticle counting by simply switching the low magnification objective used for label-free biomass quantification to a higher objective magnification. This modality enables size discrimination in complex human biological samples. Monroe et al. used IRIS to quantify protein levels spiked into human whole blood and serum and determined allergen sensitization in characterized human blood samples using zero sample processing. Other practical uses of this device include virus and pathogen detection.
=== Food analysis ===
There are several applications of biosensors in food analysis. In the food industry, optics coated with antibodies are commonly used to detect pathogens and food toxins. Commonly, the light system in these biosensors is fluorescence, since this type of optical measurement can greatly amplify the signal.
A range of immuno- and ligand-binding assays for the detection and measurement of small molecules such as water-soluble vitamins and chemical contaminants (drug residues) such as sulfonamides and Beta-agonists have been developed for use on SPR based sensor systems, often adapted from existing ELISA or other immunological assay. These are in widespread use across the food industry.
=== Detection/monitoring of pollutants ===
Biosensors could be used to monitor air, water, and soil pollutants such as pesticides, potentially carcinogenic, mutagenic, and/or toxic substances and endocrine disrupting chemicals.
For example, bionanotechnologists developed a viable biosensor, ROSALIND 2.0, that can detect levels of diverse water pollutants.
=== Ozone measurement ===
Because ozone filters out harmful ultraviolet radiation, the discovery of holes in the ozone layer of the earth's atmosphere has raised concern about how much ultraviolet light reaches the earth's surface. Of particular concern are the questions of how deeply into sea water ultraviolet radiation penetrates and how it affects marine organisms, especially plankton (floating microorganisms) and viruses that attack plankton. Plankton form the base of the marine food chains and are believed to affect our planet's temperature and weather by uptake of CO2 for photosynthesis.
Deneb Karentz, a researcher at the Laboratory of Radio-biology and Environmental Health (University of California, San Francisco) has devised a simple method for measuring ultraviolet penetration and intensity. Working in the Antarctic Ocean, she submerged to various depths thin plastic bags containing special strains of E. coli that are almost totally unable to repair ultraviolet radiation damage to their DNA. Bacterial death rates in these bags were compared with rates in unexposed control bags of the same organism. The bacterial "biosensors" revealed constant significant ultraviolet damage at depths of 10 m and frequently at 20 and 30 m. Karentz plans additional studies of how ultraviolet may affect seasonal plankton blooms (growth spurts) in the oceans.
=== Metastatic cancer cell detection ===
Metastasis is the spread of cancer from one part of the body to another via either the circulatory system or lymphatic system. Unlike radiology imaging tests (mammograms), which send forms of energy (x-rays, magnetic fields, etc.) through the body to only take interior pictures, biosensors have the potential to directly test the malignant power of the tumor. The combination of a biological and detector element allows for a small sample requirement, a compact design, rapid signals, rapid detection, high selectivity and high sensitivity for the analyte being studied. Compared to the usual radiology imaging tests biosensors have the advantage of not only finding out how far cancer has spread and checking if treatment is effective but also are cheaper, more efficient (in time, cost and productivity) ways to assess metastaticity in early stages of cancer.
Biological engineering researchers have created oncological biosensors for breast cancer. Breast cancer is the leading common cancer among women worldwide. An example would be a transferrin- quartz crystal microbalance (QCM). As a biosensor, quartz crystal microbalances produce oscillations in the frequency of the crystal's standing wave from an alternating potential to detect nano-gram mass changes. These biosensors are specifically designed to interact and have high selectivity for receptors on cell (cancerous and normal) surfaces. Ideally, this provides a quantitative detection of cells with this receptor per surface area instead of a qualitative picture detection given by mammograms.
Seda Atay, a biotechnology researcher at Hacettepe University, experimentally observed this specificity and selectivity between a QCM and MDA-MB 231 breast cells, MCF 7 cells, and starved MDA-MB 231 cells in vitro. With other researchers she devised a method of washing these different metastatic leveled cells over the sensors to measure mass shifts due to different quantities of transferrin receptors. Particularly, the metastatic power of breast cancer cells can be determined by Quartz crystal microbalances with nanoparticles and transferrin that would potentially attach to transferrin receptors on cancer cell surfaces. There is very high selectivity for transferrin receptors because they are over-expressed in cancer cells. If cells have high expression of transferrin receptors, which shows their high metastatic power, they have higher affinity and bind more to the QCM that measures the increase in mass. Depending on the magnitude of the nano-gram mass change, the metastatic power can be determined.
Additionally, in the last years, significant attentions have been focused to detect the biomarkers of lung cancer without biopsy. In this regard, biosensors are very attractive and applicable tools for providing rapid, sensitive, specific, stable, cost-effective and non-invasive detections for early lung cancer diagnosis. Thus, cancer biosensors consisting of specific biorecognition molecules such as antibodies, complementary nucleic acid probes or other immobilized biomolecules on a transducer surface. The biorecognition molecules interact specifically with the biomarkers (targets) and the generated biological responses are converted by the transducer into a measurable analytical signal. Depending on the type of biological response, various transducers are utilized in the fabrication of cancer biosensors such as electrochemical, optical and mass-based transducers.
=== Pathogen detection ===
Biosensors could be used for the detection of pathogenic organisms.
Embedded biosensors for pathogenic signatures – such as of SARS-CoV-2 – that are wearable have been developed – such as face masks with built-in tests. See also: COVID-19 public transport R&D
New types of biosensor-chips could enable novel methods "such as drone-deployed pathogen sensors actively surveying air or wastewater". Protein-binding aptamers could be used for testing for infectious disease pathogens. Systems of electronic skins (or robot skins) with built-in biosensors (or chemical sensors) and human-machine interfaces may enable wearable as well as remote sensed device- or robotic-sensing of pathogens (as well as of several hazardous materials and tactile perceptions).
== Types ==
=== Optical biosensors ===
Many optical biosensors are based on the phenomenon of surface plasmon resonance (SPR) techniques. This utilises a property of gold and other materials (metals); specifically that a thin layer of gold on a high refractive index glass surface can absorb laser light, producing electron waves (surface plasmons) on the gold surface. This occurs only at a specific angle and wavelength of incident light and is highly dependent on the surface of the gold, such that binding of a target analyte to a receptor on the gold surface produces a measurable signal.
Surface plasmon resonance sensors operate using a sensor chip consisting of a plastic cassette supporting a glass plate, one side of which is coated with a microscopic layer of gold. This side contacts the optical detection apparatus of the instrument. The opposite side is then contacted with a microfluidic flow system. The contact with the flow system creates channels across which reagents can be passed in solution. This side of the glass sensor chip can be modified in a number of ways, to allow easy attachment of molecules of interest. Normally it is coated in carboxymethyl dextran or similar compound.
The refractive index at the flow side of the chip surface has a direct influence on the behavior of the light reflected off the gold side. Binding to the flow side of the chip has an effect on the refractive index and in this way biological interactions can be measured to a high degree of sensitivity with some sort of energy. The refractive index of the medium near the surface changes when biomolecules attach to the surface, and the SPR angle varies as a function of this change.
Light of a fixed wavelength is reflected off the gold side of the chip at the angle of total internal reflection, and detected inside the instrument. The angle of incident light is varied in order to match the evanescent wave propagation rate with the propagation rate of the surface plasmon polaritons. This induces the evanescent wave to penetrate through the glass plate and some distance into the liquid flowing over the surface.
Other optical biosensors are mainly based on changes in absorbance or fluorescence of an appropriate indicator compound and do not need a total internal reflection geometry. For example, a fully operational prototype device detecting casein in milk has been fabricated. The device is based on detecting changes in absorption of a gold layer. A widely used research tool, the micro-array, can also be considered a biosensor.
=== Biological biosensors ===
Biological biosensors, also known as optogenetic sensors, often incorporate a genetically modified form of a native protein or enzyme. The protein is configured to detect a specific analyte and the ensuing signal is read by a detection instrument such as a fluorometer or luminometer. An example of a recently developed biosensor is one for detecting cytosolic concentration of the analyte cAMP (cyclic adenosine monophosphate), a second messenger involved in cellular signaling triggered by ligands interacting with receptors on the cell membrane. Similar systems have been created to study cellular responses to native ligands or xenobiotics (toxins or small molecule inhibitors). Such "assays" are commonly used in drug discovery development by pharmaceutical and biotechnology companies. Most cAMP assays in current use require lysis of the cells prior to measurement of cAMP. A live-cell biosensor for cAMP can be used in non-lysed cells with the additional advantage of multiple reads to study the kinetics of receptor response.
Nanobiosensors use an immobilized bioreceptor probe that is selective for target analyte molecules. Nanomaterials are exquisitely sensitive chemical and biological sensors. Nanoscale materials demonstrate unique properties. Their large surface area to volume ratio can achieve rapid and low cost reactions, using a variety of designs.
Other evanescent wave biosensors have been commercialised using waveguides where the propagation constant through the waveguide is changed by the absorption of molecules to the waveguide surface. One such example, dual polarisation interferometry uses a buried waveguide as a reference against which the change in propagation constant is measured. Other configurations such as the Mach–Zehnder have reference arms lithographically defined on a substrate. Higher levels of integration can be achieved using resonator geometries where the resonant frequency of a ring resonator changes when molecules are absorbed.
=== Electronic nose devices ===
Recently, arrays of many different detector molecules have been applied in so called electronic nose devices, where the pattern of response from the detectors is used to fingerprint a substance. In the Wasp Hound odor-detector, the mechanical element is a video camera and the biological element is five parasitic wasps who have been conditioned to swarm in response to the presence of a specific chemical. Current commercial electronic noses, however, do not use biological elements.
=== DNA biosensors ===
DNA can be the analyte of a biosensor, being detected through specific means, but it can also be used as part of a biosensor or, theoretically, even as a whole biosensor.
Many techniques exist to detect DNA, which is usually a means to detect organisms that have that particular DNA. DNA sequences can also be used as described above. But more forward-looking approaches exist, where DNA can be synthesized to hold enzymes in a biological, stable gel. Other applications are the design of aptamers, sequences of DNA that have a specific shape to bind a desired molecule. The most innovative processes use DNA origami for this, creating sequences that fold in a predictable structure that is useful for detection.
Scientists have built prototype sensors to detect DNA of animals from sucked in air, "airborne eDNA".
"Nanoantennas" made out of DNA – a novel type of nano-scale optical antenna – can be attached to proteins and produce a signal via fluorescence when these perform their biological functions, in particular for distinct conformational changes.
=== Graphene-based biosensor ===
Graphene is a two-dimensional carbon-based substance with superior optical, electrical, mechanical, thermal, and mechanical properties. The ability to absorb and immobilize a variety of proteins, particularly some with carbon ring structures, has proven graphene to be an excellent candidate as a biosensor transducer. As a result, various graphene-based biosensors have been explored and developed in recent times.
Graphene has been employed as a biosensor in various formats especially electrochemical sensors and field effect transistors. Amongst them graphene field effect transistors (GFETs) especially have shown excellent performance as rapid point of care (PoC) diagnostics as observed through a surge in number of research articles reporting COVID-19 diagnostics using GFETs. They have been reported to have some of the lowest limit of detection whilst also having a rapid turn around time of a few seconds along with multiplexing abilities. These capabilities allow for immediate disease detection especially in cases with overlapping symptoms which are hard to distinguish at the outset, thus allowing for better patient outcomes especially in resource strained medical settings.
== See also ==
== References ==
== Bibliography ==
Frieder Scheller & Florian Schubert (1989). Biosensoren. Akademie-Verlag, Berlin. ISBN 978-3-05-500659-3.
Massimo Grattarola & Giuseppe Massobrio (1998). Bioelectronics Handbook - MOSFETs, Biosensors and Neurons. McGraw-Hill, New York. ISBN 978-0070031746.
== External links ==
Scratching at the surface of biosensors – an Instant Insight discussing how surface chemistry lets porous silicon biosensors fulfil their promise from the Royal Society of Chemistry | Wikipedia/Biosensor |
Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish Aequorea victoria. Its excitation peak is 513 nm and its emission peak is 527 nm. Like the parent GFP, YFP is a useful tool in cell and molecular biology because the excitation and emission peaks of YFP are distinguishable from GFP which allows for the study of multiple processes/proteins within the same experiment.
Three improved versions of YFP are Citrine, Venus, and Ypet. They have reduced chloride sensitivity, faster maturation, and increased brightness (defined as the product of the extinction coefficient and quantum yield). Typically, YFP serves as the acceptor for genetically-encoded FRET sensors of which the most likely donor FP is monomeric cyan fluorescent protein (mCFP). The red-shift relative to GFP is caused by a Pi-Pi stacking interaction as a result of the T203Y substitution introduced by mutation, which essentially increases the polarizability of the local chromophore environment as well as providing additional electron density into the chromophore.
"Venus" contains a novel amino acid substitution –F46L– which accelerates the oxidation of the chromophore at 37°C, the rate limiting step of maturation. The protein has other substitutions (F64L/ M153T/ V163A/ S175G), permitting Venus to fold well and giving it relative tolerance to acidosis and Cl−.
== Evolution of YFP from GFP ==
Four protein mutations from the wild-type GFP found in Aequorea Victoria jellyfish were needed to create the YFP mutant. The most important mutation was the replacement of threonine with tyrosine at residue position 203 (the substitution is denoted by T203Y, where T and Y represent the single letter code for the amino acids threonine and tyrosine, respectively).
== See also ==
Red fluorescent protein
== References ==
== External links ==
Introduction to fluorescent proteins
EYFP on FPbase
Miyawaki, Atsushi; Llopis, Juan; Heim, Roger; McCaffery, J. Michael; Adams, Joseph A.; Ikura, Mitsuhiko; Tsien, Roger Y. (1997). "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin". Nature. 388 (6645): 882–7. doi:10.1038/42264. PMID 9278050. | Wikipedia/Yellow_fluorescent_protein |
Fisheries science is the academic discipline of managing and understanding fisheries. It is a multidisciplinary science, which draws on the disciplines of limnology, oceanography, freshwater biology, marine biology, meteorology, conservation, ecology, population dynamics, economics, statistics, decision analysis, management, and many others in an attempt to provide an integrated picture of fisheries. In some cases new disciplines have emerged, as in the case of bioeconomics and fisheries law. Because fisheries science is such an all-encompassing field, fisheries scientists often use methods from a broad array of academic disciplines. Over the most recent several decades, there have been declines in fish stocks (populations) in many regions along with increasing concern about the impact of intensive fishing on marine and freshwater biodiversity.
Fisheries science is typically taught in a university setting, and can be the focus of an undergraduate, master's or Ph.D. program. Some universities offer fully integrated programs in fisheries science. Graduates of university fisheries programs typically find employment as scientists, fisheries managers of both recreational and commercial fisheries, researchers, aquaculturists, educators, environmental consultants and planners, conservation officers, and many others.
== Fisheries research ==
Because fisheries take place in a diverse set of aquatic environments (i.e., high seas, coastal areas, large and small rivers, and lakes of all sizes), research requires different sampling equipment, tools, and techniques. For example, studying trout populations inhabiting mountain lakes requires a very different set of sampling tools than, say, studying salmon in the high seas. Ocean fisheries research vessels (FRVs) often require platforms which are capable of towing different types of fishing nets, collecting plankton or water samples from a range of depths, and carrying acoustic fish-finding equipment. Fisheries research vessels are often designed and built along the same lines as a large fishing vessel, but with space given over to laboratories and equipment storage, as opposed to storage of the catch. In addition to a diverse set of sampling gear, fisheries scientists often use scientific techniques from many different professional disciplines.
Other important areas of fisheries research are population dynamics, economics, social studies and genetics.
== Notable contributors ==
Members of this list meet one or more of the following criteria: 1) Author of widely cited peer-reviewed articles on fisheries, 2) Author of major reference work in fisheries, 3) Founder of major fisheries journal, museum or other related organisation 4) Person most notable for other reasons who has also worked in fisheries science.
== Journals ==
Some journals about fisheries are
== Professional societies ==
World Council of Fisheries Societies
American Fisheries Society
The International Council for the Exploration of the Sea (ICES)
The Fisheries Society of the British Isles
The Japanese Society of Fisheries Science
The Australian Society for Fish Biology
== See also ==
Aquaculture – Farming of aquatic organisms
Fisheries management – Regulation of fishing
International Council for the Exploration of the Sea – Intergovernmental science organization
Fisheries Law – Regulations regarding fishing activitiesPages displaying short descriptions of redirect targets
The Fisheries Law Centre
Categories:
Fisheries and aquaculture research institutes
== Notes ==
== References ==
== External links ==
The Sea Ahead... learning from the past. A web site of the Peter Wall Institute for Advanced Studies promoting ecosystem-based fisheries science.
What is fisheries science? (PDF) | Wikipedia/Fisheries_science |
Industrial agriculture is a form of modern farming that refers to the industrialized production of crops and animals and animal products like eggs or milk. The methods of industrial agriculture include innovation in agricultural machinery and farming methods, genetic technology, techniques for achieving economies of scale in production, the creation of new markets for consumption, the application of patent protection to genetic information, and global trade. These methods are widespread in developed nations and increasingly prevalent worldwide. Most of the meat, dairy, eggs, fruits and vegetables available in supermarkets are produced in this way.
== Historical development and future prospects ==
Industrial agriculture arose hand in hand with the Industrial Revolution in general. The identification of nitrogen, potassium and phosphorus (referred to by the acronym NPK) as critical factors in plant growth led to the manufacture of synthetic fertilizers, making possible more intensive types of agriculture. The discovery of vitamins and their role in animal nutrition, in the first two decades of the 20th century, led to vitamin supplements, which in the 1920s allowed certain livestock to be raised indoors, reducing their exposure to adverse natural elements. The discovery of antibiotics and vaccines facilitated raising livestock in concentrated, controlled animal feed operations by reducing diseases caused by crowding. Chemicals developed for use in World War II gave rise to synthetic pesticides. Developments in shipping networks and technology have made long-distance distribution of agricultural produce feasible.
Agricultural production across the world doubled four times between 1820 and 1975 (it doubled between 1820 and 1920; between 1920 and 1950; between 1950 and 1965; and again between 1965 and 1975) to feed a global population of one billion human beings in 1800 and 6.5 billion in 2002.: 29 During the same period, the number of people involved in farming dropped as the process became more automated. In the 1930s, 24 percent of the American population worked in agriculture compared to 1.5 percent in 2002; in 1940, each farm worker supplied 11 consumers, whereas in 2002, each worker supplied 90 consumers.: 29 The number of farms has also decreased, and their ownership is more concentrated. For example, in the 2000s, the price of farmland in the United States increased due to the Midwest farming crisis. The number of small- and medium-scale farming operations decreased due to the increased production and farmland costs. This forced farmers to find alternatives by taking advantage of new products of industrial agriculture such as financialization.
Financialization takes place through the process of ongoing monetization. An example of monetization involves financial institutions expanding and gain authority in the market. Financialization affects all aspects of farm operations, including the structure of the work, the value of it and the social organizations. Farmers turned to land availability in the Brazilian Cerrado through the help of investors and other capital gaining methods needed for financialization. investors wanted to get involved because the investment appears low-risk with high rewards. For example, investors would gain inside information on the market in Brazil. In the article Financialization of work, value, and social organization among transnational soy farmers in the Brazilian Cerrado Ofstehage gives examples of how industrialized farming has evolved into a management model.
A management model entails the structure and rules that ensure work of management is completed. Work is reliant on outsourcing in order to complete labor farming tasks, but is also an essential part in the way management and financial work is completed. Social value system of farming changed when using a management model. Farmers have to take into consideration the division between good and bad farming tactics under the new management model. Many farmers were reluctant to mobilize because of the effect this would have on their family business. The separation between the management styles of farmers comes down to two approaches; farming as a lifestyle versus farming solely for profit. In the Brazilian Cerrado the farming model is strictly based on increased profit margins which dictates decisions involving management and labor related work.
In the U.S., four companies produce 81 percent of cows, 73 percent of sheep, 57 percent of pigs, and produce 50 percent of chickens, cited as an example of "vertical integration" by the president of the U.S. National Farmers' Union. In 1967, there were one million pig farms in America; as of 2002, there were 114,000: 29 with 80 million pigs (out of 95 million) produced each year on factory farms, according to the U.S. National Pork Producers Council.: 29 According to the Worldwatch Institute, 74 percent of the world's poultry, 43 percent of beef and 68 percent of eggs are produced this way.: 26
=== British agricultural revolution ===
The British agricultural revolution describes a period of agricultural development in Britain between the 16th century and the mid-19th century, which saw a massive increase in agricultural productivity and net output. This in turn supported unprecedented population growth, freeing up a significant percentage of the workforce, and thereby helped drive the Industrial Revolution. How this came about is not entirely clear. In recent decades, historians cited four key changes in agricultural practices, enclosure, mechanization, four-field crop rotation and selective breeding, and gave credit to a relatively few individuals.
== Challenges and issues ==
The challenges and issues of industrial agriculture for global and local society, for the industrial agriculture sector, for the individual industrial agriculture farm, and for animal rights include the costs and benefits of both current practices and proposed changes to those practices. This is a continuation of thousands of years of the invention and use of technologies in feeding ever growing populations.
[W]hen hunter-gatherers with growing populations depleted the stocks of game and wild foods across the Near East, they were forced to introduce agriculture. But agriculture brought much longer hours of work and a less rich diet than hunter-gatherers enjoyed. Further population growth among shifting slash-and-burn farmers led to shorter fallow periods, falling yields and soil erosion. Plowing and fertilizers were introduced to deal with these problems—but once again involved longer hours of work and degradation of soil resources(Boserup, The Conditions of Agricultural Growth, Allen and Unwin, 1965, expanded and updated in Population and Technology, Blackwell, 1980.).
While the point of industrial agriculture is lower cost products to create greater productivity thus a higher standard of living as measured by available goods and services, industrial methods have side effects both good and bad. Further, industrial agriculture is not some single indivisible thing, but instead is composed of numerous separate elements, each of which can be modified, and in fact is modified in response to market conditions, government regulation and scientific advances. So the question then becomes for each specific element that goes into an industrial agriculture method or technique or process: What bad side effects are bad enough that the financial gain and good side effects are outweighed? Different interest groups not only reach different conclusions on this, but also recommend differing solutions, which then become factors in changing both market conditions and government regulations.
=== Society ===
The major challenges and issues faced by society concerning industrial agriculture include:
Maximizing the benefits:
Cheap and abundant food
Convenience for the consumer
The contribution to our economy on many levels, from growers to harvesters to processors to sellers
while minimizing the downsides:
Environmental and social costs
Antibiotic resistance
Damage to fisheries
Cleanup of surface and groundwater polluted with animal waste
Increased health risks from pesticides
Increased ozone pollution via methane byproducts of animals
Global warming from heavy use of fossil fuels
==== Benefits ====
An example of industrial agriculture providing cheap and plentiful food is the U.S.'s "most successful program of agricultural development of any country in the world". Between 1930 and 2000 U.S. agricultural productivity (output divided by all inputs) rose by an average of about 2 percent annually causing food prices paid by consumers to decrease. "The percentage of U.S. disposable income spent on food prepared at home decreased, from 22 percent as late as 1950 to 7 percent by the end of the century."
==== Liabilities ====
===== Economic =====
Economic liabilities for industrial agriculture include the dependence on finite non-renewable fossil fuel energy resources, as an input in farm mechanization (equipment, machinery), for food processing and transportation, and as an input in agricultural chemicals. A future increase in energy prices as projected by the International Energy Agency is therefore expected to result in increase in food prices; and there is therefore a need to 'de-couple' non-renewable energy usage from agricultural production. Other liabilities include peak phosphate as finite phosphate reserves are currently a key input into chemical fertilizer for industrial agriculture.
===== Environment =====
Industrial agriculture uses huge amounts of water, energy, and industrial chemicals; increasing pollution in the arable land, usable water and atmosphere. Herbicides, insecticides, fertilizers and animal waste products are accumulating in ground and surface waters. "Many of the negative effects of industrial agriculture are remote from fields and farms. Nitrogen compounds from the Midwest, for example, travel down the Mississippi to degrade coastal fisheries in the Gulf of Mexico. But other adverse effects are showing up within agricultural production systems—for example, the rapidly developing resistance among pests is rendering our arsenal of herbicides and insecticides increasingly ineffective.".
Chemicals used in industrial agriculture, as well as the practice of monoculture, have also been implicated in Colony Collapse Disorder which has led to a collapse in bee populations. Agricultural production is highly dependent on bee pollination to pollinate many varieties of plants, fruits and vegetables.
===== Social =====
A study done for the U.S. Office of Technology Assessment conducted by the UC Davis Macrosocial Accounting Project
concluded that industrial agriculture is associated with substantial deterioration of human living conditions in nearby rural communities.
Future increase in food commodity prices, driven by the energy price rises under peak oil and dependency of industrial agriculture on fossil fuels is expected to lead to increase in food prices which has particular impacts on poor people. An example of this can be seen in the 2007–2008 world food price crisis. Food price increases have a disproportionate impact on the poor as they spend a large proportion of their income on food.
===== Vulnerability against shocks =====
Industrial agriculture is very reliant on a steady stream of inputs like fertilizers and pesticides. If this supply of inputs would be disrupted by conflict or large catastrophes, this would decrease yields in industrial agriculture considerably. It has been estimated that this could result in a drop of 35-48 % for agricultural yields globally, and up to 75 % in highly industrialized areas like Central Europe.
== Animals ==
"Concentrated animal feeding operations" or "intensive livestock operations", can hold large numbers (some up to hundreds of thousands) of animals, often indoors. These animals are typically cows, hogs, turkeys, or chickens. The distinctive characteristics of such farms is the concentration of livestock in a given space. The aim of the operation is to produce as much meat, eggs, or milk at the lowest possible cost and with the greatest level of food safety.
Food and water are supplied in place, and artificial methods are often employed to maintain animal health and improve production, such as therapeutic use of antimicrobial agents, vitamin supplements and growth hormones. Growth hormones are not used in chicken meat production nor are they used in the European Union for any animal. In meat production, methods are also sometimes employed to control undesirable behaviours often related to stresses of being confined in restricted areas with other animals. More docile breeds are sought (with natural dominant behaviours bred out for example), physical restraints to stop interaction, such as individual cages for chickens, or animals physically modified, such as the de-beaking of chickens to reduce the harm of fighting. Weight gain is encouraged by the provision of plentiful supplies of food to animals breed for weight gain.
The designation "confined animal feeding operation" in the U.S. resulted from that country's 1972 Federal Clean Water Act, which was enacted to protect and restore lakes and rivers to a "fishable, swimmable" quality. The United States Environmental Protection Agency (EPA) identified certain animal feeding operations, along with many other types of industry, as point source polluters of groundwater. These operations were designated as CAFOs and subject to special anti-pollution regulation.
In 17 states in the U.S., isolated cases of groundwater contamination has been linked to CAFOs. For example, the ten million hogs in North Carolina generate 19 million tons of waste per year. The U.S. federal government acknowledges the waste disposal issue and requires that animal waste be stored in lagoons. These lagoons can be as large as 7.5 acres (30,000 m2). Lagoons not protected with an impermeable liner can leak waste into groundwater under some conditions, as can runoff from manure spread back onto fields as fertilizer in the case of an unforeseen heavy rainfall. A lagoon that burst in 1995 released 25 million gallons of nitrous sludge in North Carolina's New River. The spill allegedly killed eight to ten million fish.
The large concentration of animals, animal waste and dead animals in a small space poses ethical issues to some consumers. Animal rights and animal welfare activists have charged that intensive animal rearing is cruel to animals. As they become more common, so do concerns about air pollution and ground water contamination, and the effects on human health of the pollution and the use of antibiotics and growth hormones.
According to the U.S. Centers for Disease Control and Prevention (CDC), farms on which animals are intensively reared can cause adverse health reactions in farm workers. Workers may develop acute and chronic lung disease, musculoskeletal injuries, and may catch infections that transmit from animals to human beings. These type of transmissions, however, are extremely rare, as zoonotic diseases are uncommon.
== Crops ==
The projects within the Green Revolution spread technologies that had already existed, but had not been widely used outside of industrialized nations. These technologies included pesticides, irrigation projects and synthetic nitrogen fertilizer.
The novel technological development of the Green Revolution was the production of what some referred to as "miracle seeds." Scientists created strains of maize, wheat and rice that are generally referred to as HYVs or "high-yielding varieties." HYVs have an increased nitrogen-absorbing potential compared to other varieties. Since cereals that absorbed extra nitrogen would typically lodge, or fall over before harvest, semi-dwarfing genes were bred into their genomes. Norin 10 wheat, a variety developed by Orville Vogel from Japanese dwarf wheat varieties, was instrumental in developing Green Revolution wheat cultivars. IR8, the first widely implemented HYV rice to be developed by the International Rice Research Institute, was created through a cross between an Indonesian variety named "Peta" and a Chinese variety named "Dee Geo Woo Gen."
With the availability of molecular genetics in Arabidopsis and rice the mutant genes responsible (reduced height(rh), gibberellin insensitive (gai1) and slender rice (slr1)) have been cloned and identified as cellular signaling components of gibberellic acid, a phytohormone involved in regulating stem growth via its effect on cell division. Stem growth in the mutant background is significantly reduced leading to the dwarf phenotype. Photosynthetic investment in the stem is reduced dramatically as the shorter plants are inherently more stable mechanically. Assimilates become redirected to grain production, amplifying in particular the effect of chemical fertilizers on commercial yield.
HYVs significantly outperform traditional varieties in the presence of adequate irrigation, pesticides and fertilizers. In the absence of these inputs, traditional varieties may outperform HYVs. One criticism of HYVs is that they were developed as F1 hybrids, meaning they need to be purchased by a farmer every season rather than saved from previous seasons, thus increasing a farmer's cost of production.
== Sustainable agriculture ==
The idea and practice of sustainable agriculture has arisen in response to the problems of industrial agriculture. Sustainable agriculture integrates three main goals: environmental stewardship, farm profitability and prosperous farming communities. These goals have been defined by a variety of disciplines and may be looked at from the vantage point of the farmer or the consumer.
=== Organic farming methods ===
Organic farming methods combine some aspects of scientific knowledge and highly limited modern technology with traditional farming practices; accepting some of the methods of industrial agriculture while rejecting others. Organic methods rely on naturally occurring biological processes, which often take place over extended periods of time, and a holistic approach; while chemical-based farming focuses on immediate, isolated effects and reductionist strategies.
Integrated Multi-Trophic Aquaculture is an example of this holistic approach. Integrated Multi-Trophic Aquaculture (IMTA) is a practice in which the by-products (wastes) from one species are recycled to become inputs (fertilizers, food) for another. Fed aquaculture (e.g. fish, shrimp) is combined with inorganic extractive (e.g. seaweed) and organic extractive (e.g. shellfish) aquaculture to create balanced systems for environmental sustainability (bio-mitigation), economic stability (product diversification and risk reduction) and social acceptability (better management practices).
== See also ==
Agrochemical
Agroextractivism
== References == | Wikipedia/Industrial_agriculture |
Crop Science is a bimonthly peer-reviewed scientific journal covering agronomy. It was established in 1961 by founding editor-in-chief H.L. Hamilton and is published by ACSESS (Alliance of Crop, Soil, and Environmental Science Societies) in partnership with Wiley. It is the official journal of the Crop Science Society of America. Since 2013, it is available online only.
Crop Science is the originator of two spin-off journals, The Plant Genome and the Journal of Plant Registrations. The former was published as a supplement to Crop Science from 2006 to 2008, and launched as a separate open access journal later that year. The Journal of Plant Registrations was established as a separate journal in 2007, featuring an expanded format for crop registrations describing newly developed plant varieties, parental lines, germplasms, genetic stocks, and populations.
As with most other professional scientific journals, papers undergo an initial screening by the editor, followed by peer review (where other scientists chosen by the associate editor with expertise in the subject matter will evaluate the paper), before publication. The identity of the reviewers is not known to the authors.
== Abstracting and indexing ==
The journal is abstracted and indexed in:
According to the Journal Citation Reports, the journal has a 2020 impact factor of 2.319.
== References == | Wikipedia/Crop_Science_(journal) |
The following outline is provided as an overview of and topical guide to agriculture:
Agriculture – cultivation of animals, plants, fungi and other life forms for food, fiber, and other products used to sustain life.
== What type of thing is agriculture? ==
Agriculture can be described as all of the following:
A type of work
An academic discipline
A science
An applied science
An industry
=== Agricultural activities ===
Agricultural cycle – annual cycle of activities related to the growth and harvest of a crop.
Land use – management and modification of natural environment or wilderness into built environment such as fields, pastures, and settlements.
=== Agricultural production ===
Cash crop – agricultural crop which is grown for sale for profit.
Agricultural products
Food – any substance consumed to provide nutritional support for the body.
Natural fibers – class of hair-like materials that are continuous filaments or are in discrete elongated pieces, similar to pieces of thread. They can be spun into filaments, thread, or rope. Natural fibers are made from plant, animal and mineral sources.
Lumber – wood in any of its stages from felling to readiness for use as structural material for construction, or wood pulp for paper production.
Paper – sheet material used for writing on or printing on (or as a non-waterproof container), usually made by draining cellulose fibres from a suspension in water.
Medicine great quantity of herbal and animal parts are used for medicinal purposes worldwide, especially in traditional and naturopathic medicines.
Biofuels wide range of plants and plant products are used for converting to fuels, such as firewood, biodiesel, methane gas etc.
=== Agricultural resources ===
Agricultural land – denotes the land suitable for agricultural production, both crops and livestock. It is one of the main resources in agriculture.
Labor (economics) – measure of the work done by human beings.
Water – chemical substance with the chemical formula H2O.
Agricultural machinery – machinery used in the operation of an agricultural area or farm.
Fertilizers – any organic or inorganic material of natural or synthetic origin (other than liming materials) that is added to a soil to supply one or more plant nutrients essential to the growth of plants.
== Branches of agriculture ==
=== By type of life form produced or harvested ===
Agronomy – science and technology of producing and using plants for food, fuel, feed, fiber, and reclamation.
Organic gardening – science and art of growing fruits, vegetables, flowers, or ornamental plants by following the essential principles of organic agriculture in soil building and conservation, pest management, and heirloom variety preservation.
Animal husbandry – agricultural practice of breeding and raising livestock.
=== By industry ===
==== Aquafarming ====
Aquaculture – farming of aquatic organisms such as fish, crustaceans, molluscs and aquatic plants.
Mariculture – specialized branch of aquaculture involving the cultivation of marine organisms for food and other products in the open ocean, an enclosed section of the ocean, or in tanks, ponds or raceways which are filled with seawater.
==== Farming ====
===== Types of farming =====
Alligator farming – establishment for breeding and raising of crocodilians in order to produce meat, leather, and other goods.
Aquaculture – farming of aquatic organisms such as fish, crustaceans, molluscs and aquatic plants.
Contract farming – agricultural production carried out according to an agreement between a buyer and farmers
Dairy farming – class of agricultural, or an animal husbandry, enterprise, for long-term production of milk, usually from dairy cows but also from goats and sheep, which may be either processed on-site or transported to a dairy factory for processing and eventual retail sale.
Integrated farming – more integrated approach to farming as compared to existing monoculture approaches. It refers to agricultural systems that integrate livestock and crop production and may sometimes be known as Integrated Biosystems.
Orchardry – managing orchards, intentional planting of trees or shrubs that are maintained for food production. Orchards comprise fruit or nut-producing trees which are grown for commercial production.
Organic farming – form of agriculture that relies on techniques such as crop rotation, green manure, compost and biological pest control.
Pig farming –
Poultry farming
Sericulture – silk farming, the rearing of silkworms for the production of raw silk.
Sheep husbandry – specifically dealing with the raising and breeding of domestic sheep.
Viticulture – the cultivation and harvesting of grapes.
===== Farming facilities =====
Crops – non-animal species or variety that is grown to be harvested as food, livestock fodder, fuel or for any other economic purpose.
Orchard – intentional planting of trees or shrubs that is maintained for food production.
Farm – an area of land, together with the buildings on it, that is used for growing crops or raising animals, usually in order to sell them.
Greenhouse – building in which plants are grown.
===== Farming equipment =====
Farm equipment – any kind of machinery used on a farm to help with farming.
Baler – piece of farm machinery used to compress a cut and raked crop (such as hay, cotton, straw, or silage) into compact bales that are easy to handle, transport and store.
Combine harvester – or simply combine, is a machine that harvests grain crops.
Farm tractor – vehicle specifically designed to deliver a high tractive effort (or torque) at slow speeds, for the purposes of hauling a trailer or machinery used in agriculture or construction.
Manure spreader used to distribute manure over a field as a fertilizer.
Mower – machine for cutting grass or other plants that grow on the ground. Usually mowing is distinguished from reaping, which uses similar implements, but is the traditional term for harvesting grain crops, e.g. with reapers and combines.
Pickup truck – is a light motor vehicle with an open-top rear cargo area (bed).
Plough – is a tool (or machine) used in farming for initial cultivation of soil in preparation for sowing seed or planting. It has been a basic instrument for most of recorded history, and represents one of the major advances in agriculture.
===== Farming products =====
Livestock – domesticated animals raised in an agricultural setting to produce commodities such as food, fiber and labor. The term "livestock" as used in this article does not include poultry or farmed fish; however the inclusion of these, especially poultry, within the meaning of "livestock" is common.
Cattle – most common type of large domesticated ungulates.
Pigs – any of the animals in the genus Sus.
Poultry – category of domesticated birds kept by humans for the purpose of collecting their eggs, or killing for their meat and/or feathers.
Sheep – are quadrupedal, ruminant mammals typically kept as livestock.
Produce – farm-produced goods, not limited to fruits and vegetables (i.e. meats, grains, oats, etc.).
Grains – grasses (members of the monocot family Poaceae, also known as Gramineae) cultivated for the edible components of their grain (botanically, a type of fruit called a caryopsis), composed of the endosperm, germ, and bran.
Fruits – part of a flowering plant that derives from specific tissues of the flower, mainly one or more ovaries.
Legumes – plant in the family Fabaceae (or Leguminosae), or a fruit of these specific plants. A legume fruit is a simple dry fruit that develops from a simple carpel and usually dehisces (opens along a seam) on two sides.
Nut (fruit)s – hard-shelled indehiscent fruit of some plants. While a wide variety of dried seeds and fruits are called nuts in English, only a certain number of them are considered by biologists to be true nuts.
Vegetables – edible plant or part of a plant, but usually excludes seeds and most sweet fruit. This typically means the leaf, stem, or root of a plant.
===== Farming methods and practices =====
Aeroponics – the process of growing plants in an air or mist environment without the use of soil or an aggregate medium.
Aquaponics – combines aquaculture with hydroponics in a symbiotic environment.
Artificial selection – describes intentional breeding for certain traits, or combination of traits.
Field day (agriculture) – related to a show is the "field day", with elements of a trade show for machinery, equipment and skills required for broadacre farming.
Grazing – a method of feeding in which a herbivore feeds on plants such as grasses.
Hydroponics – a method of growing plants without soil.
Intercropping – practice of growing two or more crops in proximity.
Irrigation – artificial application of water to the land or soil.
Permaculture – theory of ecological design which attempts to develop sustainable human settlements and agricultural systems modeled from natural ecosystems.
Pollination management – horticultural practices that accomplish or enhance pollination of a crop, to improve yield or quality, by understanding of the particular crop's pollination needs, and by knowledgeable management of pollenizers, pollinators, and pollination conditions.
Sustainable agriculture – practice of farming using principles of ecology, the study of relationships between organisms and their environment.
==== Apiculture (Beekeeping) ====
Apiary – place where beehives of honey bees are kept.
Apiology – scientific study of honey bees
Bee – flying insects closely related to wasps and ants, and are known for their role in pollination and for producing honey and beeswax.
Beehive – enclosed structure in which some honey bee species of the subgenus Apis live and raise their young.
Beekeeper – person who keeps honey bees.
Honey – sweet food made by bees using nectar from flowers.
==== Fishery ====
Fishing – activity of trying to catch fish. Fish are normally caught in the wild. Techniques for catching fish include hand gathering, spearing, netting, angling and trapping.
Fishery – facility engaged in raising or harvesting fish
==== Forestry ====
Forestry – interdisciplinary profession embracing the science, art, and craft of creating, managing, using, and conserving forests and associated resources in a sustainable manner to meet desired goals, needs, and values for human benefit.
Agroforestry – integrated approach of using the interactive benefits from combining trees and shrubs with crops and/or livestock.
Analog forestry – system of planned, managed forests, primarily employed in tropical or subtropical areas.
Forest gardening – low-maintenance organic plant-based food production and agroforestry system based on woodland ecosystems, incorporating fruit and nut trees, shrubs, herbs, vines and perennial vegetables which have yields directly useful to humans.
Forest farming – agroforestry practice characterized by the four "I's"- Intentional, Integrated, Intensive and Interactive management of an existing forested ecosystem wherein forest health is of paramount concern. Moreover, agriculture means cultivation of crops and rearing of animals, processing of farm produce and selling.
==== Ranching ====
Ranching – practice of raising grazing livestock such as cattle or sheep for meat or wool.
=== Climate-based agriculture ===
Arid-zone agriculture – agriculture practiced in desert areas of any sort.
Greenhouse gas emissions from agriculture
Tropical agriculture – agriculture practiced in the tropics.
=== Agricultural Disciplines ===
==== Agricultural chemistry ====
Agricultural chemistry – study of both chemistry and biochemistry which are important in agricultural production, the processing of raw products into foods and beverages, and in environmental monitoring and remediation.
==== Agricultural communication ====
Agricultural communication – field of study and work that focuses on communication about agricultural related information among agricultural stakeholders and between agricultural and non-agricultural stakeholders.
==== Agricultural economics ====
Agricultural economics – originally applied the principles of economics to the production of crops and livestock – a discipline known as agronomics. Agronomics was a branch of economics that specifically dealt with land usage. It focused on maximizing the crop yield while maintaining a good soil ecosystem. Throughout the 20th century the discipline expanded and the current scope of the discipline is much broader. Agricultural economics today includes a variety of applied areas, having considerable overlap with conventional economics.
Agrarian system – the economic and technological factors that affect agricultural practices.
Agribusiness – the various businesses involved in food production, including farming and contract farming, seed supply, agrichemicals, farm machinery, wholesale and distribution, processing, marketing, and retail sales.
Agricultural extension – once known as the application of scientific research and new knowledge to agricultural practices through farmer education. The field of extension now encompasses a wider range of communication and learning activities organised for rural people by professionals from different disciplines, including agriculture, agricultural marketing, health, and business studies.
Agricultural marketing – covers the services involved in moving an agricultural product from the farm to the consumer. This may include transferring of agricultural products either directly or indirectly through middleman to consumers.
Custom harvesting – business of harvesting of crops for others. Custom harvesters usually own their own combines and work for the same farms every harvest season. Custom harvesting relieves farmers from having to invest capital in expensive equipment while at the same time maximizing the machinery's use.
Economic development – sustained, concerted actions of policymakers and communities that promote the standard of living and economic health of a specific area.
Rural Community Development – range of approaches and activities which aim to improve the welfare and livelihoods of people which live in rural area and through improving activities in rural areas it helps to maintain a population balance by reducing rural to urban migration.
==== Agricultural education ====
Agricultural education – instruction about crop production, livestock management, soil and water conservation, and various other aspects of agriculture. Farmers acquire adequate knowledge required on the correct amount use of agrochemicals and other agriculture related technologies.
Agricultural universities and colleges – tertiary agricultural educational institutions around the world
Agricultural universities in India
Agricultural universities in Indonesia
==== Agricultural engineering ====
Agricultural engineering – engineering discipline that applies engineering science and technology to agricultural production and processing.
Agricultural Machinery – machinery used in the operation of an agricultural area or farm.
Bioprocess engineering – specialization of biotechnology, chemical engineering or of agricultural engineering. It deals with the design and development of equipment and processes for the manufacturing of products such as food, feed, pharmaceuticals, nutraceuticals, chemicals, and polymers and paper from biological materials.
Electrical energy efficiency on United States farms – covers the use of electricity on farms and the methods and incentives for improving the efficiency of that use.
Electronics – branch of physics, engineering and technology dealing with electrical circuits that involve active electrical components such as vacuum tubes, transistors, diodes and integrated circuits, and associated passive interconnection technologies.
Energy – ability a physical system has to do work on other physical systems.
Farm equipment – any kind of machinery used on a farm to help with farming.
Food engineering – multidisciplinary field of applied physical sciences which combines science, microbiology, and engineering education for food and related industries.
Irrigation and drainage engineering –
Natural resource – occur naturally within environments that exist relatively undisturbed by mankind, in a natural form. A natural resource is often characterized by amounts of biodiversity and geodiversity existent in various ecosystems.
System engineering – interdisciplinary field of engineering focusing on how complex engineering projects should be designed and managed over their life cycles.
Workshop – room or building which provides both the area and tools (or machinery) that may be required for the manufacture or repair of goods.
Structures – buildings
==== Agricultural philosophy ====
Agricultural philosophy – discipline devoted to the systematic critique of the philosophical frameworks (or ethical world views) that are the foundation for decisions regarding agriculture.
==== Agricultural policy ====
Agricultural policy – set of laws relating to domestic agriculture and imports of foreign agricultural products.
Agricultural science – broad multidisciplinary field that encompasses the parts of exact, natural, economic and social sciences that are used in the practice and understanding of agriculture.
Agricultural economics – originally applied the principles of economics to the production of crops and livestock – a discipline known as agronomics. Agronomics was a branch of economics that specifically dealt with land usage. It focused on maximizing the crop yield while maintaining a good soil ecosystem. Throughout the 20th century the discipline expanded and the current scope of the discipline is much broader. Agricultural economics today includes a variety of applied areas, having considerable overlap with conventional economics.
Agricultural engineering – engineering discipline that applies engineering science and technology to agricultural production and processing.
Agricultural philosophy – discipline devoted to the systematic critique of the philosophical frameworks (or ethical world views) that are the foundation for decisions regarding agriculture.
Agrophysics – branch of science bordering on agronomy and physics, whose objects of study are the agroecosystem – the biological objects, biotope and biocoenosis affected by human activity, studied and described using the methods of physical sciences.
Animal science – studying the biology of animals that are under the control of mankind.
Animal breeding – branch of animal science that addresses the evaluation (using best linear unbiased prediction and other methods) of the genetic value (estimated breeding value, EBV) of domestic livestock.
Animal nutrition – focuses on the dietary needs of domesticated animals, primarily those in agriculture and food production.
Fisheries science – academic discipline of managing and understanding fisheries.
Poultry science – animal science applied to poultry – chickens, ducks, geese, turkeys, quail, etc.
Aquaculture – is the farming of aquatic organisms such as fish, crustaceans, molluscs and aquatic plants.
Biological engineering –
Genetic engineering – deliberate modification of the genetic structure of an organism.
Microbiology – branch of biology that deals with microorganisms, especially their effects on man and other living organisms.
Environmental science – integrated study of factors that influence the environment and environmental systems, especially the interaction of the physical, chemical, and biological components of the environment
Conservation – preservation and wise use of resources
Wildlife management – attempts to balance the needs of wildlife with the needs of people using the best available science.
Wildlife range management –
Resources management – efficient and effective deployment of an organization's resources when they are needed.
Food science – study concerned with all technical aspects of foods, beginning with harvesting or slaughtering, and ending with its cooking and consumption, an ideology commonly referred to as "from field to fork". It is considered one of the life sciences and is usually considered distinct from the field of nutrition.
Human nutrition – provision to obtain the materials necessary to support life.
Food technology – branch of food science which deals with the actual production processes to make foods.
===== Agronomy =====
Agronomy – science and technology of producing and using plants for food, fuel, feed, fiber, and reclamation.
Plant science – science of plant life.
Crop science – broad multidisciplinary field that encompasses the parts of exact, natural, economic and social sciences that are used in the practice and understanding of agriculture.
Plant pathology – scientific study of plant diseases caused by pathogens (infectious diseases) and environmental conditions (physiological factors).
Forestry – interdisciplinary profession embracing the science, art, and craft of creating, managing, using, and conserving forests and associated resources in a sustainable manner to meet desired goals, needs, and values for human benefit.
Outline of wood science –
Theoretical production ecology – quantitatively studies the growth of crops.
Horticulture – art, science, technology and business of intensive plant cultivation for human use.
Plant breeding – art and science of changing the genetics of plants in order to produce desired characteristics.
fertilizer – any organic or inorganic material of natural or synthetic origin (other than liming materials) that is added to a soil to supply one or more plant nutrients essential to the growth of plants.
====== Horticulture ======
Horticulture – art, science, technology and business of intensive plant cultivation for human use.
===== Agricultural soil science =====
Agricultural soil science – branch of soil science that deals with the study of edaphic conditions as they relate to the production of food and fiber.
Agrogeology – study of minerals of importance to farming and horticulture, especially with regard to soil fertility and fertilizer components. These minerals are usually essential plant nutrients and are referred to as agrominerals.
Agrology – branch of soil science dealing with the production of crops.
Agrominerals – minerals of importance to agriculture and horticulture, and are usually essential plant nutrients.
Land degradation – process in which the value of the biophysical environment is affected by one or more combination of human-induced processes acting upon the land.
Land improvement – investments making land more usable by humans.
Soil chemistry – study of the chemical characteristics of soil.
Soil amendment – material added to soil to improve plant growth and health.
Soil erosion – process by which soil is removed from the Earth's surface by natural processes such as wind or water flow, and then transported and deposited in other locations.
Soil life – collective term for all the organisms living within the soil.
Soil type – refers to the different sizes of mineral particles in a particular sample.
Soils retrogression and degradation – two regressive evolution processes associated with the loss of equilibrium of a stable soil.
==== Agroecology ====
Agroecology – application of ecological principles to the production of food, fuel, fiber, and pharmaceuticals and the management of agroecosystems.
Agroecosystem analysis – thorough analysis of an agricultural environment which considers aspects from ecology, sociology, economics, and politics with equal weight.
Agrophysics – branch of science bordering on agronomy and physics, whose objects of study are the agroecosystem – the biological objects, biotope and biocoenosis affected by human activity, studied and described using the methods of physical sciences.
Biodiversity – degree of variation of life forms within a given species, ecosystem, biome, or an entire planet.
Effects of climate change on agriculture – interrelated processes, both of which take place on a global scale.
Composting – Compost is organic matter that has been decomposed and recycled as a fertilizer and soil amendment.
Ecology – scientific study of the relations that living organisms have with respect to each other and their natural environment.
Ecosystem – biological system consisting of all the living organisms or biotic components in a particular area and the nonliving or abiotic component with which the organisms interact, such as air, mineral soil, water and sunlight.
Environmental Economics – subfield of economics concerned with environmental issues.
Green manure – type of cover crop grown primarily to add nutrients and organic matter to the soil.
Natural resources – occur naturally within environments that exist relatively undisturbed by mankind, in a natural form.
Recycling – is processing used materials (waste) into new products to prevent waste of potentially useful materials, reduce the consumption of fresh raw materials, reduce energy usage, reduce air pollution (from incineration) and water pollution (from landfilling) by reducing the need for "conventional" waste disposal, and lower greenhouse gas emissions as compared to virgin production.
Rural Sociology – field of sociology associated with the study of social life in non-metropolitan areas.
Soil Science – study of soil as a natural resource on the surface of the earth including soil formation, classification and mapping; physical, chemical, biological, and fertility properties of soils; and these properties in relation to the use and management of soils.
Sustainable agriculture – practice of farming using principles of ecology, the study of relationships between organisms and their environment.
Wildculture – umbrella term used to include all aspects and styles of "hunting and gathering" food harvesting.
== History of agriculture ==
History of agriculture – developed at least 10,000 years ago, although some forms of agriculture such as forest gardening and fire-stick farming date back even earlier to prehistoric times.
Agriculture in ancient Greece
Agriculture in Mesoamerica
Ancient Egyptian agriculture
Arab Agricultural Revolution –
British Agricultural Revolution –
Columbian Exchange
Domestication
Eastern Agricultural Complex
Genomics of domestication – study of the structure, content, and evolution of genomes, or the entire genetic information of organisms.
Green Revolution –
History of agricultural science – began with Gregor Mendel's genetic work
History of agriculture in Palestine
History of organic farming –
Neolithic Revolution – wide-scale transition of many human cultures from a lifestyle of hunting and gathering to agriculture and settlement.
Incan agriculture
Roman agriculture
Selective breeding
== Agriculturally based manufacturing industries ==
=== Food industry ===
Food industry – complex, global collective of diverse businesses that together supply much of the food energy consumed by the world population.
Bakery – establishment which produces and sells flour-based food baked in an oven such as bread, cakes, pastries and pies.
Brewing – production of beer through steeping a starch source (commonly cereal grains) in water and then fermenting with yeast.
Brewing industry – brewery is a dedicated building for the making of beer, though beer can be made at home, and has been for much of beer's history.
Dairy – business enterprise established for the harvesting of animal milk – mostly from cows or goats, but also from buffalo, sheep, horses or camels – for human consumption.
Distribution center – warehouse or other specialized building, often with refrigeration or air conditioning, which is stocked with products (goods) to be redistributed to retailers, to wholesalers, or directly to consumers.
Food processing – set of methods and techniques used to transform raw ingredients into food or to transform food into other forms for consumption by humans or animals either in the home or by the food processing industry.
Food additive – substances added to food to preserve flavor or enhance its taste and appearance.
Food preservation – process of treating and handling food to stop or slow down spoilage (loss of quality, edibility or nutritional value) and thus allow for longer storage.
Food safety – scientific discipline describing handling, preparation, and storage of food in ways that prevent foodborne illness.
Food science – study concerned with all technical aspects of foods, beginning with harvesting or slaughtering, and ending with its cooking and consumption, an ideology commonly referred to as "from field to fork".
Foodborne illness – any illness resulting from the consumption of contaminated food, pathogenic bacteria, viruses, or parasites that contaminate food, as well as chemical or natural toxins such as poisonous mushrooms.
Mandatory labelling – requirement of consumer products to state their ingredients or components.
Packaging – science, art, and technology of enclosing or protecting products for distribution, storage, sale, and use.
Pasteurization – process of heating a food, usually a liquid, to a specific temperature for a definite length of time and then cooling it immediately.
Quality assurance – planned and systematic activities implemented in a quality system so that quality requirements for a product or service will be fulfilled
Sterilization (microbiology) – term referring to any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media.
Warehouse – commercial building for storage of goods.
Yeast – eukaryotic micro-organisms classified in the kingdom fungi, with 1,500 species currently described
=== Pulp and paper industry ===
Pulp and paper industry – comprises companies that use wood as raw material and produce pulp, paper, board and other cellulose-based products.
== Agricultural markets ==
=== Food distribution ===
Agricultural marketing – services involved in moving an agricultural product from the farm to the consumer.
Wholesale marketing – transactions made through wholesale markets
=== Food outlets ===
Supermarket – self-service store offering a wide variety of food and household merchandise, organized into departments.
Farmers' market – consists of individual vendors—mostly farmers—who set up booths, tables or stands, outdoors or indoors, to sell produce, meat products, fruits and sometimes prepared foods and beverages.
Grocery store – store that retails food.
Street market – outdoor market such as traditionally held in a market square or in a market town, and often held only on particular days of the week.
== Prominent agricultural scientists ==
Robert Bakewell (farmer) – first to implement systematic selective breeding of livestock.
Norman Borlaug – American agronomist, humanitarian, and Nobel laureate who has been called "the father of the Green Revolution".
Luther Burbank – American botanist, horticulturist and am pioneer in agricultural science. He developed more than 800 strains and varieties of plants over his 55-year career.
George Washington Carver – American scientist, botanist, educator, and inventor. Carver's reputation is based on his research into and promotion of alternative crops to cotton, such as peanuts, soybeans and sweet potatoes, which also aided nutrition for farm families.
René Dumont – French engineer in agronomy, a sociologist, and an environmental politician.
Charles Roy Henderson – statistician and a pioneer in animal breeding – the application of quantitative methods for the genetic evaluation of domestic livestock.
Ronald Fisher – English statistician, evolutionary biologist, eugenicist and geneticist.
Jay Lush – pioneering animal geneticist who made important contributions to livestock breeding. He is sometimes known as the father of modern scientific animal breeding.
Gregor Mendel – Austrian scientist and Augustinian friar who gained posthumous fame as the founder of the new science of genetics. Mendel demonstrated that the inheritance of certain traits in pea plants follows particular patterns, now referred to as the laws of Mendelian inheritance.
Louis Pasteur – French chemist and microbiologist born in Dole. He was best known to the general public for inventing a method to stop milk and wine from causing sickness, a process that came to be called pasteurization.
M. S. Swaminathan – Indian agricultural scientist. Swaminathan is known as the "Father of the Green Revolution in India", for his leadership and success in introducing and further developing high-yielding varieties of wheat in India.
== See also ==
Agribusiness –
Extensive farming –
Factory farming –
Free range –
Industrial agriculture –
Mechanised agriculture –
Intensive farming –
Stock-free agriculture –
Subsistence agriculture –
Urban agriculture –
Lists
Largest producing countries of agricultural commodities
List of agricultural organizations
List of agricultural universities and colleges
List of sustainable agriculture topics
== References ==
== External links ==
Free Federal Resources for Educational Excellence – Agriculture
UKAgriculture.com – Advance the education of the public in all aspects of agriculture, the countryside and the rural economy
National Institute for Occupational Safety and Health – Agriculture Page
Research on the role of Agriculture in Poverty Reduction from the Overseas Development Institute | Wikipedia/Agricultural_sciences_basic_topics |
Agricultural science (or agriscience for short) is a broad multidisciplinary field of biology that encompasses the parts of exact, natural, economic and social sciences that are used in the practice and understanding of agriculture. Professionals of the agricultural science are called agricultural scientists or agriculturists.
== History ==
In the 18th century, Johann Friedrich Mayer conducted experiments on the use of gypsum (hydrated calcium sulfate) as a fertilizer.
In 1843, John Bennet Lawes and Joseph Henry Gilbert began a set of long-term field experiments at Rothamsted Research in England, some of which are still running as of 2018.
In the United States, a scientific revolution in agriculture began with the Hatch Act of 1887, which used the term "agricultural science". The Hatch Act was driven by farmers' interest in knowing the constituents of early artificial fertilizer. The Smith–Hughes Act of 1917 shifted agricultural education back to its vocational roots, but the scientific foundation had been built. For the next 44 years after 1906, federal expenditures on agricultural research in the United States outpaced private expenditures.: xxi
== Prominent agricultural scientists ==
Wilbur Olin Atwater
Robert Bakewell
Norman Borlaug
Luther Burbank
George Washington Carver
Carl Henry Clerk
George C. Clerk
René Dumont
Sir Albert Howard
Kailas Nath Kaul
Thomas Lecky
Justus von Liebig
Jay Laurence Lush
Gregor Mendel
Louis Pasteur
M. S. Swaminathan
Jethro Tull
Artturi Ilmari Virtanen
Sewall Wright
== Fields or related disciplines ==
== Scope ==
Agriculture, agricultural science, and agronomy are closely related. However, they cover different concepts:
Agriculture is the set of activities that transform the environment for the production of animals and plants for human use. Agriculture concerns techniques, including the application of agronomic research.
Agronomy is research and development related to studying and improving plant-based crops.
Geoponics is the science of cultivating the earth.
Hydroponics involves growing plants without soil, by using water-based mineral nutrient solutions in an artificial environment.
== Research topics ==
Agricultural sciences include research and development on:
Improving agricultural productivity in terms of quantity and quality (e.g., selection of drought-resistant crops and animals, development of new pesticides, yield-sensing technologies, simulation models of crop growth, in-vitro cell culture techniques)
Minimizing the effects of pests (weeds, insects, pathogens, mollusks, nematodes) on crop or animal production systems.
Transformation of primary products into end-consumer products (e.g., production, preservation, and packaging of dairy products)
Prevention and correction of adverse environmental effects (e.g., soil degradation, waste management, bioremediation)
Theoretical production ecology, relating to crop production modeling
Traditional agricultural systems, sometimes termed subsistence agriculture, which feed most of the poorest people in the world. These systems are of interest as they sometimes retain a level of integration with natural ecological systems greater than that of industrial agriculture, which may be more sustainable than some modern agricultural systems.
Food production and demand globally, with particular attention paid to the primary producers, such as China, India, Brazil, the US, and the EU.
Various sciences relating to agricultural resources and the environment (e.g. soil science, agroclimatology); biology of agricultural crops and animals (e.g. crop science, animal science and their included sciences, e.g. ruminant nutrition, farm animal welfare); such fields as agricultural economics and rural sociology; various disciplines encompassed in agricultural engineering.
== See also ==
Agricultural Research Council
Agricultural sciences basic topics
Agriculture ministry
Agroecology
American Society of Agronomy
Consultative Group on International Agricultural Research (CGIAR)
Crop Science Society of America
Genomics of domestication
History of agricultural science
Indian Council of Agricultural Research
Institute of Food and Agricultural Sciences
International Assessment of Agricultural Science and Technology for Development
International Food Policy Research Institute, IFPRI
International Institute of Tropical Agriculture
International Livestock Research Institute
List of agriculture topics
National Agricultural Library (NAL)
National FFA Organization
Research Institute of Crop Production (RICP) (in the Czech Republic)
Soil Science Society of America
USDA Agricultural Research Service
University of Agricultural Sciences
== References ==
== Further reading ==
Agricultural Research, Livelihoods, and Poverty: Studies of Economic and Social Impacts in Six Countries Edited by Michelle Adato and Ruth Meinzen-Dick (2007), Johns Hopkins University Press Food Policy Report
Claude Bourguignon, Regenerating the Soil: From Agronomy to Agrology, Other India Press, 2005
Pimentel David, Pimentel Marcia, Computer les kilocalories, Cérès, n. 59, sept-oct. 1977
Russell E. Walter, Soil conditions and plant growth, Longman group, London, New York 1973
Salamini, Francesco; Özkan, Hakan; Brandolini, Andrea; Schäfer-Pregl, Ralf; Martin, William (2002). "Genetics and geography of wild cereal domestication in the near east". Nature Reviews Genetics. 3 (6): 429–441. doi:10.1038/nrg817. PMID 12042770. S2CID 25166879.
Saltini Antonio, Storia delle scienze agrarie, 4 vols, Bologna 1984–89, ISBN 88-206-2412-5, ISBN 88-206-2413-3, ISBN 88-206-2414-1, ISBN 88-206-2415-X
Vavilov Nicolai I. (Starr Chester K. editor), The Origin, Variation, Immunity and Breeding of Cultivated Plants. Selected Writings, in Chronica botanica, 13: 1–6, Waltham, Mass., 1949–50
Vavilov Nicolai I., World Resources of Cereals, Leguminous Seed Crops and Flax, Academy of Sciences of Urss, National Science Foundation, Washington, Israel Program for Scientific Translations, Jerusalem 1960
Winogradsky Serge, Microbiologie du sol. Problèmes et methodes. Cinquante ans de recherches, Masson & c.ie, Paris 1949 | Wikipedia/Crop_Science |
Cultural methods are agriculture practices used to enhance crop and livestock health and prevent weed, pest or disease problems without the use of chemical substances. Examples include the selection of appropriate varieties and planting sites; selection of appropriate breeds of livestock; providing livestock facilities designed to meet requirements of species or type of livestock; proper timing and density of plantings; irrigation; and extending a growing season by manipulating the microclimate with green houses, cold frames, or wind breaks. Helps in proper farming methods
== History ==
In the period preceding chemical fertilizer and pesticide use, agriculture played an important part in society. Agricultural output represented the strength of a country, considered directly proportional to its military power and the produce available to support the army in sustained operation. Critical to resources, soil fertility is critical to a successful agricultural economy.
Cultural methods were divided into active composting, fertilizing, and slash and burn farming. Farmers practiced letting their land rest and allowing the wild vegetation to restore the soil.In densely populated areas, fields are fertilized with green manure, organic waste from different sources, kitchen waste and ashes. In sparsely populated areas, a slash and burn strategy created greater labor demands.One extension of active composting is the addition of charcoal and terra cotta bits; see Terra preta.
Chemical fertilizer and pesticides: Chemical fertilizer and pesticides became available and the practice of improper tillage ushered in a period of lesser quality farming practices.
Industrial Agri-business/Enterprise: With the success of the introduction of chemicals and mechanized farm operations, farms became larger and farmers equated chemicals and machines as a substitute for labor input. Farms worker numbers decreased dramatically. (e.g. from 48% of the population to about 2% in the 20th century.) The soil was depleted by imbalanced fertilizing and productivity was reduced further by improper tillage. Weeds are not permitted to grow, therefore inhibiting the buffer available in the subsoil.
Organic movement: A movement towards organic farming that sees chemically-based production as adverse towards soil health.
== See also ==
History of agriculture
Push–pull technology
== References ==
This article incorporates public domain material from Jasper Womach. Report for Congress: Agriculture: A Glossary of Terms, Programs, and Laws, 2005 Edition (PDF). Congressional Research Service. | Wikipedia/Cultural_methods |
Bayer AG (English: , commonly pronounced ; German: [ˈbaɪɐ]) is a German multinational pharmaceutical and biotechnology company and is one of the largest pharmaceutical companies and biomedical companies in the world. Headquartered in Leverkusen, Bayer's areas of business include: pharmaceuticals, consumer healthcare products, agricultural chemicals, seeds and biotechnology products. The company is a component of the EURO STOXX 50 stock market index.
Bayer was founded in 1863 in Barmen as a partnership between dye salesman Friedrich Bayer (1825–1880) and dyer Friedrich Weskott (1821–1876). The company was established as a dyestuffs producer, but the versatility of aniline chemistry led Bayer to expand its business into other areas. In 1899, Bayer launched the compound acetylsalicylic acid under the trademarked name Aspirin. Aspirin is on the World Health Organization's List of Essential Medicines. In 2021, it was the 34th most commonly prescribed medication in the United States, with more than 17 million prescriptions.
In 1904, Bayer received a trademark for the "Bayer Cross" logo, which was subsequently stamped onto each aspirin tablet, creating an iconic product that is still sold by Bayer. Other commonly known products initially commercialized by Bayer include heroin, phenobarbital, polyurethanes, and polycarbonates.
In 1925, Bayer merged with five other German companies to form IG Farben, creating the world's largest chemical and pharmaceutical company. The first sulfonamide and the first systemically active antibacterial drug, forerunner of antibiotics, Prontosil, was developed by a research team led by Gerhard Domagk in 1932 or 1933 at the Bayer Laboratories. Following World War II, the Allied Control Council seized IG Farben's assets because of its role in the Nazi war effort and involvement in the Holocaust, including using slave labour from concentration camps and humans for dangerous medical testing, and production of Zyklon B, a chemical used in gas chambers. In 1951, IG Farben was split into its constituent companies, and Bayer was reincorporated as Farbenfabriken Bayer AG. After the war, Bayer re-hired several former Nazis to high-level positions, including convicted Nazi war criminals found guilty at the IG Farben Trial like Fritz ter Meer. Bayer played a key role in the Wirtschaftswunder in post-war West Germany, quickly regaining its position as one of the world's largest chemical and pharmaceutical corporations.
In 2016, Bayer merged with the American multinational Monsanto in what was the biggest acquisition by a German company to date. However, owing to the massive financial and reputational blows caused by ongoing litigation concerning Monsanto's herbicide Roundup, the deal is considered one of the worst corporate mergers in history.
Bayer owns the Bundesliga football club Bayer 04 Leverkusen.
== Early history ==
=== Foundation ===
Bayer AG was founded as a dyestuffs factory in 1863 in Barmen (later part of Wuppertal), Germany, by Friedrich Bayer and his partner, Johann Friedrich Weskott, a master dyer. Bayer was responsible for the commercial tasks. Fuchsine and aniline became the company's most important products.
The headquarters and most production facilities moved from Barmen to a larger area in Elberfeld in 1866. Friedrich Bayer (1851–1920), the son of the company's founder, was a chemist and joined the company in 1873. After the death of his father in 1880, the company became a joint-stock company, Farbenfabriken vorm. Friedr. Bayer & Co, also known as Elberfelder Farbenfabriken.
A further expansion in Elberfeld was impossible, so the company moved to the village Wiesdorf at Rhein and settled in the area of the alizarin producer Leverkus and Sons. A new city, Leverkusen, was founded there in 1930 and became home to Bayer AG's headquarters. The company's corporate logo, the Bayer cross, was introduced in 1904, consisting of the word BAYER written vertically and horizontally, sharing the Y and enclosed in a circle. An illuminated version of the logo is a landmark in Leverkusen.
=== Aspirin ===
Bayer's first major product was acetylsalicylic acid—first described by French chemist Charles Frederic Gerhardt in 1853—a modification of salicylic acid or salicin, a folk remedy found in the bark of the willow plant. By 1899, Bayer's trademark Aspirin was registered worldwide for Bayer's brand of acetylsalicylic acid, but it lost its trademark status in the United States, France and the United Kingdom after the confiscation of Bayer's US assets and trademarks during World War I by the United States, and because of the subsequent widespread usage of the word.
The term aspirin continued to be used in the US, UK and France for all brands of the drug, but it is still a registered trademark of Bayer in over 80 countries, including Canada, Mexico, Germany and Switzerland. As of 2011, approximately 40,000 tons of aspirin were produced each year and 10–20 billion tablets consumed in the United States alone for prevention of cardiovascular events. It is on the WHO Model List of Essential Medicines, the most important medications needed in a basic health system.
There is an unresolved controversy over the roles played by Bayer scientists in the development of aspirin. Arthur Eichengrün, a Bayer chemist, said he was the first to discover an aspirin formulation that did not have the unpleasant side effects of nausea and gastric pain. He also said he had invented the name aspirin and was the first person to use the new formulation to test its safety and efficacy. Bayer contends that aspirin was discovered by Felix Hoffmann to help his father, who had arthritis. Various sources support the conflicting claims. Most mainstream historians attribute the invention of aspirin to Hoffmann and/or Eichengrün.
=== Heroin ===
Heroin (diacetylmorphine), now illegal as an addictive drug, was introduced as a non-addictive substitute for morphine, and trademarked and marketed by Bayer from 1898 to 1910 as a cough suppressant and over-the-counter treatment for other common ailments, including pneumonia and tuberculosis. While Bayer scientists were not the first to make heroin, the company did lead the way in commercializing it. Heroin was a Bayer trademark until after World War I. Bayer's director of pharmacology did not want the drug to have "too complicated a name" so Bayer settled on heroisch, the German word for heroic.
=== Phenobarbital ===
In 1903, Bayer licensed the patent for the hypnotic drug diethylbarbituric acid from its inventors Emil Fischer and Joseph von Mering. It was marketed under the trade name Veronal as a sleep aid beginning in 1904. Systematic investigations of the effect of structural changes on potency and duration of action at Bayer led to the discovery of phenobarbital in 1911 and the discovery of its potent anti-epileptic activity in 1912. Phenobarbital was among the most widely used drugs for the treatment of epilepsy through the 1970s, and as of 2014 it remains on the World Health Organization's list of essential medications.
=== World War I ===
During World War I (1914–1918), Bayer's assets, including the rights to its name and trademarks, were confiscated in the United States, Canada and several other countries. In the United States and Canada, Bayer's assets and trademarks, including the well-known Bayer cross, were acquired by Sterling Drug, a predecessor of Sterling Winthrop and were not reclaimed until 1994.
Throughout the war, Bayer was involved in production and development of various chemical weapons. In 1914, Bayer manufactured dianisidine chlorosulfate for use in 105 mm artillery shell, intended as a lung irritant against British forces.
In 1916, Bayer scientists discovered suramin, an anti-parasite drug that is still sold by Bayer under the brand name Germanin. The formula of suramin was kept secret by Bayer for commercial reasons, but it was elucidated and published in 1924 by Ernest Fourneau and his team at the Pasteur Institute. It is on the World Health Organization's List of Essential Medicines.
=== IG Farben ===
In 1925, Bayer became part of IG Farben, a German conglomerate formed from the merger of six chemical companies: BASF, Bayer, Hoechst (including Cassella and Kalle & Co.), Agfa, Chemische Fabrik Griesheim-Elektron, and Chemische Fabrik vorm. Weiler Ter Meer. In the 1930s, Gerhard Domagk, director of Bayer's Institute of Pathology and Bacteriology, working with chemists Fritz Mietzsch and Joseph Klarer, discovered prontosil, the first commercially available antibacterial drug. The discovery and development of this first sulfonamide drug opened a new era in medicine. Domagk won the Nobel Prize in Physiology or Medicine in 1939 "for the discovery of the antibacterial effects of prontosil". He was forced by the Nazi Party to relinquish the reward; German citizens had been forbidden from accepting Nobel prizes since the Nobel committee had awarded the 1935 Nobel Peace Prize to a German pacifist, Carl von Ossietzky.
=== World War II and the Holocaust ===
IG Farben, Bayer's parent company, used slave labour in factories it built in Nazi concentration camps, most notably in the Monowitz concentration camp (known as Auschwitz III), part of the Auschwitz camp complex in German-occupied Poland. By 1943, almost half of IG Farben's 330,000-strong workforce consisted of slave labour or conscripts, including 30,000 Auschwitz prisoners.
Helmuth Vetter, an Auschwitz camp physician, SS captain and employee of the Bayer group within IG Farben conducted medical experiments on inmates at Auschwitz and at the Mauthausen concentration camp. In one study of an anaesthetic, the company paid RM 170 per person for the use of 150 female inmates of Auschwitz. A Bayer employee wrote to Rudolf Höss, the Auschwitz commandant: "The transport of 150 women arrived in good condition. However, we were unable to obtain conclusive results because they died during the experiments. We would kindly request that you send us another group of women to the same number and at the same price."
After the war, the Allied Control Council seized IG Farben for "knowingly and prominently ... building up and maintaining German war potential". It was split into its six constituent companies in 1951, then split again into three: BASF, Bayer and Hoechst. Bayer was at that point known as Farbenfabriken Bayer AG; it changed its name to Bayer AG in 1972. After the war, some employees of Bayer appeared in the IG Farben Trial, one of the Nuremberg Subsequent Tribunals under US jurisdiction. Among them was Fritz ter Meer, who helped to plan the Monowitz camp (Auschwitz III) and IG Farben's Buna Werke factory at Auschwitz, where medical experimentation had been conducted and where 25,000 forced laborers were deployed. Ter Meer was sentenced to seven years, but was released in 1950. Despite being a convicted Nazi war criminal, Ter Meer was elected as chairman of Bayer AG's supervisory board in 1956, a position he retained until 1964.
Helge Wehmeier, then CEO of Bayer, offered a public apology in 1995 to Elie Wiesel for the company's actions during World War II (1939–1945) and the Holocaust.
== Products ==
=== Overview ===
In 1953, Bayer brought the first neuroleptic (chlorpromazine) onto the German market. In the 1960s, Bayer
introduced a pregnancy test, Primodos, that consisted of two pills that contained norethisterone (as acetate) and ethinylestradiol. It detected pregnancy by inducing menstruation in women who were not pregnant; the presence or absence of menstrual bleeding was then used to determine whether the user was pregnant. The test became the subject of controversy when it was blamed for birth defects, and it was withdrawn from the market in the mid-1970s. Litigation in the 1980s ended inconclusively. A review of the matter by the Medicines and Healthcare products Regulatory Agency in 2014 assessed the studies performed to date and found the evidence for adverse effects to be inconclusive.
In 1978, Bayer purchased Miles Laboratories and its subsidiaries Miles Canada and Cutter Laboratories, acquiring along with them a variety of product lines including Alka-Seltzer, Flintstones vitamins and One-A-Day vitamins, and Cutter insect repellent.
Along with the purchase of Cutter, Bayer acquired Cutter's Factor VIII business. Factor VIII, a clotting agent used to treat hemophilia, was produced, at the time, by processing donated blood. In the early days of the AIDS epidemic, people with hemophilia were found to have higher rates of AIDS, and by 1983 the CDC had identified contaminated blood products as a source of infection. According to the New York Times, this was "one of the worst drug-related medical disasters in history". Companies, including Bayer, developed new ways to treat donated blood with heat to decontaminate it, and these new products were introduced early in 1984. In 1997, Bayer and the other three makers of such blood products agreed to pay $660 million to settle cases on behalf of more than 6,000 hemophiliacs infected in United States. But in 2003, documents emerged showing that Cutter had continued to sell unheated blood products in markets outside the US until 1985, including in Malaysia, Singapore, Indonesia, Japan and Argentina, to offload a product they were unable to sell in Europe and the US; they also continued manufacturing the unheated product for several months. Bayer said it did this because some countries were doubtful about the efficacy of the new product.
Bayer has been involved in other controversies regarding its drug products. In the late 1990s it introduced a statin drug, Baycol (cerivastatin), but after 52 deaths were attributed to it, Bayer discontinued it in 2001. The side effect was rhabdomyolysis, causing kidney failure, which occurred with a tenfold greater frequency in patients treated with Baycol in comparison to those prescribed alternate medications of the statin class. Trasylol (aprotinin), used to control bleeding during major surgery, was withdrawn from the market worldwide in 2007 when reports of increased mortality emerged; it was later re-introduced in Europe but not in the US.
=== Top-selling pharmaceutical products ===
In 2014, pharmaceutical products contributed €12.05 billion of Bayer's €40.15 billion in gross revenue. In 2019, identified "key growth" products were Xarelto (rivaroxaban), Eylea (aflibercept), Stivarga (regorafenib), Xofigo (radium-223), and Adempas (riociguat).: 93 Top-selling products as of 2014 included:
Kogenate (recombinant clotting factor VIII). Kogenate is a recombinant version of clotting factor VIII, the absence or deficiency of which causes the abnormal bleeding associated with haemophilia type A. Kogenate is one of several commercially available Factor VIII products having equivalent efficacy.
Xarelto (rivaroxaban) is a small molecule inhibitor of Factor Xa, a key enzyme involved in blood coagulation. In the United States, the FDA has approved rivaroxaban for the prevention of stroke in people with atrial fibrillation, for the treatment of deep vein thrombosis and pulmonary embolism, and for the prevention of deep vein thrombosis in people undergoing hip surgery. Rivaroxaban competes with other newer generation anticoagulants such as edoxaban, apixaban, and dabigatran as well as with the generic anticoagulant warfarin. It has similar efficacy to warfarin and is associated with a lower risk of intracranial bleeding, but unlike warfarin there is no established protocol for rapidly reversing its effects in the event of uncontrolled bleeding or the need for emergency surgery.
Betaseron (interferon beta-1b) is an injectable form of the protein interferon beta used to prevent relapses in the relapsing remitting form of multiple sclerosis. Betaseron competes with other injectable forms of interferon beta, glatiramer acetate, and a variety of newer multiple sclerosis drugs, some of which can be taken orally (Dimethyl fumarate, teriflunomide, others).
Yasmin / Yaz birth control pills are part of a group of birth control pill products based on the progestin drospirenone. Yaz is approved in the United States for the prevention of pregnancy, to treat symptoms of premenstrual dysphoric disorder in women who choose an oral contraceptive for contraception, and to treat moderate acne in women at least 14 years of age who choose an oral contraceptive for contraception. The FDA conducted a safety review regarding the potential of Yaz and other drospirenone-containing products to increase the risk of blood clots; Yaz and Yasmin were associated with the deaths of 23 women in Canada, leading Health Canada to issue a warning in 2011. Although conflicting results were obtained in different studies, the FDA added a warning to the label in 2012 that Yaz and related products may be associated with an increased risk of clotting relative to other birth control pill products. Subsequently, a meta analysis suggested that birth control pills of the class Yasmin belongs to raise the risk of blood clots to a greater extent than some other classes of birth control pills.
Nexavar (sorafenib) is a kinase inhibitor used in the treatment of liver cancer (hepatocellular carcinoma), kidney cancer (renal cell carcinoma), and certain types of thyroid cancer.
Trasylol (Aprotinin) Trasylol is a trypsin inhibitor used to control bleeding during major surgery. In a 2006 meeting called by the FDA to review the drug's safety, Bayer scientists failed to reveal the results of an ongoing large study suggesting that Trasylol may increase the risks of death and stroke. According to a FDA official who preferred to remain anonymous, the FDA learned of the study only through information provided to the FDA by a whistleblowing scientist who was involved in it. The study concluded Trasylol carried greater risks of death, serious kidney damage, congestive heart failure and strokes. On 15 December of the same year, the FDA restricted the use of Trasylol, and in November 2007, they requested that the company suspend marketing. In 2011, Health Canada lifted its suspension of Trasylol for its originally approved indication of limiting bleeding in coronary bypass surgery, citing flaws in the design of the studies that led to its suspension. This decision was controversial. In 2013, the European Medicines Agency lifted its suspension of the Trasylol marketing authorization for selected patients undergoing cardiac bypass surgery, citing a favorable risk-benefit ratio.
Cipro (ciprofloxacin) Ciprofloxacin was approved by the US Food and Drug Administration (FDA) in 1987. Ciprofloxacin is the most widely used of the second-generation quinolone antibiotics that came into clinical use in the late 1980s and early 1990s. In 2010, over 20 million outpatient prescriptions were written for ciprofloxacin, making it the 35th-most commonly prescribed drug, and the 5th-most commonly prescribed antibacterial, in the US.
Rennie antacid tablets, one of the biggest selling branded over-the-counter medications sold in Great Britain, with sales of £29.8 million.
=== Agricultural ===
Bayer produces various fungicides, herbicides, insecticides, and some crop varieties.
Fungicides are primarily marketed for cereal crops, fresh produce, fungal with bacteria-based pesticides, and control of mildew and rust diseases. Nativo products are a mixture of trifloxystrobin tebuconazole. XPro products are a mix of bixafen and prothioconazole, while Luna contains fluopyram and pyrimethanil.
Herbicides are marketed primarily for field crops and orchards. Liberty brands containing glufosinate (a.k.a. Liberty or Basta) are used for general weed control. Capreno containing a mixture of thiencarbazone-methyl and tembotrione is used for grass and broad-leaf control.
Insecticides are marketed according to specific crop and insect pest type. Foliar insecticides include Belt containing flubendiamide, which is marketed against Lepidopteran pests, and Movento containing spirotetramat, which is marketed against sucking insects. Neonicotinoids such as clothianidin and imidacloprid are used as systemic seed treatments products such as Poncho and Gaucho. In 2008, neonicotinoids came under increasing scrutiny over their environmental impacts starting in Germany. Neonicotinoid use has been linked in a range of studies to adverse ecological effects, including honey-bee colony collapse disorder (CCD) and loss of birds due to a reduction in insect populations. In 2013, the European Union and a few non EU countries restricted the use of certain neonicotinoids. Parathion was discovered by scientists at IG Farben in the 1940s as a cholinesterase inhibitor insecticide. Propoxur is a carbamate insecticide that was introduced by Bayer in 1959.
== Acquisitions ==
=== Overview ===
In 1994, Bayer AG purchased Sterling Winthrop's over-the-counter (OTC) drug business from SmithKline Beecham and merged it with Miles Laboratories, thereby reclaiming the U.S. and Canadian trademark rights to "Bayer" and the Bayer cross, as well as the ownership of the Aspirin trademark in Canada.
In 2004, Bayer HealthCare acquired the over-the-counter pharmaceutical division of Roche. In March 2008, Bayer HealthCare announced an agreement to acquire the portfolio and OTC division of privately owned Sagmel, Inc., a US-based company that markets OTC medications in most of the Commonwealth of Independent States countries such as Russia, Ukraine, Kazakhstan, Belarus, and others.
On 28 August 2008, an explosion occurred at the Bayer CropScience facility at Institute, West Virginia, United States. A runaway reaction ruptured a tank and the resulting explosion killed two employees. The ruptured tank was close to a methyl isocyanate tank which was undamaged by the explosion.
=== Acquisition of Schering ===
In March 2006, Merck KGaA announced a €14.6 billion bid for Schering AG, founded in 1851. By 2006, Schering had annual gross revenue of around €5 billion and employed about 26,000 people in 140 subsidiaries worldwide. Bayer responded with a white knight bid and in July acquired the majority of shares of Schering for €14.6 billion, and in 2007, Bayer took over Schering AG and formed Bayer Schering Pharma. The acquisition of Schering was the largest take-over in Bayer's history, and as of 2015, was one of the ten biggest pharma mergers of all time.
=== Other acquisitions ===
In November 2010, Bayer AG signed an agreement to buy Auckland-based animal health company Bomac Group. Bayer partnered on the development of the radiotherapeutic Xofigo with Algeta, and in 2014, moved to acquire the company for about $2.9 billion. In 2014, Bayer agreed to buy Merck's consumer health business for $14.2 billion which would provide Bayer control with brands such as Claritin, Coppertone and Dr. Scholl's. Bayer would attain second place globally in nonprescription drugs. In June 2015, Bayer agreed to sell its diabetic care business to Panasonic Healthcare Holdings for a fee of $1.02 billion.
In August 2019, the business acquired the ≈60% of BlueRock Therapeutics it didn't already own for up to $600 million.
In August 2020, Bayer announced it had acquired KaNDy Therapeutics Ltd, helping to boost its female healthcare business, for $425 million. In October, Bayer agreed to acquire Asklepios BioPharmaceuticals for $2 billion upfront.
In June 2021, the company announced it acquire Noria Therapeutics Inc. and PSMA Therapeutics Inc. gaining rights to a number of cancer-based investigational compounds based on actinium-225.
=== Spin off of Covestro ===
In September 2015, Bayer spun out its $12.3 billion materials science division into a separate, publicly traded company called Covestro in which it retained about a 70% interest. Bayer spun out the division because it had relatively low profit margins compared to its life science divisions (10.2%, compared with 24.9% for the agriculture business and 27.5% for healthcare) and because the business required high levels of investment to maintain its growth, and to more clearly focus its efforts and identity in the life sciences. Covestro shares were first offered on the Frankfurt Stock Exchange in October 2015. Effective January 2016 following the spinout of Covestro, Bayer rebranded itself as a life sciences company, and restructured into three divisions and one business unit: Pharmaceuticals, Consumer Health, Crop Science, and Animal Health.
=== Acquisition of Monsanto ===
In May 2016, Bayer offered to buy U.S. biotechnology company Monsanto for $62 billion. Shortly after Bayer's offer, Monsanto rejected the acquisition bid, seeking a higher price. In September 2016, Monsanto agreed to a $66 billion offer by Bayer. In order to receive regulatory approval, Bayer agreed to divest a significant amount of its current agricultural assets to BASF in a series of deals. On 21 March 2018 the deal was approved by the European Union, and it was approved in the United States on 20 May 2018. The sale closed on 7 June 2018. The Monsanto brand was discontinued; its products are now marketed under the Bayer name. On 16 September 2019, under the approval of National Company Law Tribunal, Bayer completed the merger of Monsanto India.
Bayer's Monsanto acquisition is the biggest acquisition by a German company to date. However, owing to ongoing litigation concerning the Monsanto's herbicide Roundup and the massive financial and reputational blows it has caused Bayer, the deal is considered one of the worst corporate mergers in history. By 2023, Bayer's market value had declined by over 60% since its 2016 merger, leaving the company's overall worth at less than half of what it paid to acquire Monsanto.
=== Acquisition history ===
== Corporate structure ==
In 2003, to separate operational and strategic managements, Bayer AG was reorganized into a holding company. The group's core businesses were transformed into limited companies, each controlled by Bayer AG. These companies were: Bayer CropScience AG; Bayer HealthCare AG; Bayer MaterialScience AG and Bayer Chemicals AG, and the three service limited companies Bayer Technology Services GmbH, Bayer Business Services GmbH and Bayer Industry Services GmbH & Co. OHG. In 2016, the company began a second restructuring with the aim of allowing it to transition to a life sciences based company. By divesting its Chemicals division in 2004 and with the aim of off-loading its Materials division by mid-2016, Bayer will be left with the four core units, as depicted below.
=== Bayer CropScience ===
Bayer CropScience has products in crop protection (i.e. pesticides), nonagricultural pest control, and seeds and plant biotechnology. In addition to conventional agrochemical business, it is involved in genetic engineering of food. In 2002, Bayer AG acquired Aventis (now part of Sanofi) CropScience and fused it with their own agrochemicals division (Bayer Pflanzenschutz or "Crop Protection") to form Bayer CropScience; the Belgian biotech company Plant Genetic Systems became part of Bayer through the Aventis acquisition. Also in 2002, Bayer AG acquired the Dutch seed company Nunhems, which at the time was one of the world's top five seed companies.: 270 In 2006, the U.S. Department of Agriculture announced that Bayer CropScience's LibertyLink genetically modified rice had contaminated the U.S. rice supply. Shortly after the public learned of the contamination, the E.U. banned imports of U.S. long-grain rice and the futures price plunged. In April 2010, a Lonoke County, Arkansas jury awarded a dozen farmers $48 million. The case was appealed to the Arkansas Supreme Court, which affirmed the judgement. On 1 July 2011, Bayer CropScience agreed to a global settlement for up to $750 million. In September 2014, the firm announced plans to invest $1 billion in the United States between 2013 and 2016. A Bayer spokesperson said that the largest investments will be made to expand the production of its herbicide Liberty. Liberty is an alternative to Monsanto's product, Roundup, which are both used to kill weeds.
In 2016, as part of the wholesale corporate restructuring, Bayer CropScience became one of the three major divisions of Bayer AG, reporting directly to the head of the division, Liam Condon. Under the terms of the merger, Bayer promised to maintain Monsanto's more than 9,000 U.S. jobs and add 3,000 new U.S. high-tech positions.
The prospective merger parties said at the time the combined agriculture business planned to spend $16 billion on research and development over the next six years and at least $8 billion on research and development in the United States.
The global headquarters of Bayer CropScience is located in St. Louis, Missouri, United States.
Bayer CropScience Limited is the Indian subsidiary of Bayer AG. It is listed on the Indian stock exchanges; the Bombay Stock Exchange and National Stock Exchange of India, and has a market capitalization of $2 billion. Bayer BioScience, headquartered in Hyderabad, India, has about 400 employees, and has research, production, and an extensive sales network spread across India.
=== Bayer Consumer Health ===
Before the 2016 restructuring, Bayer HealthCare comprised a further four subdivisions: Bayer Schering Pharma, Bayer Consumer Care, Bayer Animal Health and Bayer Medical Care. As part of the corporate restructuring, Animal Health was moved into its own business unit, leaving the division with the following categories; Allergy, Analgesics, Cardiovascular Risk Prevention, Cough & Cold, Dermatology, Foot Care, Gastrointestinals, Nutritionals and Sun Care.
Bayer Consumer Care manages Bayer's OTC medicines portfolio. Key products include analgesics such as Bayer Aspirin and Aleve, food supplements Redoxon and Berocca, and skincare products Bepanthen and Bepanthol. Women's healthcare is an example of a General Medicine business unit. Bayer Pharma produces the birth control pills Yaz and Yasmin. Both pills use a newer type of progestin hormone called drospirenone in combination with estrogen. Yaz is advertised as a treatment for premenstrual dysphoric disorder (PMDD) and moderate acne. Other key products include the cancer drug Nexavar, the multiple sclerosis drug betaferon/betaseron and the blood-clotting drug, Kogenate. In May 2014, it was announced that Bayer would buy Merck & Co's consumer health care unit for $14.2 billion. Bayer also controls Dihon Pharmaceutical Group Co., Ltd in China.
=== Bayer Pharmaceuticals ===
The Pharmaceuticals Division focuses on prescription products, especially for women's healthcare and cardiology, and also on specialty therapeutics in the areas of oncology, hematology and ophthalmology. The division also comprises the Radiology Business Unit which markets contrast-enhanced diagnostic imaging equipment together with the necessary contrast agents.
In addition to internal R&D, Bayer has participated in public–private partnerships. One example in the area of non-clinical safety assessment is the InnoMed PredTox program. Another is the Innovative Medicines Initiative of EFPIA and the European Commission.
=== Defunct business units ===
Bayer Chemicals AG (with the exception of H.C. Starck and Wolff Walsrode) was combined with certain components of the polymers segment to form the new company Lanxess on 1 July 2004; Lanxess was listed on the Frankfurt Stock Exchange in early 2005. Bayer HealthCare's Diagnostics Division was acquired by Siemens Medical Solutions in January 2007.
Bayer sold its Animal Health business to Elanco in 2020.
Bayer Diabetes Care managed Bayer's medical devices portfolio. Key products included the blood glucose monitors Contour Next EZ (XT), Contour, Contour USB and Breeze 2 used in the management of diabetes. The diabetes business unit was sold to Panasonic Healthcare Co. for $1.15 billion in June 2015. Bayer MaterialScience was a supplier of high-tech polymers, and developed solutions for a broad range of applications relevant to everyday life. On 18 September 2014, the Board of Directors of Bayer AG announced plans to float the Bayer MaterialScience business on the stock market as a separate entity. On 1 June 2015, Bayer announced that the new company would be named Covestro; Bayer formally spun out Covestro in September 2015.
== Ownership ==
The 10 largest shareholders of Bayer AG in early 2024 were:
== Finances ==
For the fiscal year 2017, Bayer reported earnings of EUR€7.3 billion, with an annual revenue of EUR€35 billion, a decrease of 25.1% over the previous fiscal cycle. Bayer's shares traded at over €69 per share, and its market capitalization was valued at US€65.4 billion in November 2018. In September 2019, Bayer announced to reduce the number of management board members from seven to five to reduce overall costs.
The key trends of Bayer are (as at the financial year ending December 31):
* without Covestro from 2017 on
== Bayer 04 Leverkusen ==
In 1904, the company founded the sports club TuS 04 ("Turn- und Spielverein der Farbenfabriken vorm. Friedr. Bayer & Co."), later SV Bayer 04 ("Sportvereinigung Bayer 04 Leverkusen"), finally becoming TSV Bayer 04 Leverkusen ("Turn- und Sportverein") in 1984, generally, however, known simply as Bayer 04 Leverkusen. The club is best known for its football team, but has been involved in many other sports, including athletics, fencing, team handball, volleyball, boxing, and basketball. TSV Bayer 04 Leverkusen is one of the largest sports clubs in Germany. The company also supports similar clubs at other company sites, including Dormagen (particularly handball), Wuppertal (particularly volleyball), and Krefeld-Uerdingen (featuring another former Bundesliga football club, SC Bayer 05 Uerdingen, now KFC Uerdingen 05).
== Awards and recognition ==
In October 2008, Bayer's Canadian division was named one of "Canada's Top 100 Employers" by Mediacorp Canada Inc. The Canadian division was named one of Greater Toronto's Top Employers by the Toronto Star newspaper. Bayer USA was given a score of 85 (out of 100) in the Human Rights Campaign's 2011 Corporate Equality Index, a measure of gay and lesbian workplace equality.
In 2016, Standard Ethics Aei gave a rating to Bayer in order to include the company in its Standard Ethics German Index. Bayer received an EE− rating, the fourth tier in an eight-tier ranking.
Ranked third in Access to Seeds Index in 2016.
== Litigation ==
=== Roundup ===
In August 2018, two months after Bayer acquired Monsanto, a U.S. jury ordered Monsanto to pay $289 million to a school groundskeeper who claimed his Non-Hodgkin's lymphoma was caused by regularly using Roundup, a glyphosate-based herbicide produced by Monsanto. Following the Johnson v. Monsanto Co. verdict, Bayer's share price dropped by around 14% or $14 Billion in market capitalization. The company filed an appeal on 18 September 2018. Pending appeal, the award was later reduced to $78.5 million. In November 2018, Monsanto appealed the judgement, asking an appellate court to consider a motion for a new trial. A verdict on the appeal was delivered in June 2020 upholding the verdict but further reducing the award to $21.5 million. On 13 May 2019, a United States Superior Court Judge ordered Bayer to pay more than $ 2.5 billion in damages to a couple in California, both of whom contracted non-Hodgkin's Lymphoma, later cut to $87 million on appeal.
In June 2020, the company agreed to pay $9.6 billion to settle more than 10,000 lawsuits claiming harm from Roundup, saying this action will result in the resolution of 75% of those claims. Bayer will also assign $1.25 billion for future claims, an action that needs approval from the US District Court, Northern District of California. The settlement, according to the company, does not admit either liability or wrongdoing, but brings an end to irresolution in the case. The settlement does not include three cases that have already gone to jury trials and are being appealed. In July 2020, the California Court of Appeals denied the appeal but reduced the damages owed to $20.4 million. As of 2023, around 165,000 claims, more than 50,000 of which still pending, have been made against Roundup, mostly alleging that it had caused cancer.
The general consensus among national regulatory agencies, and the European Commission is that labeled usage of the herbicide poses no carcinogenic or genotoxic risk to humans. In January 2020, the US Environmental Protection Agency (EPA) finalized its interim registration review for Roundup, stating that it "...did not identify any risks of concern" for cancer and other risks to humans from glyphosate exposure." On 17 June 2022, California-based United States Court of Appeals for the Ninth Circuit ordered the Environmental Protection Agency to reexamine this 2020 finding that glyphosate did not pose a health risk for people exposed to it by any means.
In 2024, legislation was introduced in Iowa, Missouri and Idaho with language supplied by Bayer that experts say could shield the company from any lawsuits related to cancer risk. Bayer leads a group called Modern Ag Alliance which produces advertisements claiming that lawsuits threaten the availability of glyphosate. In 2025, flyers from a dark money source were sent to constituents of Missouri senators who oppose the bill, claiming that the lawmakers' opposition would cause "Chinese Communist Party chemicals" to enter the food supply. The targeted senators allege that Bayer is behind the mailers, which Bayer denies.
=== Xarelto ===
In 2019, Bayer and Johnson & Johnson (who market Xarelto together) settled around 25,000 lawsuits on the blood thinning drug Xarelto (rivaroxaban) by agreeing to disburse $775 million (US) to federal and state plaintiffs who said the companies had not properly warned patients about possible fatal bleeding as a result of ingesting the drug. There was no admission of liability from the companies in the settlement as they noted they had prevailed in six previous trials. The settlement will be divided evenly between the companies.
=== One A Day Vitamins ===
In 2019, a federal jury in San Francisco CA sided with Bayer in a $600 million (US) class action suit alleging that the company misinformed consumers by promoting its One A Day vitamins as supporting cardiac health, vigorous immune systems and boosting user energy. The suit was first filed as a nationwide class action; in 2017, the US District Court in San Francisco said subclasses of purchasers of the vitamin in Florida, New York, and California could act together.
The jury found that the plaintiffs failed to prove that Bayer misrepresented its One A Day claims, and also did not demonstrate that any of the class representative consumers who purchased One A Day relied on the so-called false information as part of their buying decision.
=== HIV contamination ===
In the mid-1980s, when Bayer's Cutter Laboratories realized that their blood products, the clotting agents Factor VIII and IX, were contaminated with HIV, the financial investment in the product was considered too high to destroy the inventory. Bayer misrepresented the results of its own research and knowingly supplied hemophilia medication tainted with HIV to patients in Asia and Latin America, without the precaution of heat treating the product, recommended for eliminating the risk. As a consequence, thousands who infused the product tested positive for HIV and later developed AIDS.
=== Dicamba ===
On 14 February 2020, Bayer and BASF were ordered to pay Missouri peach farmer Bill Bader $15 million in damages as a result of destruction of his peach trees which was caused by the usage of dicamba by nearby farmers. Dicamba was another product which Bayer acquired from Monsanto. Bayer also inherited the lawsuit from Monsanto as well. On 15 February 2020, Bayer—representing Monsanto—and BASF were ordered to pay not only the $15 million in damages, but an additional $250 million in punitive damages. Bayer and BASF afterwards announced plans to appeal the $265 million fine.
In June 2020, Bayer agreed to a settlement of up to $400 million for all 2015 to 2020 crop year dicamba claims, not including the $265 million judgement. On 25 November 2020, U.S. District Judge Stephen Limbaugh Jr. reduced the punitive damage amount in the Bader Farms case to $60 million.
=== PCB pollution ===
In June 2020, Bayer agreed to pay $800 million to settle lawsuits in a variety of jurisdictions which claimed contamination of public waterways with PCBs by Monsanto before 1978. On 25 November 2020, however, U.S. District Judge Fernando M. Olguin rejected Bayer's settlement offer, which was now at $650 million, and allowed Monsanto-related lawsuits involving PCB to proceed.
=== Talc-related liabilities ===
On 4 April 2023, a Delaware judge dismissed a lawsuit by Merck & Co’s seeking to hold Bayer AG responsible for more talc-related liabilities stemming from its $14.2 billion purchase of Merck’s consumer care business in 2014. The judge called Bayer’s interpretation of the purchase agreement “the only reasonable one,” and said letting Merck “dump” cases would give the Rahway, New Jersey–based company an incentive to prolong or stall lawsuits. Bayer said in a statement, it welcomed the decision, and it "will continue to defend itself against any further efforts by Merck to avoid or improperly transfer its liabilities to Bayer”.
== See also ==
List of German companies
List of pharmaceutical companies
== Notes ==
== References ==
=== Works cited ===
== Further reading ==
"The original Bayer Aspirin". wonderdrug.com. Bayer AG.
Blaschke, Stefan (1999). Unternehmen und Gemeinde: Das Bayerwerk im Raum Leverkusen 1891–1914. Cologne: SH-Verlag. ISBN 3-89498-068-0
Cornwell J (2004). Hitler's Scientists: Science, War, and the Devil's Pact. London: Penguin Books.
Lesch JE, ed. (2000). The German Chemical Industry in the Twentieth Century. Dordrecht: Springer Netherlands.
Plumpe G (1990). Die I.G. Farbenindustrie AG: Wirtschaft, Technik und Politik 1904–1945. Berlin: Duncker & Humblot.
Stokes R (1988). Divide and Prosper: The Heirs of I.G. Farben under Allied Authority, 1945–1951. Berkeley: University of California Press.
Stokes R (1994). Opting for Oil: The Political Economy of Technological Change in the West German Chemical Industry, 1945–1961. New York: Cambridge University Press.
Tenfelde, Klaus (2007). Stimmt die Chemie? : Mitbestimmung und Sozialpolitik in der Geschichte des Bayer-Konzerns. Essen: Klartext. ISBN 978-3-89861-888-5
Tully J (2011). The Devil's Milk: A Social History of Rubber. New York: Monthly Review Press.
== External links ==
Official website
Documents and clippings about Bayer in the 20th Century Press Archives of the ZBW
Product Portfolio | Wikipedia/Bayer_CropScience |
Animal science is described as "studying the biology of animals that are under the control of humankind". It can also be described as the production and management of farm animals. Historically, the degree was called animal husbandry and the animals studied were livestock species, like cattle, sheep, pigs, poultry, and horses. Today, courses available look at a broader area, including companion animals, like dogs and cats, and many exotic species. Degrees in Animal Science are offered at a number of colleges and universities. Animal science degrees are often offered at land-grant universities, which will often have on-campus farms to give students hands-on experience with livestock animals.
== Education ==
Professional education in animal science prepares students for careers in areas such as animal breeding, food and fiber production, nutrition, animal agribusiness, animal behavior, and welfare. Courses in a typical Animal Science program may include genetics, microbiology, animal behavior, nutrition, physiology, and reproduction. Courses in support areas, such as genetics, soils, agricultural economics and marketing, legal aspects, and the environment also are offered.
=== Bachelor degree ===
At many universities, a Bachelor of Science (BS) degree in Animal Science allows emphasis in certain areas. Typical areas are species-specific or career-specific. Species-specific areas of emphasis prepare students for a career in dairy management, beef management, swine management, sheep or small ruminant management, poultry production, or the horse industry. Other career-specific areas of study include pre-veterinary medicine studies, livestock business and marketing, animal welfare and behavior, animal nutrition science, animal reproduction science, or genetics. Youth programs are also an important part of animal science programs.
==== Pre-veterinary emphasis ====
Many schools that offer a degree option in Animal Science also offer a pre-veterinary emphasis such as Iowa State University, the University of Nebraska–Lincoln, and the University of Minnesota, for example. This option provides knowledge of the biological and physical sciences including nutrition, reproduction, physiology, and genetics. This can prepare students for graduate studies in animal science, veterinary school, and pharmaceutical or animal science industries.
=== Graduate studies ===
In a Master of Science degree option, students take required courses in areas that support their main interest. These courses are above courses normally required for a Bachelor of Science degree in the Animal Science major. For example, in a Ph.D. degree program students take courses related to their major that are more in-depth than those for the Master of Science degree, with an emphasis on research or teaching.
Graduate studies in animal sciences are considered preparation for upper-level positions in production, management, education, research, or agri-services. Professional study in veterinary medicine, law, and business administration are among the most commonly chosen programs by graduates. Other areas of study include growth biology, physiology, nutrition, and production systems.
== Careers in Animal Science ==
There are a variety of careers available to someone with an animal science degree. Including, but not limited to, Academic researcher, Animal nutritionist, Animal physiotherapist technician, Nature conservation officer, Zookeeper, and Zoologist.
== Areas of study ==
=== Animal Behavior ===
Animal behavior is the study of how animals interact with their environment, interact with each other socially, and how they may achieve understanding of their environment. Animal behavior is examined with the framework of its development, mechanism, adaptive value, and evolution.
=== Animal Genetics ===
Animal genetics is the study of an animal genes and how they effect an animal's appearance, health, and function. The information gained from such studies is often applied to livestock breeding.
=== Veterinary Medicine ===
Veterinary medicine is a specialization within the field of medicine focusing on the diagnosis, prevention, control, and treatment of diseases that effect both wild and domesticated animals. There are three main medical positions within veterinary medicine, veterinarians, veterinary technicians, and veterinary assistants.
== See also ==
American Registry of Professional Animal Scientists
List of animal science degree-granting institutions
Zoology, the interest of all animals.
Veterinary science
== References ==
== External links ==
"Career Information." American Society of Animal Science. ASAS, 2009. Web. 29 September 2011.
http://www.asas.org American Society of Animal Science
"UNL Animal Science Department." University of Nebraska-Lincoln. UNL Institute of Agriculture and Natural Resources, 27 January 2015.
"MSU Department of Animal Science." Michigan State University. Michigan State University Department of Animal Science, 28 December 2013.
"Animal Industry Careers." Purdue University. Purdue University, 11 August 2005. Web. 5 October 2011.
http://www.ansc.purdue.edu Purdue University Animal Science | Wikipedia/Animal_science |
In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.
Transformation is one of three processes that lead to horizontal gene transfer, in which exogenous genetic material passes from one bacterium to another, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium). In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.
As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative bacteria; the number might be an overestimate since several of the reports are supported by single papers.
"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection".
== History ==
Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist Frederick Griffith. Griffith was interested in determining whether injections of heat-killed bacteria could be used to vaccinate mice against pneumonia. However, he discovered that a non-virulent strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming principle" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of S. pneumoniae and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation" (See Avery-MacLeod-McCarty experiment) The results of Avery et al.'s experiments were at first skeptically received by the scientific community and it was not until the development of genetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery's experiments were accepted.
It was originally thought that Escherichia coli, a commonly used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko Higa showed that E. coli may be induced to take up DNA from bacteriophage λ without the use of helper phage after treatment with calcium chloride solution. Two years later in 1972, Stanley Norman Cohen, Annie Chang and Leslie Hsu showed that CaCl2 treatment is also effective for transformation of plasmid DNA. The method of transformation by Mandel and Higa was later improved upon by Douglas Hanahan. The discovery of artificially induced competence in E. coli created an efficient and convenient procedure for transforming bacteria which allows for simpler molecular cloning methods in biotechnology and research, and it is now a routinely used laboratory procedure.
Transformation using electroporation was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains that could be transformed. Transformation of animal and plant cells was also investigated with the first transgenic mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982. In 1897 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor-inducing agent was found to be a DNA plasmid called the Ti plasmid. By removing the genes in the plasmid that caused the tumor and adding in novel genes, researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants. Not all plant cells are susceptible to infection by A. tumefaciens, so other methods were developed, including electroporation and micro-injection. Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s.
== Definitions ==
Transformation is one of three forms of horizontal gene transfer that occur in nature among bacteria, in which DNA encoding for a trait passes from one bacterium to another and is integrated into the recipient genome by homologous recombination; the other two are transduction, carried out by means of a bacteriophage, and conjugation, in which a gene is passed through direct contact between bacteria. In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.
Competence refers to a temporary state of being able to take up exogenous DNA from the environment; it may be induced in a laboratory.
It appears to be an ancient process inherited from a common prokaryotic ancestor that is a beneficial adaptation for promoting recombinational repair of DNA damage, especially damage acquired under stressful conditions. Natural genetic transformation appears to be an adaptation for repair of DNA damage that also generates genetic diversity.
Transformation has been studied in medically important Gram-negative bacteria species such as Helicobacter pylori, Legionella pneumophila, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae and Vibrio cholerae. It has also been studied in Gram-negative species found in soil such as Pseudomonas stutzeri, Acinetobacter baylyi, and Gram-negative plant pathogens such as Ralstonia solanacearum and Xylella fastidiosa. Transformation among Gram-positive bacteria has been studied in medically important species such as Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus and Streptococcus sanguinis and in Gram-positive soil bacterium Bacillus subtilis. It has also been reported in at least 30 species of Pseudomonadota distributed in several different classes. The best studied Pseudomonadota with respect to transformation are the medically important human pathogens Neisseria gonorrhoeae, Haemophilus influenzae, and Helicobacter pylori.
"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection".
== Natural competence and transformation ==
Naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogenous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.
Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase. Only single-stranded DNA may pass through, the other strand being degraded by nucleases in the process. The translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence, but its role is uncertain. The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.
=== Natural transformation ===
Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes whose products appear to be responsible for this process. In general, transformation is a complex, energy-requiring developmental process. In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a special physiological state. Competence development in Bacillus subtilis requires expression of about 40 genes. The DNA integrated into the host chromosome is usually (but with rare exceptions) derived from another bacterium of the same species, and is thus homologous to the resident chromosome.
In B. subtilis the length of the transferred DNA is greater than 1271 kb (more than 1 million bases). The length transferred is likely double stranded DNA and is often more than a third of the total chromosome length of 4215 kb. It appears that about 7-9% of the recipient cells take up an entire chromosome.
The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67 prokaryotic species (in seven different phyla) are known to undergo this process.
Competence for transformation is typically induced by high cell density and/or nutritional limitation, conditions associated with the stationary phase of bacterial growth. Transformation in Haemophilus influenzae occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase. Transformation in Streptococcus mutans, as well as in many other streptococci, occurs at high cell density and is associated with biofilm formation. Competence in B. subtilis is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation. Similarly, in Micrococcus luteus (a representative of the less well studied Actinomycetota phylum), competence develops during the mid-late exponential growth phase and is also triggered by amino acids starvation.
By releasing intact host and plasmid DNA, certain bacteriophages are thought to contribute to transformation.
=== Transformation, as an adaptation for DNA repair ===
Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced in Streptococcus pneumoniae by the DNA damaging agents mitomycin C (a DNA cross-linking agent) and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks). In B. subtilis, transformation is increased by UV light, a DNA damaging agent. In Helicobacter pylori, ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation Using Legionella pneumophila, Charpentier et al. tested 64 toxic molecules to determine which of these induce competence. Of these, only six, all DNA damaging agents, caused strong induction. These DNA damaging agents were mitomycin C (which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid (inhibitors of DNA gyrase that cause double-strand breaks), bicyclomycin (causes single- and double-strand breaks), and hydroxyurea (induces DNA base oxidation). UV light also induced competence in L. pneumophila. Charpentier et al. suggested that competence for transformation probably evolved as a DNA damage response. Natural transformation in the extraordinarily radiation resistant bacterium Deinococcus radiodurans is associated with the repair of DNA damage under stressful conditions.
Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of genome copies present in the cell, and this has implications for the capability to carry out an important DNA repair process. During logarithmic growth, two or more copies of any particular region of the chromosome may be present in a bacterial cell, as cell division is not precisely matched with chromosome replication. The process of homologous recombinational repair (HRR) is a key DNA repair process that is especially effective for repairing double-strand damages, such as double-strand breaks. This process depends on a second homologous chromosome in addition to the damaged chromosome. During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence information from the other homologous chromosome. Once cells approach stationary phase, however, they typically have just one copy of the chromosome, and HRR requires input of homologous template from outside the cell by transformation.
To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using B. subtilis irradiated by UV light as the damaging agent (reviewed by Michod et al. and Bernstein et al.) The results of these experiments indicated that transforming DNA acts to repair potentially lethal DNA damages introduced by UV light in the recipient DNA. The particular process responsible for repair was likely HRR. Transformation in bacteria can be viewed as a primitive sexual process, since it involves interaction of homologous DNA from two individuals to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes (see Evolution of sexual reproduction; Meiosis.)
== Methods and mechanisms of transformation in laboratory ==
=== Bacterial ===
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature. Typically the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. Cells that are able to take up the DNA are called competent cells.
It has been found that growth of Gram-negative bacteria in 20 mM Mg reduces the number of protein-to-lipopolysaccharide bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation. The role of lipopolysaccharides here are verified from the observation that shorter O-side chains are more effectively transformed – perhaps because of improved DNA accessibility.
The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.
DNA entry into E. coli cells is through channels known as zones of adhesion or Bayer's junction, with a typical cell carrying as many as 400 such zones. Their role was established when cobalamine (which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.
It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.
Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms.
=== Yeast ===
Most species of yeast, including Saccharomyces cerevisiae, may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab.
Yeast cells may be treated with enzymes to degrade their cell walls, yielding spheroplasts. These cells are very fragile but take up foreign DNA at a high rate.
Exposing intact yeast cells to alkali cations such as those of caesium or lithium allows the cells to take up plasmid DNA. Later protocols adapted this transformation method, using lithium acetate, polyethylene glycol, and single-stranded DNA. In these protocols, the single-stranded DNA preferentially binds to the yeast cell wall, preventing plasmid DNA from doing so and leaving it available for transformation.
Electroporation: Formation of transient holes in the cell membranes using electric shock; this allows DNA to enter as described above for bacteria.
Enzymatic digestion or agitation with glass beads may also be used to transform yeast cells.
Efficiency – Different yeast genera and species take up foreign DNA with different efficiencies. Also, most transformation protocols have been developed for baker's yeast, S. cerevisiae, and thus may not be optimal for other species. Even within one species, different strains have different transformation efficiencies, sometimes different by three orders of magnitude. For instance, when S. cerevisiae strains were transformed with 10 ug of plasmid YEp13, the strain DKD-5D-H yielded between 550 and 3115 colonies while strain OS1 yielded fewer than five colonies.
=== Plants ===
A number of methods are available to transfer DNA into plant cells. Some vector-mediated methods are:
Agrobacterium-mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for ten minutes in a fluid containing suspended Agrobacterium. The bacteria will attach to many of the plant cells exposed by the cut. The plant cells secrete wound-related phenolic compounds which in turn act to upregulate the virulence operon of the Agrobacterium. The virulence operon includes many genes that encode for proteins that are part of a Type IV secretion system that exports from the bacterium proteins and DNA (delineated by specific recognition motifs called border sequences and excised as a single strand from the virulence plasmid) into the plant cell through a structure called a pilus. The transferred DNA (called T-DNA) is piloted to the plant cell nucleus by nuclear localization signals present in the Agrobacterium protein VirD2, which is covalently attached to the end of the T-DNA at the Right border (RB). Exactly how the T-DNA is integrated into the host plant genomic DNA is an active area of plant biology research. Assuming that a selection marker (such as an antibiotic resistance gene) was included in the T-DNA, the transformed plant tissue can be cultured on selective media to produce shoots. The shoots are then transferred to a different medium to promote root formation. Once roots begin to grow from the transgenic shoot, the plants can be transferred to soil to complete a normal life cycle (make seeds). The seeds from this first plant (called the T1, for first transgenic generation) can be planted on a selective (containing an antibiotic), or if an herbicide resistance gene was used, could alternatively be planted in soil, then later treated with herbicide to kill wildtype segregants. Some plants species, such as Arabidopsis thaliana can be transformed by dipping the flowers or whole plant, into a suspension of Agrobacterium tumefaciens, typically strain C58 (C=Cherry, 58=1958, the year in which this particular strain of A. tumefaciens was isolated from a cherry tree in an orchard at Cornell University in Ithaca, New York). Though many plants remain recalcitrant to transformation by this method, research is ongoing that continues to add to the list the species that have been successfully modified in this manner.
Viral transformation (transduction): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However, genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus-free and also free of the inserted gene.
Some vector-less methods include:
Gene gun: Also referred to as particle bombardment, microprojectile bombardment, or biolistics. Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The transformation efficiency is lower than in Agrobacterium-mediated transformation, but most plants can be transformed with this method.
Electroporation: Formation of transient holes in cell membranes using electric pulses of high field strength; this allows DNA to enter as described above for bacteria.
=== Fungi ===
There are some methods to produce transgenic fungi most of them being analogous to those used for plants. However, fungi have to be treated differently due to some of their microscopic and biochemical traits:
A major issue is the dikaryotic state that parts of some fungi are in; dikaryotic cells contain two haploid nuclei, one of each parent fungus. If only one of these gets transformed, which is the rule, the percentage of transformed nuclei decreases after each sporulation.
Fungal cell walls are quite thick hindering DNA uptake so (partial) removal is often required; complete degradation, which is sometimes necessary, yields protoplasts.
Mycelial fungi consist of filamentous hyphae, which are, if at all, separated by internal cell walls interrupted by pores big enough to enable nutrients and organelles, sometimes even nuclei, to travel through each hypha. As a result, individual cells usually cannot be separated. This is problematic as neighbouring transformed cells may render untransformed ones immune to selection treatments, e.g. by delivering nutrients or proteins for antibiotic resistance.
Additionally, growth (and thereby mitosis) of these fungi exclusively occurs at the tip of their hyphae which can also deliver issues.
As stated earlier, an array of methods used for plant transformation do also work in fungi:
Agrobacterium is not only capable of infecting plants but also fungi, however, unlike plants, fungi do not secrete the phenolic compounds necessary to trigger Agrobacterium so that they have to be added, e.g. in the form of acetosyringone.
Thanks to development of an expression system for small RNAs in fungi the introduction of a CRISPR/CAS9-system in fungal cells became possible. In 2016 the USDA declared that it will not regulate a white button mushroom strain edited with CRISPR/CAS9 to prevent fruit body browning causing a broad discussion about placing CRISPR/CAS9-edited crops on the market.
Physical methods like electroporation, biolistics ("gene gun"), sonoporation that uses cavitation of gas bubbles produced by ultrasound to penetrate the cell membrane, etc. are also applicable to fungi.
=== Animals ===
Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.
== Practical aspects of transformation in molecular biology ==
The discovery of artificially induced competence in bacteria allow bacteria such as Escherichia coli to be used as a convenient host for the manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation in E. coli. In order to be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the replication of the cell's own chromosome.
The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as transformation efficiency and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1×108 cfu/μg for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.
In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca2+ (in CaCl2 solution), making the cell become permeable to plasmid DNA. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42 °C for 30–120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/μg or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of plasmid. Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 109. The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.
The transformation efficiency using the CaCl2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA. Cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the electroporation process.
=== Selection and screening in plasmid transformation ===
Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable marker such that those cells without the plasmid may be killed or have their growth arrested. Antibiotic resistance is the most commonly used marker for prokaryotes. The transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis or utility of a particular biomolecule, and the transformed cells are cultured in a medium that allows only cells containing the plasmid to grow.
In a cloning experiment, a gene may be inserted into a plasmid used for transformation. However, in such experiment, not all the plasmids may contain a successfully inserted gene. Additional techniques may therefore be employed further to screen for transformed cells that contain plasmid with the insert. Reporter genes can be used as markers, such as the lacZ gene which codes for β-galactosidase used in blue-white screening. This method of screening relies on the principle of α-complementation, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ-α is transformed into a lacZΔM15 cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be detected when cells are grown in plates containing X-gal, forming characteristic blue colonies. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα gene, and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.
Other commonly used reporter genes are green fluorescent protein (GFP), which produces cells that glow green under blue light, and the enzyme luciferase, which catalyzes a reaction with luciferin to emit light. The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods.
== References ==
== External links ==
Bacterial Transformation (a Flash Animation)
"Ready, aim, fire!" At the Max Planck Institute for Molecular Plant Physiology in Potsdam-Golm plant cells are 'bombarded' using a particle gun | Wikipedia/Genetic_transformation |
Oceanography (from Ancient Greek ὠκεανός (ōkeanós) 'ocean' and γραφή (graphḗ) 'writing'), also known as oceanology, sea science, ocean science, and marine science, is the scientific study of the ocean, including its physics, chemistry, biology, and geology.
It is an Earth science, which covers a wide range of topics, including ocean currents, waves, and geophysical fluid dynamics; fluxes of various chemical substances and physical properties within the ocean and across its boundaries; ecosystem dynamics; and plate tectonics and seabed geology.
Oceanographers draw upon a wide range of disciplines to deepen their understanding of the world’s oceans, incorporating insights from astronomy, biology, chemistry, geography, geology, hydrology, meteorology and physics.
== History ==
=== Early history ===
Humans first acquired knowledge of the waves and currents of the seas and oceans in pre-historic times. Observations on tides were recorded by Aristotle and Strabo in 384–322 BC. Early exploration of the oceans was primarily for cartography and mainly limited to its surfaces and of the animals that fishermen brought up in nets, though depth soundings by lead line were taken.
The Portuguese campaign of Atlantic navigation is the earliest example of a systematic scientific large project, sustained over many decades, studying the currents and winds of the Atlantic.
The work of Pedro Nunes (1502–1578) is remembered in the navigation context for the determination of the loxodromic curve: the shortest course between two points on the surface of a sphere represented onto a two-dimensional map. When he published his "Treatise of the Sphere" (1537), mostly a commentated translation of earlier work by others, he included a treatise on geometrical and astronomic methods of navigation. There he states clearly that Portuguese navigations were not an adventurous endeavour:
"nam se fezeram indo a acertar: mas partiam os nossos mareantes muy ensinados e prouidos de estromentos e regras de astrologia e geometria que sam as cousas que os cosmographos ham dadar apercebidas (...) e leuaua cartas muy particularmente rumadas e na ja as de que os antigos vsauam" (were not done by chance: but our seafarers departed well taught and provided with instruments and rules of astrology (astronomy) and geometry which were matters the cosmographers would provide (...) and they took charts with exact routes and no longer those used by the ancient).
His credibility rests on being personally involved in the instruction of pilots and senior seafarers from 1527 onwards by Royal appointment, along with his recognized competence as mathematician and astronomer.
The main problem in navigating back from the south of the Canary Islands (or south of Boujdour) by sail alone, is due to the change in the regime of winds and currents: the North Atlantic gyre and the Equatorial counter current will push south along the northwest bulge of Africa, while the uncertain winds where the Northeast trades meet the Southeast trades (the doldrums) leave a sailing ship to the mercy of the currents. Together, prevalent current and wind make northwards progress very difficult or impossible. It was to overcome this problem and clear the passage to India around Africa as a viable maritime trade route, that a systematic plan of exploration was devised by the Portuguese. The return route from regions south of the Canaries became the 'volta do largo' or 'volta do mar'. The 'rediscovery' of the Azores islands in 1427 is merely a reflection of the heightened strategic importance of the islands, now sitting on the return route from the western coast of Africa (sequentially called 'volta de Guiné' and 'volta da Mina'); and the references to the Sargasso Sea (also called at the time 'Mar da Baga'), to the west of the Azores, in 1436, reveals the western extent of the return route. This is necessary, under sail, to make use of the southeasterly and northeasterly winds away from the western coast of Africa, up to the northern latitudes where the westerly winds will bring the seafarers towards the western coasts of Europe.
The secrecy involving the Portuguese navigations, with the death penalty for the leaking of maps and routes, concentrated all sensitive records in the Royal Archives, completely destroyed by the Lisbon earthquake of 1775. However, the systematic nature of the Portuguese campaign, mapping the currents and winds of the Atlantic, is demonstrated by the understanding of the seasonal variations, with expeditions setting sail at different times of the year taking different routes to take account of seasonal predominate winds. This happens from as early as late 15th century and early 16th: Bartolomeu Dias followed the African coast on his way south in August 1487, while Vasco da Gama would take an open sea route from the latitude of Sierra Leone, spending three months in the open sea of the South Atlantic to profit from the southwards deflection of the southwesterly on the Brazilian side (and the Brazilian current going southward - Gama departed in July 1497); and Pedro Álvares Cabral (departing March 1500) took an even larger arch to the west, from the latitude of Cape Verde, thus avoiding the summer monsoon (which would have blocked the route taken by Gama at the time he set sail). Furthermore, there were systematic expeditions pushing into the western Northern Atlantic (Teive, 1454; Vogado, 1462; Teles, 1474; Ulmo, 1486).
The documents relating to the supplying of ships, and the ordering of sun declination tables for the southern Atlantic for as early as 1493–1496, all suggest a well-planned and systematic activity happening during the decade long period between Bartolomeu Dias finding the southern tip of Africa, and Gama's departure; additionally, there are indications of further travels by Bartolomeu Dias in the area. The most significant consequence of this systematic knowledge was the negotiation of the Treaty of Tordesillas in 1494, moving the line of demarcation 270 leagues to the west (from 100 to 370 leagues west of the Azores), bringing what is now Brazil into the Portuguese area of domination. The knowledge gathered from open sea exploration allowed for the well-documented extended periods of sail without sight of land, not by accident but as pre-determined planned route; for example, 30 days for Bartolomeu Dias culminating on Mossel Bay, the three months Gama spent in the South Atlantic to use the Brazil current (southward), or the 29 days Cabral took from Cape Verde up to landing in Monte Pascoal, Brazil.
The Danish expedition to Arabia 1761–67 can be said to be the world's first oceanographic expedition, as the ship Grønland had on board a group of scientists, including naturalist Peter Forsskål, who was assigned an explicit task by the king, Frederik V, to study and describe the marine life in the open sea, including finding the cause of mareel, or milky seas. For this purpose, the expedition was equipped with nets and scrapers, specifically designed to collect samples from the open waters and the bottom at great depth.
Although Juan Ponce de León in 1513 first identified the Gulf Stream, and the current was well known to mariners, Benjamin Franklin made the first scientific study of it and gave it its name. Franklin measured water temperatures during several Atlantic crossings and correctly explained the Gulf Stream's cause. Franklin and Timothy Folger printed the first map of the Gulf Stream in 1769–1770.
Information on the currents of the Pacific Ocean was gathered by explorers of the late 18th century, including James Cook and Louis Antoine de Bougainville. James Rennell wrote the first scientific textbooks on oceanography, detailing the current flows of the Atlantic and Indian oceans. During a voyage around the Cape of Good Hope in 1777, he mapped "the banks and currents at the Lagullas". He was also the first to understand the nature of the intermittent current near the Isles of Scilly, (now known as Rennell's Current). The tides and currents of the ocean are distinct. Tides are the rise and fall of sea levels created by the combination of the gravitational forces of the Moon along with the Sun (the Sun just in a much lesser extent) and are also caused by the Earth and Moon orbiting each other. An ocean current is a continuous, directed movement of seawater generated by a number of forces acting upon the water, including wind, the Coriolis effect, breaking waves, cabbeling, and temperature and salinity differences.
Sir James Clark Ross took the first modern sounding in deep sea in 1840, and Charles Darwin published a paper on reefs and the formation of atolls as a result of the second voyage of HMS Beagle in 1831–1836. Robert FitzRoy published a four-volume report of Beagle's three voyages. In 1841–1842 Edward Forbes undertook dredging in the Aegean Sea that founded marine ecology.
The first superintendent of the United States Naval Observatory (1842–1861), Matthew Fontaine Maury devoted his time to the study of marine meteorology, navigation, and charting prevailing winds and currents. His 1855 textbook Physical Geography of the Sea was one of the first comprehensive oceanography studies. Many nations sent oceanographic observations to Maury at the Naval Observatory, where he and his colleagues evaluated the information and distributed the results worldwide.
=== Modern oceanography ===
Knowledge of the oceans remained confined to the topmost few fathoms of the water and a small amount of the bottom, mainly in shallow areas. Almost nothing was known of the ocean depths. The British Royal Navy's efforts to chart all of the world's coastlines in the mid-19th century reinforced the vague idea that most of the ocean was very deep, although little more was known. As exploration ignited both popular and scientific interest in the polar regions and Africa, so too did the mysteries of the unexplored oceans.
The seminal event in the founding of the modern science of oceanography was the 1872–1876 Challenger expedition. As the first true oceanographic cruise, this expedition laid the groundwork for an entire academic and research discipline. In response to a recommendation from the Royal Society, the British Government announced in 1871 an expedition to explore world's oceans and conduct appropriate scientific investigation. Charles Wyville Thomson and Sir John Murray launched the Challenger expedition. Challenger, leased from the Royal Navy, was modified for scientific work and equipped with separate laboratories for natural history and chemistry. Under the scientific supervision of Thomson, Challenger travelled nearly 70,000 nautical miles (130,000 km) surveying and exploring. On her journey circumnavigating the globe, 492 deep sea soundings, 133 bottom dredges, 151 open water trawls and 263 serial water temperature observations were taken. Around 4,700 new species of marine life were discovered. The result was the Report Of The Scientific Results of the Exploring Voyage of H.M.S. Challenger during the years 1873–76. Murray, who supervised the publication, described the report as "the greatest advance in the knowledge of our planet since the celebrated discoveries of the fifteenth and sixteenth centuries". He went on to found the academic discipline of oceanography at the University of Edinburgh, which remained the centre for oceanographic research well into the 20th century. Murray was the first to study marine trenches and in particular the Mid-Atlantic Ridge, and map the sedimentary deposits in the oceans. He tried to map out the world's ocean currents based on salinity and temperature observations, and was the first to correctly understand the nature of coral reef development.
In the late 19th century, other Western nations also sent out scientific expeditions (as did private individuals and institutions). The first purpose-built oceanographic ship, Albatros, was built in 1882. In 1893, Fridtjof Nansen allowed his ship, Fram, to be frozen in the Arctic ice. This enabled him to obtain oceanographic, meteorological and astronomical data at a stationary spot over an extended period.
In 1881 the geographer John Francon Williams published a seminal book, Geography of the Oceans. Between 1907 and 1911 Otto Krümmel published the Handbuch der Ozeanographie, which became influential in awakening public interest in oceanography. The four-month 1910 North Atlantic expedition headed by John Murray and Johan Hjort was the most ambitious research oceanographic and marine zoological project ever mounted until then, and led to the classic 1912 book The Depths of the Ocean.
The first acoustic measurement of sea depth was made in 1914. Between 1925 and 1927 the "Meteor" expedition gathered 70,000 ocean depth measurements using an echo sounder, surveying the Mid-Atlantic Ridge.
In 1934, Easter Ellen Cupp, the first woman to have earned a PhD (at Scripps) in the United States, completed a major work on diatoms that remained the standard taxonomy in the field until well after her death in 1999. In 1940, Cupp was let go from her position at Scripps. Sverdrup specifically commended Cupp as a conscientious and industrious worker and commented that his decision was no reflection on her ability as a scientist. Sverdrup used the instructor billet vacated by Cupp to employ Marston Sargent, a biologist studying marine algae, which was not a new research program at Scripps. Financial pressures did not prevent Sverdrup from retaining the services of two other young post-doctoral students, Walter Munk and Roger Revelle. Cupp's partner, Dorothy Rosenbury, found her a position teaching high school, where she remained for the rest of her career. (Russell, 2000)
Sverdrup, Johnson and Fleming published The Oceans in 1942, which was a major landmark. The Sea (in three volumes, covering physical oceanography, seawater and geology) edited by M.N. Hill was published in 1962, while Rhodes Fairbridge's Encyclopedia of Oceanography was published in 1966.
The Great Global Rift, running along the Mid Atlantic Ridge, was discovered by Maurice Ewing and Bruce Heezen in 1953 and mapped by Heezen and Marie Tharp using bathymetric data; in 1954 a mountain range under the Arctic Ocean was found by the Arctic Institute of the USSR. The theory of seafloor spreading was developed in 1960 by Harry Hammond Hess. The Ocean Drilling Program started in 1966. Deep-sea vents were discovered in 1977 by Jack Corliss and Robert Ballard in the submersible DSV Alvin.
In the 1950s, Auguste Piccard invented the bathyscaphe and used the bathyscaphe Trieste to investigate the ocean's depths. The United States nuclear submarine Nautilus made the first journey under the ice to the North Pole in 1958. In 1962 the FLIP (Floating Instrument Platform), a 355-foot (108 m) spar buoy, was first deployed.
In 1968, Tanya Atwater led the first all-woman oceanographic expedition. Until that time, gender policies restricted women oceanographers from participating in voyages to a significant extent.
From the 1970s, there has been much emphasis on the application of large scale computers to oceanography to allow numerical predictions of ocean conditions and as a part of overall environmental change prediction. Early techniques included analog computers (such as the Ishiguro Storm Surge Computer) generally now replaced by numerical methods (e.g. SLOSH.) An oceanographic buoy array was established in the Pacific to allow prediction of El Niño events.
1990 saw the start of the World Ocean Circulation Experiment (WOCE) which continued until 2002. Geosat seafloor mapping data became available in 1995.
Study of the oceans is critical to understanding shifts in Earth's energy balance along with related global and regional changes in climate, the biosphere and biogeochemistry. The atmosphere and ocean are linked because of evaporation and precipitation as well as thermal flux (and solar insolation). Recent studies have advanced knowledge on ocean acidification, ocean heat content, ocean currents, sea level rise, the oceanic carbon cycle, the water cycle, Arctic sea ice decline, coral bleaching, marine heatwaves, extreme weather, coastal erosion and many other phenomena in regards to ongoing climate change and climate feedbacks.
In general, understanding the world ocean through further scientific study enables better stewardship and sustainable utilization of Earth's resources. The Intergovernmental Oceanographic Commission reports that 1.7% of the total national research expenditure of its members is focused on ocean science.
== Branches ==
The study of oceanography is divided into these five branches:
=== Biological oceanography ===
Biological oceanography investigates the ecology and biology of marine organisms in the context of the physical, chemical and geological characteristics of their ocean environment.
=== Chemical oceanography ===
Chemical oceanography is the study of the chemistry of the ocean. Whereas chemical oceanography is primarily occupied with the study and understanding of seawater properties and its changes, ocean chemistry focuses primarily on the geochemical cycles. The following is a central topic investigated by chemical oceanography.
==== Ocean acidification ====
Ocean acidification describes the decrease in ocean pH that is caused by anthropogenic carbon dioxide (CO2) emissions into the atmosphere. Seawater is slightly alkaline and had a preindustrial pH of about 8.2. More recently, anthropogenic activities have steadily increased the carbon dioxide content of the atmosphere; about 30–40% of the added CO2 is absorbed by the oceans, forming carbonic acid and lowering the pH (now below 8.1) through ocean acidification. The pH is expected to reach 7.7 by the year 2100.
An important element for the skeletons of marine animals is calcium, but calcium carbonate becomes more soluble with pressure, so carbonate shells and skeletons dissolve below the carbonate compensation depth. Calcium carbonate becomes more soluble at lower pH, so ocean acidification is likely to affect marine organisms with calcareous shells, such as oysters, clams, sea urchins and corals, and the carbonate compensation depth will rise closer to the sea surface. Affected planktonic organisms will include pteropods, coccolithophorids and foraminifera, all important in the food chain. In tropical regions, corals are likely to be severely affected as they become less able to build their calcium carbonate skeletons, in turn adversely impacting other reef dwellers.
The current rate of ocean chemistry change seems to be unprecedented in Earth's geological history, making it unclear how well marine ecosystems will adapt to the shifting conditions of the near future. Of particular concern is the manner in which the combination of acidification with the expected additional stressors of higher ocean temperatures and lower oxygen levels will impact the seas.
=== Geological oceanography ===
Geological oceanography is the study of the geology of the ocean floor including plate tectonics and paleoceanography.
=== Physical oceanography ===
Physical oceanography studies the ocean's physical attributes including temperature-salinity structure, mixing, surface waves, internal waves, surface tides, internal tides, and currents. The following are central topics investigated by physical oceanography.
==== Seismic Oceanography ====
==== Ocean currents ====
Since the early ocean expeditions in oceanography, a major interest was the study of ocean currents and temperature measurements. The tides, the Coriolis effect, changes in direction and strength of wind, salinity, and temperature are the main factors determining ocean currents. The thermohaline circulation (THC) (thermo- referring to temperature and -haline referring to salt content) connects the ocean basins and is primarily dependent on the density of sea water. It is becoming more common to refer to this system as the 'meridional overturning circulation' because it more accurately accounts for other driving factors beyond temperature and salinity.
Examples of sustained currents are the Gulf Stream and the Kuroshio Current which are wind-driven western boundary currents.
==== Ocean heat content ====
Oceanic heat content (OHC) refers to the extra heat stored in the ocean from changes in Earth's energy balance. The increase in the ocean heat play an important role in sea level rise, because of thermal expansion. Ocean warming accounts for 90% of the energy accumulation associated with global warming since 1971.
=== Paleoceanography ===
Paleoceanography is the study of the history of the oceans in the geologic past with regard to circulation, chemistry, biology, geology and patterns of sedimentation and biological productivity. Paleoceanographic studies using environment models and different proxies enable the scientific community to assess the role of the oceanic processes in the global climate by the reconstruction of past climate at various intervals. Paleoceanographic research is also intimately tied to palaeoclimatology.
== Oceanographic institutions ==
The earliest international organizations of oceanography were founded at the turn of the 20th century, starting with the International Council for the Exploration of the Sea created in 1902, followed in 1919 by the Mediterranean Science Commission. Marine research institutes were already in existence, starting with the Stazione Zoologica Anton Dohrn in Naples, Italy (1872), the Biological Station of Roscoff, France (1876), the Arago Laboratory in Banyuls-sur-mer, France (1882), the Laboratory of the Marine Biological Association in Plymouth, UK (1884), the Norwegian Institute for Marine Research in Bergen, Norway (1900), the Laboratory für internationale Meeresforschung, Kiel, Germany (1902). On the other side of the Atlantic, the Scripps Institution of Oceanography was founded in 1903, followed by the Woods Hole Oceanographic Institution in 1930, the Virginia Institute of Marine Science in 1938, the Lamont–Doherty Earth Observatory at Columbia University in 1949, and later the School of Oceanography at University of Washington. In Australia, the Australian Institute of Marine Science (AIMS), established in 1972 soon became a key player in marine tropical research.
In 1921 the International Hydrographic Bureau, called since 1970 the International Hydrographic Organization, was established to develop hydrographic and nautical charting standards.
== Related disciplines ==
== See also ==
== References ==
=== Sources and further reading ===
Boling Guo, Daiwen Huang. Infinite-Dimensional Dynamical Systems in Atmospheric and Oceanic Science, 2014, World Scientific Publishing, ISBN 978-981-4590-37-2. Sample Chapter
Hamblin, Jacob Darwin (2005) Oceanographers and the Cold War: Disciples of Marine Science. University of Washington Press. ISBN 978-0-295-98482-7
Lang, Michael A., Ian G. Macintyre, and Klaus Rützler, eds. Proceedings of the Smithsonian Marine Science Symposium. Smithsonian Contributions to the Marine Sciences, no. 38. Washington, D.C.: Smithsonian Institution Scholarly Press (2009)
Roorda, Eric Paul, ed. The Ocean Reader: History, Culture, Politics (Duke University Press, 2020) 523 pp. online review
Steele, J., K. Turekian and S. Thorpe. (2001). Encyclopedia of Ocean Sciences. San Diego: Academic Press. (6 vols.) ISBN 0-12-227430-X
Sverdrup, Keith A., Duxbury, Alyn C., Duxbury, Alison B. (2006). Fundamentals of Oceanography, McGraw-Hill, ISBN 0-07-282678-9
Russell, Joellen Louise. Easter Ellen Cupp, 2000, Regents of the University of California.
== External links ==
NASA Jet Propulsion Laboratory – Physical Oceanography Distributed Active Archive Center (PO.DAAC). A data centre responsible for archiving and distributing data about the physical state of the ocean.
Scripps Institution of Oceanography. One of the world's oldest, largest, and most important centres for ocean and Earth science research, education, and public service.
Woods Hole Oceanographic Institution (WHOI). One of the world's largest private, non-profit ocean research, engineering and education organizations.
British Oceanographic Data Centre. A source of oceanographic data and information.
NOAA Ocean and Weather Data Navigator. Plot and download ocean data.
Freeview Video 'Voyage to the Bottom of the Deep Deep Sea' Oceanography Programme by the Vega Science Trust and the BBC/Open University.
Atlas of Spanish Oceanography by InvestigAdHoc.
Glossary of Physical Oceanography and Related Disciplines by Steven K. Baum, Department of Oceanography, Texas A&M University
Barcelona-Ocean.com . Inspiring Education in Marine Sciences
CFOO: Sea Atlas. A source of oceanographic live data (buoy monitoring) and education for South African coasts.
Oceanography on In Our Time at the BBC
Memorial website for USNS Bowditch, USNS Dutton, USNS Michelson and USNS H. H. Hess | Wikipedia/Oceanographer |
Renewable energy (also called green energy) is energy made from renewable natural resources that are replenished on a human timescale. The most widely used renewable energy types are solar energy, wind power, and hydropower. Bioenergy and geothermal power are also significant in some countries. Some also consider nuclear power a renewable power source, although this is controversial, as nuclear energy requires mining uranium, a nonrenewable resource. Renewable energy installations can be large or small and are suited for both urban and rural areas. Renewable energy is often deployed together with further electrification. This has several benefits: electricity can move heat and vehicles efficiently and is clean at the point of consumption. Variable renewable energy sources are those that have a fluctuating nature, such as wind power and solar power. In contrast, controllable renewable energy sources include dammed hydroelectricity, bioenergy, or geothermal power.
Renewable energy systems have rapidly become more efficient and cheaper over the past 30 years. A large majority of worldwide newly installed electricity capacity is now renewable. Renewable energy sources, such as solar and wind power, have seen significant cost reductions over the past decade, making them more competitive with traditional fossil fuels. In most countries, photovoltaic solar or onshore wind are the cheapest new-build electricity. From 2011 to 2021, renewable energy grew from 20% to 28% of global electricity supply. Power from the sun and wind accounted for most of this increase, growing from a combined 2% to 10%. Use of fossil energy shrank from 68% to 62%. In 2024, renewables accounted for over 30% of global electricity generation and are projected to reach over 45% by 2030. Many countries already have renewables contributing more than 20% of their total energy supply, with some generating over half or even all their electricity from renewable sources.
The main motivation to use renewable energy instead of fossil fuels is to slow and eventually stop climate change, which is mostly caused by their greenhouse gas emissions. In general, renewable energy sources pollute much less than fossil fuels. The International Energy Agency estimates that to achieve net zero emissions by 2050, 90% of global electricity will need to be generated by renewables. Renewables also cause much less air pollution than fossil fuels, improving public health, and are less noisy.
The deployment of renewable energy still faces obstacles, especially fossil fuel subsidies, lobbying by incumbent power providers, and local opposition to the use of land for renewable installations. Like all mining, the extraction of minerals required for many renewable energy technologies also results in environmental damage. In addition, although most renewable energy sources are sustainable, some are not.
== Overview ==
=== Definition ===
Renewable energy is usually understood as energy harnessed from continuously occurring natural phenomena. The International Energy Agency defines it as "energy derived from natural processes that are replenished at a faster rate than they are consumed". Solar power, wind power, hydroelectricity, geothermal energy, and biomass are widely agreed to be the main types of renewable energy. Renewable energy often displaces conventional fuels in four areas: electricity generation, hot water/space heating, transportation, and rural (off-grid) energy services.
Although almost all forms of renewable energy cause much fewer carbon emissions than fossil fuels, the term is not synonymous with low-carbon energy. Some non-renewable sources of energy, such as nuclear power,generate almost no emissions, while some renewable energy sources can be very carbon-intensive, such as the burning of biomass if it is not offset by planting new plants. Renewable energy is also distinct from sustainable energy, a more abstract concept that seeks to group energy sources based on their overall permanent impact on future generations of humans. For example, biomass is often associated with unsustainable deforestation.
=== Role in addressing climate change ===
As part of the global effort to limit climate change, most countries have committed to net zero greenhouse gas emissions. In practice, this means phasing out fossil fuels and replacing them with low-emissions energy sources. This much needed process, coined as "low-carbon substitutions" in contrast to other transition processes including energy additions, needs to be accelerated multiple times in order to successfully mitigate climate change. At the 2023 United Nations Climate Change Conference, around three-quarters of the world's countries set a goal of tripling renewable energy capacity by 2030. The European Union aims to generate 40% of its electricity from renewables by the same year.
=== Other benefits ===
Renewable energy is more evenly distributed around the world than fossil fuels, which are concentrated in a limited number of countries. It also brings health benefits by reducing air pollution caused by the burning of fossil fuels. The potential worldwide savings in health care costs have been estimated at trillions of dollars annually.
=== Intermittency ===
The two most important forms of renewable energy, solar and wind, are intermittent energy sources: they are not available constantly, resulting in lower capacity factors. In contrast, fossil fuel power plants, nuclear power plants and hydropower are usually able to produce precisely the amount of energy an electricity grid requires at a given time. Solar energy can only be captured during the day, and ideally in cloudless conditions. Wind power generation can vary significantly not only day-to-day, but even month-to-month. This poses a challenge when transitioning away from fossil fuels: energy demand will often be higher or lower than what renewables can provide.
In the medium-term, this variability may require keeping some gas-fired power plants or other dispatchable generation on standby until there is enough energy storage, demand response, grid improvement, or base load power from non-intermittent sources. In the long-term, energy storage is an important way of dealing with intermittency. Using diversified renewable energy sources and smart grids can also help flatten supply and demand.
Sector coupling of the power generation sector with other sectors may increase flexibility: for example the transport sector can be coupled by charging electric vehicles and sending electricity from vehicle to grid. Similarly the industry sector can be coupled by hydrogen produced by electrolysis, and the buildings sector by thermal energy storage for space heating and cooling.
Building overcapacity for wind and solar generation can help ensure sufficient electricity production even during poor weather. In optimal weather, it may be necessary to curtail energy generation if it is not possible to use or store excess electricity.
==== Electrical energy storage ====
Electrical energy storage is a collection of methods used to store electrical energy. Electrical energy is stored during times when production (especially from intermittent sources such as wind power, tidal power, solar power) exceeds consumption, and returned to the grid when production falls below consumption. Pumped-storage hydroelectricity accounts for more than 85% of all grid power storage. Batteries are increasingly being deployed for storage and grid ancillary services and for domestic storage. Green hydrogen is a more economical means of long-term renewable energy storage, in terms of capital expenditures compared to pumped hydroelectric or batteries.
==== Energy supply security ====
Two main renewable energy sources - solar power and wind power - are usually deployed in distributed generation architecture, which offers specific benefits and comes with specific risks. Notable risks are associated with centralisation of 90% of the supply chains in a single country (China) in the photovoltaic sector. Mass-scale installation of photovoltaic power inverters with remote control, security vulnerabilities and backdoors results in cyberattacks that can disable generation from millions of physically decentralised panels, resulting in disappearance of hundreds of gigawatts of installed power from the grid in one moment. Similar attacks have targeted wind power farms through vulnerabilities in their remote control and monitoring systems. The European NIS2 directive partially responds to these challenges by extending the scope of cybersecurity regulations to the energy generation market.
== Mainstream technologies ==
=== Solar energy ===
Solar power produced around 1.3 terrawatt-hours (TWh) worldwide in 2022, representing 4.6% of the world's electricity. Almost all of this growth has happened since 2010. Solar energy can be harnessed anywhere that receives sunlight; however, the amount of solar energy that can be harnessed for electricity generation is influenced by weather conditions, geographic location and time of day.
There are two mainstream ways of harnessing solar energy: solar thermal, which converts solar energy into heat; and photovoltaics (PV), which converts it into electricity. PV is far more widespread, accounting for around two thirds of the global solar energy capacity as of 2022. It is also growing at a much faster rate, with 170 GW newly installed capacity in 2021, compared to 25 GW of solar thermal.
Passive solar refers to a range of construction strategies and technologies that aim to optimize the distribution of solar heat in a building. Examples include solar chimneys, orienting a building to the sun, using construction materials that can store heat, and designing spaces that naturally circulate air.
From 2020 to 2022, solar technology investments almost doubled from USD 162 billion to USD 308 billion, driven by the sector's increasing maturity and cost reductions, particularly in solar photovoltaic (PV), which accounted for 90% of total investments. China and the United States were the main recipients, collectively making up about half of all solar investments since 2013. Despite reductions in Japan and India due to policy changes and COVID-19, growth in China, the United States, and a significant increase from Vietnam's feed-in tariff program offset these declines. Globally, the solar sector added 714 gigawatts (GW) of solar PV and concentrated solar power (CSP) capacity between 2013 and 2021, with a notable rise in large-scale solar heating installations in 2021, especially in China, Europe, Turkey, and Mexico.
==== Photovoltaics ====
A photovoltaic system, consisting of solar cells assembled into panels, converts light into electrical direct current via the photoelectric effect. PV has several advantages that make it by far the fastest-growing renewable energy technology. It is cheap, low-maintenance and scalable; adding to an existing PV installation as demanded arises is simple. Its main disadvantage is its poor performance in cloudy weather.
PV systems range from small, residential and commercial rooftop or building integrated installations, to large utility-scale photovoltaic power station. A household's solar panels can either be used for just that household or, if connected to an electrical grid, can be aggregated with millions of others.
The first utility-scale solar power plant was built in 1982 in Hesperia, California by ARCO. The plant was not profitable and was sold eight years later. However, over the following decades, PV cells became significantly more efficient and cheaper. As a result, PV adoption has grown exponentially since 2010. Global capacity increased from 230 GW at the end of 2015 to 890 GW in 2021. PV grew fastest in China between 2016 and 2021, adding 560 GW, more than all advanced economies combined. Four of the ten biggest solar power stations are in China, including the biggest, Golmud Solar Park in China.
Solar panels are recycled to reduce electronic waste and create a source for materials that would otherwise need to be mined, but such business is still small and work is ongoing to improve and scale-up the process.
==== Solar thermal ====
Unlike photovoltaic cells that convert sunlight directly into electricity, solar thermal systems convert it into heat. They use mirrors or lenses to concentrate sunlight onto a receiver, which in turn heats a water reservoir. The heated water can then be used in homes. The advantage of solar thermal is that the heated water can be stored until it is needed, eliminating the need for a separate energy storage system. Solar thermal power can also be converted to electricity by using the steam generated from the heated water to drive a turbine connected to a generator. However, because generating electricity this way is much more expensive than photovoltaic power plants, there are very few in use today.
==== Floatovoltaics ====
Floatovoltiacs, or floating solar panels, are solar panels floating on bodies of water. There are both positive and negative points to this. Some positive points are increased efficiency and price decrease of water space compared to land space. A negative point is that making floating solar panels could be more expensive.
==== Agrivoltaics ====
Agrivoltaics is where there is simultaneous use of land for energy production and agriculture. There are again both positive and negative points. A positive viewpoint is there is a better use of land, which leads to lower land costs. A negative viewpoint is it the plants grown underneath would have to be plants that can grow well under shade, such as Polka Dot Plant, Pineapple Sage, and Begonia. Agrivoltaics not only optimizes land use and reduces costs by enabling dual revenue streams from both energy production and agriculture, but it can also help moderate temperatures beneath the panels, potentially reducing water loss and improving microclimates for crop growth. However, careful design and crop selection are crucial, as the shading effect may limit the types of plants that can thrive, necessitating the use of shade-tolerant species and innovative management practices.
=== Wind power ===
Humans have harnessed wind energy since at least 3500 BC. Until the 20th century, it was primarily used to power ships, windmills and water pumps. Today, the vast majority of wind power is used to generate electricity using wind turbines. Modern utility-scale wind turbines range from around 600 kW to 9 MW of rated power. The power available from the wind is a function of the cube of the wind speed, so as wind speed increases, power output increases up to the maximum output for the particular turbine. Areas where winds are stronger and more constant, such as offshore and high-altitude sites, are preferred locations for wind farms.
Wind-generated electricity met nearly 4% of global electricity demand in 2015, with nearly 63 GW of new wind power capacity installed. Wind energy was the leading source of new capacity in Europe, the US and Canada, and the second largest in China. In Denmark, wind energy met more than 40% of its electricity demand while Ireland, Portugal and Spain each met nearly 20%.
Globally, the long-term technical potential of wind energy is believed to be five times total current global energy production, or 40 times current electricity demand, assuming all practical barriers needed were overcome. This would require wind turbines to be installed over large areas, particularly in areas of higher wind resources, such as offshore, and likely also industrial use of new types of VAWT turbines in addition to the horizontal axis units currently in use. As offshore wind speeds average ~90% greater than that of land, offshore resources can contribute substantially more energy than land-stationed turbines.
Investments in wind technologies reached USD 161 billion in 2020, with onshore wind dominating at 80% of total investments from 2013 to 2022. Offshore wind investments nearly doubled to USD 41 billion between 2019 and 2020, primarily due to policy incentives in China and expansion in Europe. Global wind capacity increased by 557 GW between 2013 and 2021, with capacity additions increasing by an average of 19% each year.
=== Hydropower ===
Since water is about 800 times denser than air, even a slow flowing stream of water, or moderate sea swell, can yield considerable amounts of energy. Water can generate electricity with a conversion efficiency of about 90%, which is the highest rate in renewable energy. There are many forms of water energy:
Historically, hydroelectric power came from constructing large hydroelectric dams and reservoirs, which are still popular in developing countries. The largest of them are the Three Gorges Dam (2003) in China and the Itaipu Dam (1984) built by Brazil and Paraguay.
Small hydro systems are hydroelectric power installations that typically produce up to 50 MW of power. They are often used on small rivers or as a low-impact development on larger rivers. China is the largest producer of hydroelectricity in the world and has more than 45,000 small hydro installations.
Run-of-the-river hydroelectricity plants derive energy from rivers without the creation of a large reservoir. The water is typically conveyed along the side of the river valley (using channels, pipes or tunnels) until it is high above the valley floor, whereupon it can be allowed to fall through a penstock to drive a turbine. A run-of-river plant may still produce a large amount of electricity, such as the Chief Joseph Dam on the Columbia River in the United States. However many run-of-the-river hydro power plants are micro hydro or pico hydro plants.
Much hydropower is flexible, thus complementing wind and solar, as it not intermittent. In 2021, the world renewable hydropower capacity was 1,360 GW. Only a third of the world's estimated hydroelectric potential of 14,000 TWh/year has been developed. New hydropower projects face opposition from local communities due to their large impact, including relocation of communities and flooding of wildlife habitats and farming land. High cost and lead times from permission process, including environmental and risk assessments, with lack of environmental and social acceptance are therefore the primary challenges for new developments. It is popular to repower old dams thereby increasing their efficiency and capacity as well as quicker responsiveness on the grid. Where circumstances permit existing dams such as the Russell Dam built in 1985 may be updated with "pump back" facilities for pumped-storage which is useful for peak loads or to support intermittent wind and solar power. Because dispatchable power is more valuable than VRE countries with large hydroelectric developments such as Canada and Norway are spending billions to expand their grids to trade with neighboring countries having limited hydro.
=== Bioenergy ===
Biomass is biological material derived from living, or recently living organisms. Most commonly, it refers to plants or plant-derived materials. As an energy source, biomass can either be used directly via combustion to produce heat, or converted to a more energy-dense biofuel like ethanol. Wood is the most significant biomass energy source as of 2012 and is usually sourced from a trees cleared for silvicultural reasons or fire prevention. Municipal wood waste – for instance, construction materials or sawdust – is also often burned for energy. The biggest per-capita producers of wood-based bioenergy are heavily forested countries like Finland, Sweden, Estonia, Austria, and Denmark.
Bioenergy can be environmentally destructive if old-growth forests are cleared to make way for crop production. In particular, demand for palm oil to produce biodiesel has contributed to the deforestation of tropical rainforests in Brazil and Indonesia. In addition, burning biomass still produces carbon emissions, although much less than fossil fuels (39 grams of CO2 per megajoule of energy, compared to 75 g/MJ for fossil fuels).
Some biomass sources are unsustainable at current rates of exploitation (as of 2017).
==== Biofuel ====
Biofuels are primarily used in transportation, providing 3.5% of the world's transport energy demand in 2022, up from 2.7% in 2010. Biojet is expected to be important for short-term reduction of carbon dioxide emissions from long-haul flights.
Aside from wood, the major sources of bioenergy are bioethanol and biodiesel. Bioethanol is usually produced by fermenting the sugar components of crops like sugarcane and maize, while biodiesel is mostly made from oils extracted from plants, such as soybean oil and corn oil. Most of the crops used to produce bioethanol and biodiesel are grown specifically for this purpose, although used cooking oil accounted for 14% of the oil used to produce biodiesel as of 2015. The biomass used to produce biofuels varies by region. Maize is the major feedstock in the United States, while sugarcane dominates in Brazil. In the European Union, where biodiesel is more common than bioethanol, rapeseed oil and palm oil are the main feedstocks. China, although it produces comparatively much less biofuel, uses mostly corn and wheat. In many countries, biofuels are either subsidized or mandated to be included in fuel mixtures.
There are many other sources of bioenergy that are more niche, or not yet viable at large scales. For instance, bioethanol could be produced from the cellulosic parts of crops, rather than only the seed as is common today. Sweet sorghum may be a promising alternative source of bioethanol, due to its tolerance of a wide range of climates. Cow dung can be converted into methane. There is also a great deal of research involving algal fuel, which is attractive because algae is a non-food resource, grows around 20 times faster than most food crops, and can be grown almost anywhere.
=== Geothermal energy ===
Geothermal energy is thermal energy (heat) extracted from the Earth's crust. It originates from several different sources, of which the most significant is slow radioactive decay of minerals contained in the Earth's interior, as well as some leftover heat from the formation of the Earth. Some of the heat is generated near the Earth's surface in the crust, but some also flows from deep within the Earth from the mantle and core. Geothermal energy extraction is viable mostly in countries located on tectonic plate edges, where the Earth's hot mantle is more exposed. As of 2023, the United States has by far the most geothermal capacity (2.7 GW, or less than 0.2% of the country's total energy capacity), followed by Indonesia and the Philippines. Global capacity in 2022 was 15 GW.
Geothermal energy can be either used directly to heat homes, as is common in Iceland where almost all of its energy is renewable, or to generate electricity. Iceland is a global leader in renewable energy, relying almost entirely on its abundant geothermal and hydroelectric resources derived from volcanic activity and glaciers. At smaller scales, geothermal power can be generated with geothermal heat pumps, which can extract heat from ground temperatures of under 30 °C (86 °F), allowing them to be used at relatively shallow depths of a few meters. Electricity generation requires large plants and ground temperatures of at least 150 °C (302 °F). In some countries, electricity produced from geothermal energy accounts for a large portion of the total, such as Kenya (43%) and Indonesia (5%).
Technical advances may eventually make geothermal power more widely available. For example, enhanced geothermal systems involve drilling around 10 kilometres (6.2 mi) into the Earth, breaking apart hot rocks and extracting the heat using water. In theory, this type of geothermal energy extraction could be done anywhere on Earth.
== Emerging technologies ==
There are also other renewable energy technologies that are still under development, including enhanced geothermal systems, concentrated solar power, cellulosic ethanol, and marine energy. These technologies are not yet widely demonstrated or have limited commercialization. Some may have potential comparable to other renewable energy technologies, but still depend on further breakthroughs from research, development and engineering.
=== Enhanced geothermal systems ===
Enhanced geothermal systems (EGS) are a new type of geothermal power which does not require natural hot water reservoirs or steam to generate power. Most of the underground heat within drilling reach is trapped in solid rocks, not in water. EGS technologies use hydraulic fracturing to break apart these rocks and release the heat they contain, which is then harvested by pumping water into the ground. The process is sometimes known as "hot dry rock" (HDR). Unlike conventional geothermal energy extraction, EGS may be feasible anywhere in the world, depending on the cost of drilling. EGS projects have so far primarily been limited to demonstration plants, as the technology is capital-intensive due to the high cost of drilling.
=== Marine energy ===
Marine energy (also sometimes referred to as ocean energy) is the energy carried by ocean waves, tides, salinity, and ocean temperature differences. Technologies to harness the energy of moving water include wave power, marine current power, and tidal power. Reverse electrodialysis (RED) is a technology for generating electricity by mixing fresh water and salty sea water in large power cells. Most marine energy harvesting technologies are still at low technology readiness levels and not used at large scales. Tidal energy is generally considered the most mature, but has not seen wide deployment. The world's largest tidal power station is on Sihwa Lake, South Korea, which produces around 550 gigawatt-hours of electricity per year.
=== Earth infrared thermal radiation ===
Earth emits roughly 1017 W of infrared thermal radiation that flows toward the cold outer space. Solar energy hits the surface and atmosphere of the earth and produces heat. Using various theorized devices like emissive energy harvester (EEH) or thermoradiative diode, this energy flow can be converted into electricity. In theory, this technology can be used during nighttime.
=== Others ===
==== Algae fuels ====
Producing liquid fuels from oil-rich (fat-rich) varieties of algae is an ongoing research topic. Various microalgae grown in open or closed systems are being tried including some systems that can be set up in brownfield and desert lands.
==== Space-based solar power ====
There have been numerous proposals for space-based solar power, in which very large satellites with photovoltaic panels would be equipped with microwave transmitters to beam power back to terrestrial receivers. A 2024 study by the NASA Office of Science and Technology Policy examined the concept and concluded that with current and near-future technologies it would be economically uncompetitive.
==== Water vapor ====
Collection of static electricity charges from water droplets on metal surfaces is an experimental technology that would be especially useful in low-income countries with relative air humidity over 60%.
==== Nuclear energy ====
Breeder reactors could, in principle, depending on the fuel cycle employed, extract almost all of the energy contained in uranium or thorium, decreasing fuel requirements by a factor of 100 compared to widely used once-through light water reactors, which extract less than 1% of the energy in the actinide metal (uranium or thorium) mined from the earth. The high fuel-efficiency of breeder reactors could greatly reduce concerns about fuel supply, energy used in mining, and storage of radioactive waste. With seawater uranium extraction (currently too expensive to be economical), there is enough fuel for breeder reactors to satisfy the world's energy needs for 5 billion years at 1983's total energy consumption rate, thus making nuclear energy effectively a renewable energy. In addition to seawater the average crustal granite rocks contain significant quantities of uranium and thorium with which breeder reactors can supply abundant energy for the remaining lifespan of the sun on the main sequence of stellar evolution.
==== Artificial photosynthesis ====
Artificial photosynthesis uses techniques including nanotechnology to store solar electromagnetic energy in chemical bonds by splitting water to produce hydrogen and then using carbon dioxide to make methanol. Researchers in this field strived to design molecular mimics of photosynthesis that use a wider region of the solar spectrum, employ catalytic systems made from abundant, inexpensive materials that are robust, readily repaired, non-toxic, stable in a variety of environmental conditions and perform more efficiently allowing a greater proportion of photon energy to end up in the storage compounds, i.e., carbohydrates (rather than building and sustaining living cells). However, prominent research faces hurdles, Sun Catalytix a MIT spin-off stopped scaling up their prototype fuel-cell in 2012 because it offers few savings over other ways to make hydrogen from sunlight.
Recent research emphasizes that while artificial photosynthesis shows promise in splitting water to generate hydrogen, its broader significance lies in the ability to produce dense, carbon-based solar fuels suitable for transport applications, such as aviation and long-haul shipping. These fuels, if derived from carbon dioxide and water using sunlight, could close the carbon loop and reduce reliance on fossil-based hydrocarbons. However, realizing this potential requires overcoming major technical hurdles, including the development of efficient, durable catalysts for water oxidation and CO₂ reduction, and careful attention to land use and public perception.
== Market and industry trends ==
Most new renewables are solar, followed by wind then hydro then bioenergy. Investment in renewables, especially solar, tends to be more effective in creating jobs than coal, gas or oil. Worldwide, renewables employ about 12 million people as of 2020, with solar PV being the technology employing the most at almost 4 million. However, as of February 2024, the world's supply of workforce for solar energy is lagging greatly behind demand as universities worldwide still produce more workforce for fossil fuels than for renewable energy industries.
In 2021, China accounted for almost half of the global increase in renewable electricity.
There are 3,146 gigawatts installed in 135 countries, while 156 countries have laws regulating the renewable energy sector.
Globally in 2020 there are over 10 million jobs associated with the renewable energy industries, with solar photovoltaics being the largest renewable employer. The clean energy sectors added about 4.7 million jobs globally between 2019 and 2022, totaling 35 million jobs by 2022.: 5
=== Usage by sector or application ===
Some studies say that a global transition to 100% renewable energy across all sectors – power, heat, transport and industry – is feasible and economically viable.
One of the efforts to decarbonize transportation is the increased use of electric vehicles (EVs). Despite that and the use of biofuels, such as biojet, less than 4% of transport energy is from renewables. Occasionally hydrogen fuel cells are used for heavy transport. Meanwhile, in the future electrofuels may also play a greater role in decarbonizing hard-to-abate sectors like aviation and maritime shipping.
Solar water heating makes an important contribution to renewable heat in many countries, most notably in China, which now has 70% of the global total (180 GWth). Most of these systems are installed on multi-family apartment buildings and meet a portion of the hot water needs of an estimated 50–60 million households in China. Worldwide, total installed solar water heating systems meet a portion of the water heating needs of over 70 million households.
Heat pumps provide both heating and cooling, and also flatten the electric demand curve and are thus an increasing priority. Renewable thermal energy is also growing rapidly. About 10% of heating and cooling energy is from renewables.
=== Cost comparison ===
The International Renewable Energy Agency (IRENA) stated that ~86% (187 GW) of renewable capacity added in 2022 had lower costs than electricity generated from fossil fuels. IRENA also stated that capacity added since 2000 reduced electricity bills in 2022 by at least $520 billion, and that in non-OECD countries, the lifetime savings of 2022 capacity additions will reduce costs by up to $580 billion.
* = 2018. All other values for 2019.
=== Growth of renewables ===
The results of a recent review of the literature concluded that as greenhouse gas (GHG) emitters begin to be held liable for damages resulting from GHG emissions resulting in climate change, a high value for liability mitigation would provide powerful incentives for deployment of renewable energy technologies.
In the decade of 2010–2019, worldwide investment in renewable energy capacity excluding large hydropower amounted to US$2.7 trillion, of which the top countries China contributed US$818 billion, the United States contributed US$392.3 billion, Japan contributed US$210.9 billion, Germany contributed US$183.4 billion, and the United Kingdom contributed US$126.5 billion. This was an increase of over three and possibly four times the equivalent amount invested in the decade of 2000–2009 (no data is available for 2000–2003).
As of 2022, an estimated 28% of the world's electricity was generated by renewables. This is up from 19% in 1990.
==== Future projections ====
A December 2022 report by the IEA forecasts that over 2022-2027, renewables are seen growing by almost 2,400 GW in its main forecast, equal to the entire installed power capacity of China in 2021. This is an 85% acceleration from the previous five years, and almost 30% higher than what the IEA forecast in its 2021 report, making its largest ever upward revision. Renewables are set to account for over 90% of global electricity capacity expansion over the forecast period. To achieve net zero emissions by 2050, IEA believes that 90% of global electricity generation will need to be produced from renewable sources.
In June 2022, IEA Executive Director Fatih Birol said that countries should invest more in renewables to "ease the pressure on consumers from high fossil fuel prices, make our energy systems more secure, and get the world on track to reach our climate goals."
China's five year plan to 2025 includes increasing direct heating by renewables such as geothermal and solar thermal.
REPowerEU, the EU plan to escape dependence on fossil Russian gas, is expected to call for much more green hydrogen.
After a transitional period, renewable energy production is expected to make up most of the world's energy production. In 2018, the risk management firm, DNV GL, forecasts that the world's primary energy mix will be split equally between fossil and non-fossil sources by 2050.
Middle eastern nations are also planning on reducing their reliance fossil fuel. Many planned green projects will contribute in 26% of energy supply for the region by 2050 achieving emission reductions equal to 1.1 Gt CO2/year.
Massive Renewable Energy Projects in the Middle East:
Mohammed bin Rashid Al Maktoum Solar Park in Dubai, UAE
Shuaibah Two (2) Solar Facility in Mecca Province, Saudi Arabia
NEOM Green Hydrogen Project in NEOM, Saudi Arabia
Gulf of Suez Wind Power Project in Suez, Egypt
Al-Ajban Solar Park in Abu Dhabi, UAE
Besides future energy carriers like hydrogen, research indicates that ammonia also has significant potential as an energy carrier. Its high energy density and ease of storage make it a promising option for large-scale energy applications. The underground storage of ammonia on a wide scale could provide a safe and efficient method for energy storage, but this avenue still requires more comprehensive investigation to address associated challenges and ensure its viability for future energy systems.
=== Demand ===
In July 2014, WWF and the World Resources Institute convened a discussion among a number of major US companies who had declared their intention to increase their use of renewable energy. These discussions identified a number of "principles" which companies seeking greater access to renewable energy considered important market deliverables. These principles included choice (between suppliers and between products), cost competitiveness, longer term fixed price supplies, access to third-party financing vehicles, and collaboration.
UK statistics released in September 2020 noted that "the proportion of demand met from renewables varies from a low of 3.4 per cent (for transport, mainly from biofuels) to highs of over 20 per cent for 'other final users', which is largely the service and commercial sectors that consume relatively large quantities of electricity, and industry".
In some locations, individual households can opt to purchase renewable energy through a consumer green energy program.
=== Developing countries ===
In Kenya, the Olkaria V Geothermal Power Station is one of the largest in the world. The Grand Ethiopia Renaissance Dam project incorporates wind turbines. Once completed, Morocco's Ouarzazate Solar Power Station is projected to provide power to over a million people.
== Policy ==
Policies to support renewable energy have been vital in their expansion. Where Europe dominated in establishing energy policy in the early 2000s, most countries around the world now have some form of energy policy.
The International Renewable Energy Agency (IRENA) is an intergovernmental organization for promoting the adoption of renewable energy worldwide. It aims to provide concrete policy advice and facilitate capacity building and technology transfer. IRENA was formed in 2009, with 75 countries signing the charter of IRENA. As of April 2019, IRENA has 160 member states. The then United Nations Secretary-General Ban Ki-moon has said that renewable energy can lift the poorest nations to new levels of prosperity, and in September 2011 he launched the UN Sustainable Energy for All initiative to improve energy access, efficiency and the deployment of renewable energy.
The 2015 Paris Agreement on climate change motivated many countries to develop or improve renewable energy policies. In 2017, a total of 121 countries adopted some form of renewable energy policy. National targets that year existed in 176 countries. In addition, there is also a wide range of policies at the state/provincial, and local levels. Some public utilities help plan or install residential energy upgrades.
Many national, state and local governments have created green banks. A green bank is a quasi-public financial institution that uses public capital to leverage private investment in clean energy technologies. Green banks use a variety of financial tools to bridge market gaps that hinder the deployment of clean energy.
Global and national policies related to renewable energy can be divided based on sectors, such as agriculture, transport, buildings, industry:
Climate neutrality (net zero emissions) by the year 2050 is the main goal of the European Green Deal. For the European Union to reach their target of climate neutrality, one goal is to decarbonise its energy system by aiming to achieve "net-zero greenhouse gas emissions by 2050."
== Finance ==
The International Renewable Energy Agency's (IRENA) 2023 report on renewable energy finance highlights steady investment growth since 2018: USD 348 billion in 2020 (a 5.6% increase from 2019), USD 430 billion in 2021 (24% up from 2020), and USD 499 billion in 2022 (16% higher). This trend is driven by increasing recognition of renewable energy's role in mitigating climate change and enhancing energy security, along with investor interest in alternatives to fossil fuels. Policies such as feed-in tariffs in China and Vietnam have significantly increased renewable adoption. Furthermore, from 2013 to 2022, installation costs for solar photovoltaic (PV), onshore wind, and offshore wind fell by 69%, 33%, and 45%, respectively, making renewables more cost-effective.
Between 2013 and 2022, the renewable energy sector underwent a significant realignment of investment priorities. Investment in solar and wind energy technologies markedly increased. In contrast, other renewable technologies such as hydropower (including pumped storage hydropower), biomass, biofuels, geothermal, and marine energy experienced a substantial decrease in financial investment. Notably, from 2017 to 2022, investment in these alternative renewable technologies declined by 45%, falling from USD 35 billion to USD 17 billion.
In 2023, the renewable energy sector experienced a significant surge in investments, particularly in solar and wind technologies, totaling approximately USD 200 billion—a 75% increase from the previous year. The increased investments in 2023 contributed between 1% and 4% to the GDP in key regions including the United States, China, the European Union, and India.
The energy sector receives investments of approximately USD 3 trillion each year, with USD 1.9 trillion directed towards clean energy technologies and infrastructure. To meet the targets set in the Net Zero Emissions (NZE) Scenario by 2035, this investment must increase to USD 5.3 trillion per year.: 15
== Debates ==
=== Nuclear power proposed as renewable energy ===
=== Geopolitics ===
The geopolitical impact of the growing use of renewable energy is a subject of ongoing debate and research. Many fossil-fuel producing countries, such as Qatar, Russia, Saudi Arabia and Norway, are currently able to exert diplomatic or geopolitical influence as a result of their oil wealth. Most of these countries are expected to be among the geopolitical "losers" of the energy transition, although some, like Norway, are also significant producers and exporters of renewable energy. Fossil fuels and the infrastructure to extract them may, in the long term, become stranded assets. It has been speculated that countries dependent on fossil fuel revenue may one day find it in their interests to quickly sell off their remaining fossil fuels.
Conversely, nations abundant in renewable resources, and the minerals required for renewables technology, are expected to gain influence. In particular, China has become the world's dominant manufacturer of the technology needed to produce or store renewable energy, especially solar panels, wind turbines, and lithium-ion batteries. Nations rich in solar and wind energy could become major energy exporters. Some may produce and export green hydrogen, although electricity is projected to be the dominant energy carrier in 2050, accounting for almost 50% of total energy consumption (up from 22% in 2015). Countries with large uninhabited areas such as Australia, China, and many African and Middle Eastern countries have a potential for huge installations of renewable energy. The production of renewable energy technologies requires rare-earth elements with new supply chains.
Countries with already weak governments that rely on fossil fuel revenue may face even higher political instability or popular unrest. Analysts consider Nigeria, Angola, Chad, Gabon, and Sudan, all countries with a history of military coups, to be at risk of instability due to dwindling oil income.
A study found that transition from fossil fuels to renewable energy systems reduces risks from mining, trade and political dependence because renewable energy systems don't need fuel – they depend on trade only for the acquisition of materials and components during construction.
In October 2021, European Commissioner for Climate Action Frans Timmermans suggested "the best answer" to the 2021 global energy crisis is "to reduce our reliance on fossil fuels." He said those blaming the European Green Deal were doing so "for perhaps ideological reasons or sometimes economic reasons in protecting their vested interests." Some critics blamed the European Union Emissions Trading System (EU ETS) and closure of nuclear plants for contributing to the energy crisis. European Commission President Ursula von der Leyen said that Europe is "too reliant" on natural gas and too dependent on natural gas imports. According to Von der Leyen, "The answer has to do with diversifying our suppliers ... and, crucially, with speeding up the transition to clean energy."
=== Metal and mineral extraction ===
The transition to renewable energy requires increased extraction of certain metals and minerals. Like all mining, this impacts the environment and can lead to environmental conflict. For example, lithium mining uses around 65% of the water in the Salar de Atamaca desert forcing farmers and llama herders to abandon their ancestral settlements and creating environment degradation, in several African countries, the green energy transition has created a mining boom, causing deforestation, and threatening already endangered species. Wind power requires large amounts of copper and zinc, as well as smaller amounts of the rarer metal neodymium. Solar power is less resource-intensive, but still requires significant amounts of aluminum. The expansion of electrical grids requires both copper and aluminum. Batteries, which are critical to enable storage of renewable energy, use large quantities of copper, nickel, aluminum and graphite. Demand for lithium is expected to grow 42-fold from 2020 to 2040. Demand for nickel, cobalt and graphite is expected to grow by a factor of about 20–25. For each of the most relevant minerals and metals, its mining is dominated by a single country: copper in Chile, nickel in Indonesia, rare earths in China, cobalt in the Democratic Republic of the Congo (DRC), and lithium in Australia. China dominates processing of all of these.
Recycling these metals after the devices they are embedded in are spent is essential to create a circular economy and ensure renewable energy is sustainable. By 2040, recycled copper, lithium, cobalt, and nickel from spent batteries could reduce combined primary supply requirements for these minerals by around 10%.
A controversial approach is deep sea mining. Minerals can be collected from new sources like polymetallic nodules lying on the seabed. This would damage local biodiversity, but proponents point out that biomass on resource-rich seabeds is much scarcer than in the mining regions on land, which are often found in vulnerable habitats like rainforests.
Due to co-occurrence of rare-earth and radioactive elements (thorium, uranium and radium), rare-earth mining results in production of low-level radioactive waste.
=== Conservation areas ===
Installations used to produce wind, solar and hydropower are an increasing threat to key conservation areas, with facilities built in areas set aside for nature conservation and other environmentally sensitive areas. They are often much larger than fossil fuel power plants, needing areas of land up to 10 times greater than coal or gas to produce equivalent energy amounts. More than 2000 renewable energy facilities are built, and more are under construction, in areas of environmental importance and threaten the habitats of plant and animal species across the globe. The authors' team emphasized that their work should not be interpreted as anti-renewables because renewable energy is crucial for reducing carbon emissions. The key is ensuring that renewable energy facilities are built in places where they do not damage biodiversity.
In 2020 scientists published a world map of areas that contain renewable energy materials as well as estimations of their overlaps with "Key Biodiversity Areas", "Remaining Wilderness" and "Protected Areas". The authors assessed that careful strategic planning is needed.
=== Impact of climate change on renewable energy production ===
Climate change is making weather patterns less predictable. This can seriously hamper the use of renewable energy. For example, in the year 2023, in Sudan and Namibia, hydropower production dropped by more than half due to drastic reduction in rainfall, in China, India and some regions in Africa unusual weather phenomena reduced the amount of produced wind energy, heatwaves and clouds reduce the effectiveness of solar pannels, melting glaciers are creating problems to hydropower. Nuclear energy is also affected as drought create water shortage, so nuclear power plants sometimes do not have enough water for cooling.
== Society and culture ==
=== Public support ===
Solar power plants may compete with arable land, while on-shore wind farms often face opposition due to aesthetic concerns and noise. Such opponents are often described as NIMBYs ("not in my back yard"). Some environmentalists are concerned about fatal collisions of birds and bats with wind turbines. Although protests against new wind farms occasionally occur around the world, regional and national surveys generally find broad support for both solar and wind power.
Community-owned wind energy is sometimes proposed as a way to increase local support for wind farms. A 2011 UK Government document stated that "projects are generally more likely to succeed if they have broad public support and the consent of local communities. This means giving communities both a say and a stake." In the 2000s and early 2010s, many renewable projects in Germany, Sweden and Denmark were owned by local communities, particularly through cooperative structures. In the years since, more installations in Germany have been undertaken by large companies, but community ownership remains strong in Denmark.
== History ==
Prior to the development of coal in the mid 19th century, nearly all energy used was renewable. The oldest known use of renewable energy, in the form of traditional biomass to fuel fires, dates from more than a million years ago. The use of biomass for fire did not become commonplace until many hundreds of thousands of years later. Probably the second oldest usage of renewable energy is harnessing the wind in order to drive ships over water. This practice can be traced back some 7000 years, to ships in the Persian Gulf and on the Nile. From hot springs, geothermal energy has been used for bathing since Paleolithic times and for space heating since ancient Roman times. Moving into the time of recorded history, the primary sources of traditional renewable energy were human labor, animal power, water power, wind, in grain crushing windmills, and firewood, a traditional biomass.
In 1885, Werner Siemens, commenting on the discovery of the photovoltaic effect in the solid state, wrote:
In conclusion, I would say that however great the scientific importance of this discovery may be, its practical value will be no less obvious when we reflect that the supply of solar energy is both without limit and without cost, and that it will continue to pour down upon us for countless ages after all the coal deposits of the earth have been exhausted and forgotten.
Max Weber mentioned the end of fossil fuel in the concluding paragraphs of his Die protestantische Ethik und der Geist des Kapitalismus (The Protestant Ethic and the Spirit of Capitalism), published in 1905. Development of solar engines continued until the outbreak of World War I. The importance of solar energy was recognized in a 1911 Scientific American article: "in the far distant future, natural fuels having been exhausted [solar power] will remain as the only means of existence of the human race".
The theory of peak oil was published in 1956. In the 1970s environmentalists promoted the development of renewable energy both as a replacement for the eventual depletion of oil, as well as for an escape from dependence on oil, and the first electricity-generating wind turbines appeared. Solar had long been used for heating and cooling, but solar panels were too costly to build solar farms until 1980.
New government spending, regulation and policies helped the renewables industry weather the 2008 financial crisis and the Great Recession better than many other sectors. In 2022, renewables accounted for 30% of global electricity generation, up from 21% in 1985.
=== Ancient Historical Examples ===
Among the most notable historical uses of renewable energy (in the form of ancient and traditional methods), the following examples can be highlighted:
Windmills in Europe and Asia (such as the windmills of the Netherlands and Nashtifan in Iran). The earliest discovered verified designs of windmills date back to Iran, between 700 and 900 CE.
Water mills (Ancient China and Ancient Persia).
Archimedes' burning lens.
Traditional cooling and ventilation systems based on windcatchers and Solar updraft tower (or Solar chimney).
Traditional architecture aware of natural heat transfer and natural energy transformation processes.
Gravity-based fountains.
Using animal biomass in ancient fuel bricks.
Solar ovens and furnaces in ancient China, India, Egypt, and Persia.
Solar energy applications for traditional agricultural processing (drying), engineering material properties (solar curing of pottery and ceramics), and ancient health practices (natural disinfection by solar radiation).
Long-distance gravitational water flow control in ancient qanat technology for water transport and supply.
Cargo and passenger transportation using sails on rivers, seas, and oceans.
Cargo and passenger transportation based on understanding water currents in rivers, seas, and oceans.
Using renewable vegetation (such as desert shrubs, agricultural waste, and pruned branches) for producing light and heat.
Using renewable oils (vegetable or animal-based) for producing light and heat.
Maximizing use of natural sunlight during the day and moonlight at night in building architecture for purposes such as lighting, decorative applications (e.g., reflective tilework, mirror work, and surface polishing on stone or brick), timekeeping (sundials, noon markers, prayer time indicators, seasonal change markers), etc.
== See also ==
Distributed generation – Decentralised electricity generation
Efficient energy use – Methods for higher energy efficiency
Fossil fuel phase-out – Gradual reduction of the use and production of fossil fuels
Thermal energy storage – Technologies to store thermal energy
List of countries by renewable electricity production
List of renewable energy topics by country and territory
Renewable heat – Application of renewable energy
== References ==
=== Sources ===
"Renewable Power Generation Costs in 2019" (PDF). IRENA. 2020.
"Renewable Capacity Statistics 2020". IRENA. 2020.
"Renewable Energy Statistics 2020". IRENA. 2020.
== External links ==
Energypedia – a wiki platform for collaborative knowledge exchange on renewable energy in developing countries
Renewable Energy Conference – a global platform for industry professionals, academics, and policymakers to exchange knowledge and discuss advancements in renewable energy technologies, with a focus on innovation, sustainability, and future energy solutions. | Wikipedia/Alternative_energy |
"Environmental Science" is the tenth episode of the first season of the American comedy television series Community. It aired in the United States on NBC on November 19, 2009.
== Plot ==
Dean Pelton (Jim Rash) announces that Greendale will have its annual environmentalism initiative "Green Week," culminating with a performance by Green Day.
After a small infraction by Annie (Alison Brie), Spanish teacher Ben Chang (Ken Jeong) requires that the students write twenty-page essays due the following Monday. The study group finds the assignment untimely: Shirley (Yvette Nicole Brown) has a marketing presentation due, and Abed (Danny Pudi) and Troy (Donald Glover) are conducting a biology lab experiment. The group asks Jeff (Joel McHale) to intervene with Chang to cancel the assignment.
Pierce (Chevy Chase) offers to help Shirley overcome her public speaking anxiety. She feels that his advice is strange and useless, such as using hand gestures and sexual innuendo to catch attention.
Abed and Troy are trying to train a rat named Fievel to respond to their duet of "Somewhere Out There", but during a test, the rat escapes. Troy, who is afraid of rats, is unable to help catch it, which enters the air ventilation system.
Jeff learns that Chang is bitter over his wife (Andrea de Oliveira) throwing him out. Jeff takes pity on Chang and tries to help him move on, spending the night out drinking with him in exchange for not having to do the essay, unbeknownst to his study group. The next day, the rest of the study group is infuriated to learn that Jeff claims to have already completed the assignment while Chang increases the importance of the essay on their course grade. The group catches Jeff talking to Chang about getting a good grade in exchange for spending more time with him, and insist he fix the problem. Instead, he spends the next night with Chang, ridiculing the homework of other students. Chang breaks down into tears, missing his ex-wife; Jeff realizes he still loves her.
The promised concert arrives, but Dean Pelton finds that the band is actually "Greene Daeye," an Irish folk music ensemble. As the band plays, Chang spots his wife in the crowd. Troy joins Abed as they sing "Somewhere Out There" to recover Fievel; Chang and his wife steal the spotlight dancing at the concert, and Shirley realizes the value of Pierce's advice and gives a successful presentation. After dancing, Chang announces that the essay assignment is cancelled.
== Reception ==
"Environmental Science" was met with positive reviews from critics. It first aired on NBC on November 19, 2009 to an audience of 4.86 million Americans.
Emily VanDerWerff of The A.V. Club rated the episode A−, praising the show's continued development but worrying that some viewers might find Chang's character too broad and irritating.
Jonah Krakow of IGN rated the episode a 7.6/10 described as narratively satisfying.
== References ==
== External links ==
"Environmental Science" at NBC
"Environmental Science" at IMDb | Wikipedia/Environmental_Science_(Community) |
The Annual Review of Pathology: Mechanisms of Disease is a peer-reviewed academic journal that publishes an annual volume of review articles relevant to pathology. It was established in 2006 and is published by Annual Reviews. Its co-editors have been Jon C. Aster, Mel B. Feany, and Jayanta Debnath since 2021. As of 2023, Annual Review of Pathology: Mechanisms of Disease is being published as open access, under the Subscribe to Open model. As of 2024, Journal Citation Reports gives the journal a 2023 impact factor of 28.4, ranking it first of 88 journal titles in the category "Pathology".
== History ==
The Annual Review of Pathology: Mechanisms of Disease was first published in 2006 by the nonprofit publisher Annual Reviews. The editorial goals for the journal were to cover recent advancements in the study of disease mechanism and present new analytical methodologies for pathology. Its first co-editors were Abul K. Abbas, James R. Downing, and Vinay Kumar. Though it was initially in publication with a print volume, it is now only published electronically.
== Scope and indexing ==
The Annual Review of Pathology: Mechanisms of Disease defines its scope as covering significant developments in research on the initiation and progression of human disease. It is abstracted and indexed in Scopus, Science Citation Index Expanded, EMBASE, MEDLINE, and Academic Search, among others.
== Editorial processes ==
The Annual Review of Pathology: Mechanisms of Disease is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review of accepted manuscripts is undertaken by the editorial committee.
=== Editors of volumes ===
Dates indicate publication years in which someone was credited as a lead editor or co-editor of a journal volume. The planning process for a volume begins well before the volume appears, so appointment to the position of lead editor generally occurred prior to the first year shown here. An editor who has retired or died may be credited as a lead editor of a volume that they helped to plan, even if it is published after their retirement or death.
Abul K. Abbas, James R. Downing, and Vinay Kumar (2006)
Abbas, Stephen J. Galli, and Peter M. Howley (2007–2015)
Abbas, Galli, and Jon C. Aster (2016-2018)
Abbas, Aster, and Mel B. Feany (2019–2020)
Aster and Feany (2021)
Aster, Feany, and Jayanta Debnath (present)
=== Current editorial board ===
As of 2022, the editorial committee consists of the three co-editors and the following members:
As of 2022, the committee's members were:
== See also ==
Annual Review of Genomics and Human Genetics
Annual Review of Medicine
Annual Review of Neuroscience
== References == | Wikipedia/Annual_Review_of_Pathology:_Mechanisms_of_Disease |
The Annual Review of Vision Science is an academic journal published by Annual Reviews. In publication since 2015, this journal covers significant developments in the field of vision science with an annual volume of review articles. As of 2023, it is being published as open access, under the Subscribe to Open model.
It is currently edited by David H. Brainard and John H. R. Maunsell. As of 2024, Journal Citation Reports gives the journal a 2023 impact factor of 5.0, ranking it sixth of 95 journals in "Ophthalmology".
== History ==
The Annual Review of Vision Science was first published in 2015 by Annual Reviews. Though it began with a physical edition, it is now only published electronically.
Its founding editors were J. Anthony Movshon and Brian Wandell. In 2021, Wandell was succeeded by David H. Brainard. Brainard and Movshon were joined by John H. R. Maunsell for 2023. As of October 2023, Brainard and Maunsell became the editors.
== Scope and indexing ==
The Annual Review of Vision Science is multidisciplinary, including aspects of neuroscience, genetics, computer science, cell biology, and medicine. Reviews may cover optics, retina, visual perception, eye movement, visual processing, visual development, vision models, computer vision, and the mechanisms and treatments of diseases that affect vision. As of 2024, Journal Citation Reports lists the journal's 2023 impact factor as 5.0, ranking it sixth of 95 journal titles in the category "Ophthalmology" and 49th of 310 titles in "Neurosciences". It is abstracted and indexed in Scopus, Science Citation Index Expanded, and BIOSIS Previews, among others.
== Editorial processes ==
The Annual Review of Vision Science is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review of accepted manuscripts is undertaken by the editorial committee.
== References == | Wikipedia/Annual_Review_of_Vision_Science |
The Annual Review of Biomedical Data Science is an academic journal published by Annual Reviews. In publication since 2018, this journal covers significant developments in the field of health informatics and biomedical data science with an annual volume of review articles. It is edited by Russ Altman. As of 2023, it is being published as open access, under the Subscribe to Open model. As of 2024, Journal Citation Reports lists the journal's impact factor as 7.0, ranking it second out of 65 journals.
== History ==
The Annual Review of Biomedical Data Science was first published in 2018 by the nonprofit publisher Annual Reviews. The journal focuses on biomedical data science, the development of scientific methods to acquire, annotate, organize, analyze, and interpret biomedical data and extract knowledge about life, health, and disease.
The founding co-editors were Russ B. Altman and Michael Levitt. As of 2021, Altman was the lead editor.
== Scope and indexing ==
The Annual Review of Biomedical Data Science is abstracted and indexed in Science Citation Index Expanded, Scopus and BIOSIS Previews, among others.
== Editorial processes ==
The Annual Review of Biomedical Data Science is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review of accepted manuscripts is undertaken by the editorial committee.
=== Current editorial board ===
As of 2023, the editorial committee consists of the editor and the following members:
== References == | Wikipedia/Annual_Review_of_Biomedical_Data_Science |
The Annual Review of Marine Science is an annual peer-reviewed scientific review journal published by Annual Reviews. It was established in 2009. It covers all aspects of marine science. The co-editors are Craig A. Carlson and Stephen J. Giovannoni. As of 2023, Annual Review of Marine Science is being published as open access, under the Subscribe to Open model. As of 2024, Journal Citation Reports gives the journal a 2023 impact factor of 14.3, ranking it first out of 119 in the category "Marine & Freshwater Biology", first out of 65 in the category "Oceanography", and first out of 101 in the category "Geochemistry & Geophysics".
== History ==
The Annual Review of Marine Science was first published in 2009 by nonprofit publisher Annual Reviews. Its founding editors were Craig A. Carlson and Stephen J. Giovannoni. While it was initially published with a print edition, it is now only published online.
== Scope and indexing ==
The Annual Review of Marine Science defines its scope as covering significant developments in marine science. Included subfields are chemical, biological, geological, and physical processes that occur in the coastal and oceanic zones. It also covers marine conservation, marine biology, and technologies used in the study of oceanography. It is abstracted and indexed in Scopus, Science Citation Index Expanded, EMBASE, INSPEC, CAB Abstracts, MEDLINE, and GEOBASE, among others.
== Editorial processes ==
The Annual Review of Marine Science is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review of accepted manuscripts is undertaken by the editorial committee.
=== Current editorial board ===
As of 2022, the editorial committee consists of the two co-editors and the following members:
== References == | Wikipedia/Annual_Review_of_Marine_Science |
The Annual Review of Biophysics is a peer-reviewed scientific journal published annually by Annual Reviews. It covers all aspects of biophysics with solicited review articles. Ken A. Dill has been its editor since 2013. As of 2023, Annual Review of Biophysics is being published as open access, under the Subscribe to Open model. As of 2024, according to the Journal Citation Reports, the journal has a 2023 impact factor of 10.4 ranking it fourth out of 77 journals in the category "Biophysics".
== History ==
The Annual Review of Biophysics and Bioengineering was first published in 1972 by Annual Reviews in collaboration with the Biophysical Society. Its inaugural editor was Manuel F. Morales. In 1985, the name of the journal was changed to Annual Review of Biophysics and Biophysical Chemistry, followed by another name change in 1992 to Annual Review of Biophysics and Biomolecular Structure. In 2008 the journal obtained its current title.
== Abstracting and indexing ==
The journal is abstracted and indexed in Scopus, Science Citation Index Expanded, BIOSIS Previews, Embase, MEDLINE, and Academic Search, among others.
== Editorial processes ==
The journal is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review is undertaken by the editorial committee.
=== Volume editors ===
Dates indicate publication years in which someone was credited as a lead editor or co-editor of a journal volume. The planning process for a volume begins well before the volume appears, so appointment to the position of lead editor generally occurred prior to the first year shown here. An editor who has retired or died may be credited as a lead editor of a volume that they helped to plan, even if it is published after their retirement or death.
Manuel F. Morales (1972)
Lorin J. Mullins (1973–1983)
Donald M. Engelman (1984–1993)
Robert M. Stroud (1994–2003)
Douglas C. Rees (2004–2012)
Douglas C. Rees and Ken A. Dill (2013–2014)
Ken A. Dill (2015–2024)
Jané Kondev (2024–present)
== See also ==
List of physics journals
== References ==
== External links ==
Official website | Wikipedia/Annual_Review_of_Biophysics |
The Annual Review of Condensed Matter Physics is an annual peer-reviewed review journal published by Annual Reviews. It was established in 2010 and covers advances in condensed matter physics and related subjects. The co-editors are M. Cristina Marchetti and Andrew P. Mackenzie. As of 2023, Annual Review of Condensed Matter Physics is being published as open access, under the Subscribe to Open model. As of 2024, Journal Citation Reports lists the journal's impact factor of 14.3, ranking it sixth of 79 journal titles in the category "Physics, Condensed Matter" in 2023.
== History ==
The Annual Review of Condensed Matter Physics was first published in 2010 by the nonprofit publisher Annual Reviews. Its founding editor was James S. Langer. He was joined by James P. Eisenstein in 2014. M. Cristina Marchetti and Subir Sachdev were co-editors in 2016 and 2017; in 2018, Andrew P. Mackenzie joined as the third co-editor. As of 2021, the co-editors were Marchetti and Mackenzie. Though it was initially published in print, as of 2021 it is only published electronically.
== Scope and indexing ==
The Annual Review of Condensed Matter Physics defines its scope as covering significant developments relevant to condensed matter physics. It is abstracted and indexed in Scopus, Science Citation Index Expanded, Compendex, and INSPEC.
== Editorial processes ==
The Annual Review of Condensed Matter Physics is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review of accepted manuscripts is undertaken by the editorial committee.
=== Current editorial board ===
As of 2022, the editorial committee consists of the two co-editors and the following members:
== See also ==
List of physics journals
== References == | Wikipedia/Annual_Review_of_Condensed_Matter_Physics |
The Annual Review of Neuroscience is a peer-reviewed academic journal that publishes review articles relevant to neuroscience. In publication since 1978 by Annual Reviews, founding editor W. Maxwell Cowan led the editorial committee until his death in 2002. Mary E. Hatten and Botond Roska are the current co-editors. As of 2023, it is being published as open access, under the Subscribe to Open model.
== History ==
In 1975, the nonprofit publisher Annual Reviews had two meetings in New York to coincide with the annual meeting of the Society for Neuroscience. In the meetings, neuroscientists from the US and Canada concurred that it would be useful to establish a journal that published an annual volume of review articles relevant to neuroscience. The board of directors of Annual Reviews gave the final approval for the journal in early 1976, appointing the first editorial committee with W. Maxwell Cowan appointed editor. In April 1976 the editorial committee planned the first volume of the journal, which was published in 1978. As of 2021, it was published both in print and electronically. Some of its articles are available online in advance of the volume's publication date.
== Scope and indexing ==
The Annual Review of Neuroscience defines its scope as covering significant developments in the field of neuroscience, including the subfields of molecular neuroscience, cellular neuroscience, neurodevelopment, neurogenetics, neuroplasticity, systems neuroscience, neurological disorders, and the history and ethics of neuroscience. As of 2024, Journal Citation Reports lists the journal's 2023 impact factor as 12.1, ranking it eleventh of 310 journal titles in the category "Neurosciences". It is abstracted and indexed in Scopus, CAB Abstracts, EMBASE, MEDLINE, PsycINFO, and Academic Search, among others.
== Editorial processes ==
The Annual Review of Neuroscience is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review of accepted manuscripts is undertaken by the editorial committee.
=== Editors of volumes ===
Dates indicate publication years in which someone was credited as a lead editor or co-editor of a journal volume. The planning process for a volume begins well before the volume appears, so appointment to the position of lead editor generally occurred prior to the first year shown here. An editor who has retired or died may be credited as a lead editor of a volume that they helped to plan, even if it is published after their retirement or death.
W. Maxwell Cowan (1978–2002)
Steven E. Hyman (2003–2017)
Huda Y. Zoghbi (2018–2019)
Zoghbi and Botond Roska (2020-2024)
Roska and Mary E. Hatten (2025-)
=== Current editorial committee ===
As of 2024, the editorial committee consists of two co-editors and the following members:
== See also ==
List of neuroscience journals
== References == | Wikipedia/Annual_Review_of_Neuroscience |
The Annual Review of Animal Biosciences is a peer-reviewed scientific journal published by Annual Reviews. It releases an annual volume of review articles relevant to the fields of zoology, veterinary medicine, animal husbandry, and conservation biology. It has been in publication since 2013. The co-editors are Harris A. Lewin and R. Michael Roberts. As of 2023, Annual Review of Animal Biosciences is being published as open access, under the Subscribe to Open model. As of 2024, Journal Citation Reports lists the journal's impact factor for 2023 as 8.7. It was rated number one of 180 titles in Zoology, number one of 80 in "Agriculture, Dairy, and Animal Sciences", number one of 168 in "Veterinary Science" and number three of 174 titles in Biotechnology and Applied Microbiology.
== History ==
The Annual Review of Animal Biosciences was first published in 2013, with Harris A. Lewin and R. Michael Roberts as the founding co-editors. Though it was initially published in print, as of 2021 it is only published electronically.
== Scope and indexing ==
The Annual Review of Animal Biosciences defines its scope as covering significant developments relevant to biotechnology, genomics, genetics, veterinary medicine, animal breeding, and conservation biology. The intended audience for the journal is scientists and veterinarians involved with wild and domestic animals. It is abstracted and indexed in Scopus, Science Citation Index Expanded, MEDLINE, and Embase, among others.
== References == | Wikipedia/Annual_Review_of_Animal_Biosciences |
The Annual Review of Control, Robotics, and Autonomous Systems is an annual peer-reviewed scientific journal published by Annual Reviews. In publication since 2018, the journal covers developments in the engineering of autonomous and semiautonomous systems through an annual volume of review articles. It is edited by Naomi Ehrich Leonard. As of 2023, Annual Review of Control, Robotics, and Autonomous Systems is being published as open access, under the Subscribe to Open model. As of 2024, Journal Citation Reports gives the journal an impact factor of 11.2 for the year 2023, ranking it third of 84 journal titles in the category "Automation and Control Systems" and second of 46 journal titles in the category "Robotics".
== History ==
The journal was first published in 2018 by publisher Annual Reviews, making it their 49th journal title. Its founding editor was Naomi Ehrich Leonard.
== Abstracting and indexing ==
It is abstracted and indexed in the Science Citation Index Expanded and Inspec.
== Editorial processes ==
The journal is helmed by the editor or the co-editors. The editor is assisted by the editorial committee, which includes associate editors, regular members, and occasionally guest editors. Guest members participate at the invitation of the editor, and serve terms of one year. All other members of the editorial committee are appointed by the Annual Reviews board of directors and serve five-year terms. The editorial committee determines which topics should be included in each volume and solicits reviews from qualified authors. Unsolicited manuscripts are not accepted. Peer review of invited manuscripts is undertaken by the editorial committee.
== References ==
== External links ==
Official website | Wikipedia/Annual_Review_of_Control,_Robotics,_and_Autonomous_Systems |
Single-strand DNA-binding protein (SSB) is a protein found in Escherichia coli (E. coli) bacteria, that binds to single-stranded regions of deoxyribonucleic acid (DNA). Single-stranded DNA is produced during all aspects of DNA metabolism: replication, recombination, and repair. As well as stabilizing this single-stranded DNA, SSB proteins bind to and modulate the function of numerous proteins involved in all of these processes.
Active E. coli SSB is composed of four identical 19 kDa subunits. Binding of single-stranded DNA to the tetramer can occur in different "modes", with SSB occupying different numbers of DNA bases depending on a number of factors, including salt concentration. For example, the (SSB)65 binding mode, in which approximately 65 nucleotides of DNA wrap around the SSB tetramer and contact all four of its subunits, is favoured at high salt concentrations in vitro. At lower salt concentrations, the (SSB)35 binding mode, in which about 35 nucleotides bind to only two of the SSB subunits, tends to form. Further work is required to elucidate the functions of the various binding modes in vivo.
== Bacterial SSB ==
SSB protein domains in bacteria are important in its function of maintaining DNA metabolism, more specifically DNA replication, repair, and recombination. It has a structure of three beta-strands to a single six-stranded beta-sheet to form a protein dimer.
== See also ==
DNA-binding protein
Single-stranded binding protein
Comparison of nucleic acid simulation software
== References ==
== External links ==
Single-Stranded+DNA+Binding+Proteins at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
SSB in PFAM | Wikipedia/Single-strand_DNA-binding_protein |
dnaN is the gene that codes for the DNA clamp (also known as β sliding clamp) of DNA polymerase III in prokaryotes. The β clamp physically locks Pol III onto a DNA strand during replication to help increase its processivity. The eukaryotic equivalent to the β clamp is PCNA.
== Description ==
The β clamp plays an essential role in bacterial DNA replication. DnaN is a key gene in many research findings from molecular genetics, structural biology, mutational analysis, and evolutionary studies. The dnaN gene encodes the β subunit of DNA polymerase III, the primary enzyme responsible for chromosomal replication in E. coli. The β subunit functions as a sliding clamp, forming a ring around the DNA to secure the polymerase and allow it to move efficiently along the strand without dissociating. As a processivity factor, the β clamp holds DNA polymerase III on the DNA, allowing long stretches of DNA to be synthesized without detaching from the DNA template. It also serves as a docking site for multiple protein interactions, such as facilitating the transition between replicative and translesion synthesis polymerases, coordinating DNA repair, integrating checkpoints, and allowing the cell to keep copying DNA even when problems are detected. This function is crucial for rapid and accurate replication, ensuring the entire genome is efficiently and properly duplicated in each cell cycle. Therefore, dnaN and the β sliding clamp are crucial for proper cell division.
Located directly downstream of the dnaA gene (separated by only four base pairs), dnaN forms part of a bicistronic operon, transcribed under the control of the dnaA promoter. There is no promoter between dnaA and dnaN, and the genes are transcribed as a single mRNA. Experimental data demonstrate that disruptions to this region impact both replication initiation (dnaA) and elongation (dnaN), indicating a tightly regulated and evolutionarily conserved linkage.
The translation of dnaN begins at an ATG start codon, producing a 366 amino acid protein with a molecular mass of about 40 kDa.[11] This protein folds into three domains per monomer, and two monomers assemble into a ring-shaped dimer that clamps around DNA. Specific motifs near the N-terminal and domain linkers contribute to dimer formation and stability.
== Mutations and functional analysis ==
Researchers have contacted numerous different studies to identify gene mutations and how different mutations of the dnaN gene, which makes the β sliding clamp protein, affect bacterial survival.
Mutations in dnaN, such as dnaN59 and dnaN806, reveal the exact amino acids in a protein sequence that are required for clamp function. The dnaN59 mutation produces a temperature-sensitive phenotype, halting DNA replication at non-permissive temperatures. DnaN806 results in a shortened β clamp that is unable to function unless suppressed by a tRNA suppressor allele. These mutants have been pivotal in identifying essential protein domains and interactions, as they allow researchers to observe how specific changes in the β clamp structure affect its ability to interact with DNA, polymerases, and other replication or repair proteins. By analyzing the functional consequences of these mutations, scientists can map out which regions of the protein are necessary for its stability, dimerization, and binding capacity.
A study used a technique called in vivo complementation, where they tested whether a mutant version of the gene can replace the normal one in living bacteria. The study found that some mutations in the β clamp, specifically at positions D150 and P363, are critical. When these amino acids are changed, the clamp no longer works on its own, meaning the bacteria can’t survive without the normal version. This shows that these spots are essential for the clamp to function. However, other mutations, like Q61K and M204K, do not stop bacteria survival, but they do affect how cells respond to stress, such as problems during DNA replication. The study also found if the normal β clamp (without mutations) is made in too large amounts, it actually becomes toxic to the bacteria and slows their growth. But when certain mutant clamps are made in high amounts instead, the bacteria don’t get sick, suggesting that some of the mutated versions are less disruptive.
The dnaN and dnaE genes encode for essential subunits of DNA polymerase III. The dnaN gene produces the β subunit, while dnaE encodes the α subunit (the catalytic core that builds new DNA strands). A mutation known as sueA77 in the dnaN gene showed the ability to compensate for certain defective mutations in the dnaE gene. This mutation is trans-dominant, meaning it could exert its effect over a normal copy of dnaN was present. The sueA77 mutation allowed cells with otherwise nonfunctional dnaE alleles to replicate DNA and survive at higher temperatures. This suggests that sueA77 alters the β subunit in a way that enhances or stabilizes its interaction with the α subunit, supporting the idea that direct cooperation between these subunits is essential for effective DNA replication.
== Clamp function ==
The β clamp is loaded onto DNA by a group of helper proteins called the γ-complex. This process requires energy, and is driven by ATP to open the clamp and attach it. Structural studies show that the β clamp must first open to be loaded and then close around the DNA. This loading process is precisely regulated to occurs exactly where DNA copying begins.[10] High-resolution crystallography studies have shown that DNA passes through the β clamp at a tilted angle of approximately 22°, allowing key amino acid residues like R24 and Q149 to make contact with the DNA. These interactions are essential for the clamp to close properly and recruit DNA polymerase. The αε polymerase is the actual enzyme that does the copying. On its own, it can’t stay on the DNA for long, But once it connects to the β clamp, it becomes very stable and can copy DNA without falling off. Additionally, other residues form a secondary binding pocket that may temporarily hold single-stranded DNA, helping to keep the clamp in place and facilitating polymerase switching during replication stress.[5] After loading, the clamp interacts with multiple polymerases and accessory proteins to ensure replication proceeds even if there are damaged DNA regions.
== DnaN inhibitors ==
Research has identified a new class of inhibitors that target dnaN, known as mycoplanecins, which offer promising avenues for tuberculosis antibiotic development. Mycoplanecins bind to dnaN with nanomolar affinity, meaning they interact very strongly and specifically with their target, effectively blocking its role in bacterial DNA replication. Among them, mycoplanecin E has shown exceptional potency, with an MIC of just 83 ng/mL, which is 24 times more effective than griselimycin. Structural studies, including co-crystallization with dnaN, have revealed how mycoplanecins engage the clamp, making them strong candidates for new anti-tuberculosis therapies. Another dnaN inhibitor known as, griselimycins, is a cyclic peptide derived from Streptomyces species. Griselimycins have been shown to bind to dnaN with high affinity, disrupting its interaction with DNA polymerase III. These compounds have already provided key structural insights into how dnaN can be inhibited and have served as a foundation for further tuberculosis antibiotic research. Beyond natural products, synthetic peptides and small molecules have also been developed to target dnaN. These include engineered peptides that mimic natural dnaN-binding motifs and small molecules identified through high-throughput screening. Some of these synthetic inhibitors have successfully blocked bacterial replication in lab models, demonstrating the broad potential of dnaN-targeting strategies for next-generation antibiotic development.
== References == | Wikipedia/DnaN |
Extrachromosomal DNA (abbreviated ecDNA) is any DNA that is found off the chromosomes, either inside or outside the nucleus of a cell. Most DNA in an individual genome is found in chromosomes contained in the nucleus. Multiple forms of extrachromosomal DNA exist, and, while some of these serve important biological functions, they can also play a role in diseases such as cancer.
In prokaryotes, nonviral extrachromosomal DNA is primarily found in plasmids, whereas, in eukaryotes extrachromosomal DNA is primarily found in organelles. Mitochondrial DNA is a main source of this extrachromosomal DNA in eukaryotes. The fact that this organelle contains its own DNA supports the hypothesis that mitochondria originated as bacterial cells engulfed by ancestral eukaryotic cells. Extrachromosomal DNA is often used in research into replication because it is easy to identify and isolate.
Although extrachromosomal circular DNA (eccDNA) is found in normal eukaryotic cells, extrachromosomal DNA (ecDNA) is a distinct entity that has been identified in the nuclei of cancer cells and has been shown to carry many copies of driver oncogenes. ecDNA is considered to be a primary mechanism of gene amplification, resulting in many copies of driver oncogenes and very aggressive cancers.
Extrachromosomal DNA in the cytoplasm has been found to be structurally different from nuclear DNA. Cytoplasmic DNA is less methylated than DNA found within the nucleus. It was also confirmed that the sequences of cytoplasmic DNA were different from nuclear DNA in the same organism, showing that cytoplasmic DNAs are not simply fragments of nuclear DNA. In cancer cells, ecDNA have been shown to be primarily isolated to the nucleus (reviewed in ).
In addition to DNA found outside the nucleus in cells, infection by viral genomes also provides an example of extrachromosomal DNA.
== Prokaryotic ==
Although prokaryotic organisms do not possess a membrane-bound nucleus like eukaryotes, they do contain a nucleoid region in which the main chromosome is found. Extrachromosomal DNA exists in prokaryotes outside the nucleoid region as circular or linear plasmids. Bacterial plasmids are typically short sequences, consisting of 1 to a few hundred kilobase (kb) segments, and contain an origin of replication which allows the plasmid to replicate independently of the bacterial chromosome. The total number of a particular plasmid within a cell is referred to as the copy number and can range from as few as two copies per cell to as many as several hundred copies per cell. Circular bacterial plasmids are classified according to the special functions that the genes encoded on the plasmid provide. Fertility plasmids, or f plasmids, allow for conjugation to occur whereas resistance plasmids, or r plasmids, contain genes that convey resistance to a variety of different antibiotics such as ampicillin and tetracycline. Virulence plasmids contain the genetic elements necessary for bacteria to become pathogenic. Degradative plasmids that contain genes that allow bacteria to degrade a variety of substances such as aromatic compounds and xenobiotics. Bacterial plasmids can also function in pigment production, nitrogen fixation and the resistance to heavy metals.
Naturally occurring circular plasmids can be modified to contain multiple resistance genes and several unique restriction sites, making them valuable tools as cloning vectors in biotechnology. Circular bacterial plasmids are also the basis for the production of DNA vaccines. Plasmid DNA vaccines are genetically engineered to contain a gene which encodes for an antigen or a protein produced by a pathogenic virus, bacterium or other parasites. Once delivered into the host, the products of the plasmid genes will then stimulate both the innate immune response and the adaptive immune response of the host. The plasmids are often coated with some type of adjuvant prior to delivery to enhance the immune response from the host.
Linear bacterial plasmids have been identified in several species of spirochete bacteria, including members of the genus Borrelia (to which the pathogen responsible for Lyme disease belongs), several species of the gram positive soil bacteria of the genus Streptomyces, and in the gram negative species Thiobacillus versutus, a bacterium that oxidizes sulfur. Linear plasmids of prokaryotes are found either containing a hairpin loop or a covalently bonded protein attached to the telomeric ends of the DNA molecule. The adenine-thymine rich hairpin loops of the Borrelia bacteria range in size from 5 kilobase pairs (kb) to over 200 kb and contain the genes responsible for producing a group of major surface proteins, or antigens, on the bacteria that allow it to evade the immune response of its infected host. The linear plasmids which contain a protein that has been covalently attached to the 5’ end of the DNA strands are known as invertrons and can range in size from 9 kb to over 600 kb consisting of inverted terminal repeats. The linear plasmids with a covalently attached protein may assist with bacterial conjugation and integration of the plasmids into the genome. These types of linear plasmids represent the largest class of extrachromosomal DNA as they are not only present in certain bacterial cells, but all linear extrachromosomal DNA molecules found in eukaryotic cells also take on this invertron structure with a protein attached to the 5’ end.
The long, linear "borgs" that co-occur with a species of archaeon – which may host them and shares many of their genes – could be an unknown form of extrachromosomal DNA structures.
== Eukaryotic ==
=== Mitochondrial ===
Mitochondria present in eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial matrix. In multicellular animals, including humans, the circular mtDNA chromosome contains 13 genes that encode proteins that are part of the electron transport chain and 24 genes for mitochondrial RNAs; these genes are broken down into 2 rRNA genes and 22 tRNA genes. The size of an animal mtDNA plasmid is roughly 16.6 kb and, although it contains genes for tRNA and mRNA synthesis, proteins coded for by nuclear genes are still required for the mtDNA to replicate or for mitochondrial proteins to be translated. There is only one region of the mitochondrial chromosome that does not contain a coding sequence, the 1 kb region known as the D-loop to which nuclear regulatory proteins bind. The number of mtDNA molecules per mitochondrion varies from species to species, as well as between cells with different energy demands. For example, muscle and liver cells contain more copies of mtDNA per mitochondrion than blood and skin cells do. Due to the proximity of the electron transport chain within the mitochondrial inner membrane and the production of reactive oxygen species (ROS), and due to the fact that the mtDNA molecule is not bound by or protected by histones, the mtDNA is more susceptible to DNA damage than nuclear DNA. In cases where mtDNA damage does occur, the DNA can either be repaired via base excision repair pathways, or the damaged mtDNA molecule is destroyed (without causing damage to the mitochondrion since there are multiple copies of mtDNA per mitochondrion).
The standard genetic code by which nuclear genes are translated is universal, meaning that each 3-base sequence of DNA codes for the same amino acid regardless of what species from which the DNA comes. However, this code is quite universal and is slightly different in mitochondrial DNA of fungi, animals, protists and plants. While most of the 3-base sequences (codons) in the mtDNA of these organisms do code for the same amino acids as those of the nuclear genetic code, a few are different.
The coding differences are thought to be a result of chemical modifications in the transfer RNAs that interact with the messenger RNAs produced as a result of transcribing the mtDNA sequences.
=== Chloroplast ===
Eukaryotic chloroplasts, as well as the other plant plastids, also contain extrachromosomal DNA molecules. Most chloroplasts house all of their genetic material in a single ringed chromosome, however in some species there is evidence of multiple smaller ringed plasmids. A recent theory that questions the current standard model of ring shaped chloroplast DNA (cpDNA), suggests that cpDNA may more commonly take a linear shape. A single molecule of cpDNA can contain anywhere from 100 to 200 genes and varies in size from species to species. The size of cpDNA in higher plants is around 120–160 kb. The genes found on the cpDNA code for mRNAs that are responsible for producing necessary components of the photosynthetic pathway as well as coding for tRNAs, rRNAs, RNA polymerase subunits, and ribosomal protein subunits. Like mtDNA, cpDNA is not fully autonomous and relies upon nuclear gene products for replication and production of chloroplast proteins. Chloroplasts contain multiple copies of cpDNA and the number can vary not only from species to species or cell type to cell type, but also within a single cell depending upon the age and stage of development of the cell. For example, cpDNA content in the chloroplasts of young cells, during the early stages of development where the chloroplasts are in the form of indistinct proplastids, are much higher than those present when that cell matures and expands, containing fully mature plastids.
=== Circular ===
Extrachromosomal circular DNA (eccDNA) are present in all eukaryotic cells, are usually derived from genomic DNA, and consist of repetitive sequences of DNA found in both coding and non-coding regions of chromosomes. EccDNA can vary in size from less than 2000 base pairs to more than 20,000 base pairs. In plants, eccDNA contain repeated sequences similar to those that are found in the centromeric regions of the chromosomes and in repetitive satellite DNA. In animals, eccDNA molecules have been shown to contain repetitive sequences that are seen in satellite DNA, 5S ribosomal DNA and telomere DNA. Certain organisms, such as yeast, rely on chromosomal DNA replication to produce eccDNA whereas eccDNA formation can occur in other organisms, such as mammals, independently of the replication process. The function of eccDNA have not been widely studied, but it has been proposed that the production of eccDNA elements from genomic DNA sequences add to the plasticity of the eukaryotic genome and can influence genome stability, cell aging and the evolution of chromosomes.
A distinct type of extrachromosomal DNA, denoted as ecDNA, is commonly observed in human cancer cells. ecDNA found in cancer cells contain one or more genes that confer a selective advantage. ecDNA are much larger than eccDNA, and are visible by light microscopy. ecDNA in cancers generally range in size from 1-3 MB and beyond. Large ecDNA molecules have been found in the nuclei of human cancer cells and are shown to carry many copies of driver oncogenes, which are transcribed in tumor cells. Based on this evidence it is thought that ecDNA contributes to cancer growth.
Specialized tools exist that allow ecDNA to be identified, such as
software developed by Paul Mischel and Vineet Bafna that allows ecDNA to be identified in microscopic images
"Circle-Seq, a method for physically isolating ecDNA from cells, removing any remaining linear DNA with enzymes, and sequencing the circular DNA that remains", developed by Birgitte Regenberg and her team at the University of Copenhagen.
== Viral ==
Viral DNA are an example of extrachromosomal DNA. Understanding viral genomes is very important for understanding the evolution and mutation of the virus. Some viruses, such as HIV and oncogenic viruses, incorporate their own DNA into the genome of the host cell. Viral genomes can be made up of single stranded DNA (ssDNA), double stranded DNA (dsDNA) and can be found in both linear and circular form.
One example of infection of a virus constituting as extrachromosomal DNA is the human papillomavirus (HPV). The HPV DNA genome undergoes three distinct stages of replication: establishment, maintenance and amplification. HPV infects epithelial cells in the anogenital tract and oral cavity. Normally, HPV is detected and cleared by the immune system. The recognition of viral DNA is an important part of immune responses. For this virus to persist, the circular genome must be replicated and inherited during cell division.
=== Recognition by host cell ===
Cells can recognize foreign cytoplasmic DNA. Understanding the recognition pathways has implications towards prevention and treatment of diseases. Cells have sensors that can specifically recognize viral DNA such as the Toll-like receptor (TLR) pathway.
The Toll Pathway was recognized, first in insects, as a pathway that allows certain cell types to act as sensors capable of detecting a variety of bacterial or viral genomes and PAMPS (pathogen-associated molecular patterns). PAMPs are known to be potent activators of innate immune signaling. There are approximately 10 human Toll-Like Receptors (TLRs). Different TLRs in human detect different PAMPS: lipopolysaccharides by TLR4, viral dsRNA by TLR3, viral ssRNA by TLR7/TLR8, viral or bacterial unmethylated DNA by TLR9. TLR9 has evolved to detect CpG DNA commonly found in bacteria and viruses and to initiate the production of IFN (type I interferons ) and other cytokines.
== Inheritance ==
Inheritance of extrachromosomal DNA differs from the inheritance of nuclear DNA found in chromosomes. Unlike chromosomes, ecDNA does not contain centromeres and therefore exhibits a non-Mendelian inheritance pattern that gives rise to heterogeneous cell populations. In humans, virtually all of the cytoplasm is inherited from the egg of the mother. For this reason, organelle DNA, including mtDNA, is inherited from the mother. Mutations in mtDNA or other cytoplasmic DNA will also be inherited from the mother. This uniparental inheritance is an example of non-Mendelian inheritance. Plants also show uniparental mtDNA inheritance. Most plants inherit mtDNA maternally with one noted exception being the redwood Sequoia sempervirens that inherit mtDNA paternally.
There are two theories why the paternal mtDNA is rarely transmitted to the offspring. One is simply the fact that paternal mtDNA is at such a lower concentration than the maternal mtDNA and thus it is not detectable in the offspring. A second, more complex theory, involves the digestion of the paternal mtDNA to prevent its inheritance. It is theorized that the uniparental inheritance of mtDNA, which has a high mutation rate, might be a mechanism to maintain the homoplasmy of cytoplasmic DNA.
== Clinical significance ==
Sometimes called EEs, extrachromosomal elements, have been associated with genomic instability in eukaryotes. Small polydispersed DNAs (spcDNAs), a type of eccDNA, are commonly found in conjunction with genome instability. SpcDNAs are derived from repetitive sequences such as satellite DNA, retrovirus-like DNA elements, and transposable elements in the genome. They are thought to be the products of gene rearrangements.
Extrachromosomal DNA (ecDNA) found in cancer have historically been referred to as Double minute chromosomes (DMs), which present as paired chromatin bodies under light microscopy. Double minute chromosomes represent ~30% of the cancer-containing spectrum of ecDNA, including single bodies and have been found to contain identical gene content as single bodies. The ecDNA notation encompasses all forms of the large, oncogene-containing, extrachromosomal DNA found in cancer cells. This type of ecDNA is commonly seen in cancer cells of various histologies, but virtually never in normal cells. ecDNA are thought to be produced through double-strand breaks in chromosomes or over-replication of DNA in an organism. Studies show that in cases of cancer and other genomic instability, higher levels of EEs can be observed.
Mitochondrial DNA can play a role in the onset of disease in a variety of ways. Point mutations in or alternative gene arrangements of mtDNA have been linked to several diseases that affect the heart, central nervous system, endocrine system, gastrointestinal tract, eye, and kidney. Loss of the amount of mtDNA present in the mitochondria can lead to a whole subset of diseases known as mitochondrial depletion syndromes (MDDs) which affect the liver, central and peripheral nervous systems, smooth muscle and hearing in humans. There have been mixed, and sometimes conflicting, results in studies that attempt to link mtDNA copy number to the risk of developing certain cancers. Studies have been conducted that show an association between both increased and decreased mtDNA levels and the increased risk of developing breast cancer. A positive association between increased mtDNA levels and an increased risk for developing kidney tumors has been observed but there does not appear to be a link between mtDNA levels and the development of stomach cancer.
Extrachromosomal DNA is found in Apicomplexa, which is a group of protozoa. The malaria parasite (genus Plasmodium), the AIDS-related pathogen (Taxoplasma and Cryptosporidium) are both members of the Apicomplexa group. Mitochondrial DNA (mtDNA) was found in the malaria parasite. There are two forms of extrachromosomal DNA found in the malaria parasites. One of these is 6-kb linear DNA and the second is 35-kb circular DNA. These DNA molecules have been researched as potential nucleotide target sites for antibiotics.
== Role of ecDNA in cancer ==
Gene amplification is among the most common mechanisms of oncogene activation. Gene amplifications in cancer are often on extrachromosomal, circular elements. One of the primary functions of ecDNA in cancer is to enable the tumor to rapidly reach high copy numbers, while also promoting rapid, massive cell-to-cell genetic heterogeneity. The most commonly amplified oncogenes in cancer are found on ecDNA and have been shown to be highly dynamic, re-integrating into non-native chromosomes as homogeneous staining regions (HSRs) and altering copy numbers and composition in response to various drug treatments.
ecDNA is responsible for a large number of the more advanced and most serious cancers, as well as for the resistance to anti-cancer drugs.
The circular shape of ecDNA differs from the linear structure of chromosomal DNA in meaningful ways that influence cancer pathogenesis. Oncogenes encoded on ecDNA have massive transcriptional output, ranking in the top 1% of genes in the entire transcriptome. In contrast to bacterial plasmids or mitochondrial DNA, ecDNA are chromatinized, containing high levels of active histone marks, but a paucity of repressive histone marks. The ecDNA chromatin architecture lacks the higher-order compaction that is present on chromosomal DNA and is among the most accessible DNA in the entire cancer genome.
EcDNAs could be clustered together within the nucleus, which can be referred to as ecDNA hubs. Spacially, ecDNA hubs could cause intermolecular enhancer–gene interactions to promote oncogene overexpression.
== References ==
== Further reading == | Wikipedia/Extrachromosomal_DNA |
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones.
These enzymes catalyze the chemical reaction
deoxynucleoside triphosphate + DNAn ⇌ pyrophosphate + DNAn+1.
DNA polymerase adds nucleotides to the three prime (3')-end of a DNA strand, one nucleotide at a time. Every time a cell divides, DNA polymerases are required to duplicate the cell's DNA, so that a copy of the original DNA molecule can be passed to each daughter cell. In this way, genetic information is passed down from generation to generation.
Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form, in the process breaking the hydrogen bonds between the nucleotide bases. This opens up or "unzips" the double-stranded DNA to give two single strands of DNA that can be used as templates for replication in the above reaction.
== History ==
In 1956, Arthur Kornberg and colleagues discovered DNA polymerase I (Pol I), in Escherichia coli. They described the DNA replication process by which DNA polymerase copies the base sequence of a template DNA strand. Kornberg was later awarded the Nobel Prize in Physiology or Medicine in 1959 for this work. DNA polymerase II was discovered by Thomas Kornberg (the son of Arthur Kornberg) and Malcolm E. Gefter in 1970 while further elucidating the role of Pol I in E. coli DNA replication. Three more DNA polymerases have been found in E. coli, including DNA polymerase III (discovered in the 1970s) and DNA polymerases IV and V (discovered in 1999). From 1983 on, DNA polymerases have been used in the polymerase chain reaction (PCR), and from 1988 thermostable DNA polymerases were used instead, as they do not need to be added in every cycle of a PCR.
== Function ==
The main function of DNA polymerase is to synthesize DNA from deoxyribonucleotides, the building blocks of DNA. The DNA copies are created by the pairing of nucleotides to bases present on each strand of the original DNA molecule. This pairing always occurs in specific combinations, with cytosine along with guanine, and thymine along with adenine, forming two separate pairs, respectively. By contrast, RNA polymerases synthesize RNA from ribonucleotides from either RNA or DNA.
When synthesizing new DNA, DNA polymerase can add free nucleotides only to the 3' end of the newly forming strand. This results in elongation of the newly forming strand in a 5'–3' direction.
It is important to note that the directionality of the newly forming strand (the daughter strand) is opposite to the direction in which DNA polymerase moves along the template strand. Since DNA polymerase requires a free 3' OH group for initiation of synthesis, it can synthesize in only one direction by extending the 3' end of the preexisting nucleotide chain. Hence, DNA polymerase moves along the template strand in a 3'–5' direction, and the daughter strand is formed in a 5'–3' direction. This difference enables the resultant double-strand DNA formed to be composed of two DNA strands that are antiparallel to each other.
The function of DNA polymerase is not quite perfect, with the enzyme making about one mistake for every billion base pairs copied. Error correction is a property of some, but not all DNA polymerases. This process corrects mistakes in newly synthesized DNA. When an incorrect base pair is recognized, DNA polymerase moves backwards by one base pair of DNA. The 3'–5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue forwards. This preserves the integrity of the original DNA strand that is passed onto the daughter cells.
Fidelity is very important in DNA replication. Mismatches in DNA base pairing can potentially result in dysfunctional proteins and could lead to cancer. Many DNA polymerases contain an exonuclease domain, which acts in detecting base pair mismatches and further performs in the removal of the incorrect nucleotide to be replaced by the correct one. The shape and the interactions accommodating the Watson and Crick base pair are what primarily contribute to the detection or error. Hydrogen bonds play a key role in base pair binding and interaction. The loss of an interaction, which occurs at a mismatch, is said to trigger a shift in the balance, for the binding of the template-primer, from the polymerase, to the exonuclease domain. In addition, an incorporation of a wrong nucleotide causes a retard in DNA polymerization. This delay gives time for the DNA to be switched from the polymerase site to the exonuclease site. Different conformational changes and loss of interaction occur at different mismatches. In a purine:pyrimidine mismatch there is a displacement of the pyrimidine towards the major groove and the purine towards the minor groove. Relative to the shape of DNA polymerase's binding pocket, steric clashes occur between the purine and residues in the minor groove, and important van der Waals and electrostatic interactions are lost by the pyrimidine. Pyrimidine:pyrimidine and purine:purine mismatches present less notable changes since the bases are displaced towards the major groove, and less steric hindrance is experienced. However, although the different mismatches result in different steric properties, DNA polymerase is still able to detect and differentiate them so uniformly and maintain fidelity in DNA replication. DNA polymerization is also critical for many mutagenesis processes and is widely employed in biotechnologies.
=== Structure ===
The known DNA polymerases have highly conserved structure, which means that their overall catalytic subunits vary very little from species to species, independent of their domain structures. Conserved structures usually indicate important, irreplaceable functions of the cell, the maintenance of which provides evolutionary advantages. The shape can be described as resembling a right hand with thumb, finger, and palm domains. The palm domain appears to function in catalyzing the transfer of phosphoryl groups in the phosphoryl transfer reaction. DNA is bound to the palm when the enzyme is active. This reaction is believed to be catalyzed by a two-metal-ion mechanism. The finger domain functions to bind the nucleoside triphosphates with the template base. The thumb domain plays a potential role in the processivity, translocation, and positioning of the DNA.
=== Processivity ===
DNA polymerase's rapid catalysis due to its processive nature. Processivity is a characteristic of enzymes that function on polymeric substrates. In the case of DNA polymerase, the degree of processivity refers to the average number of nucleotides added each time the enzyme binds a template. The average DNA polymerase requires about one second locating and binding a primer/template junction. Once it is bound, a nonprocessive DNA polymerase adds nucleotides at a rate of one nucleotide per second.: 207–208 Processive DNA polymerases, however, add multiple nucleotides per second, drastically increasing the rate of DNA synthesis. The degree of processivity is directly proportional to the rate of DNA synthesis. The rate of DNA synthesis in a living cell was first determined as the rate of phage T4 DNA elongation in phage infected E. coli. During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second.
DNA polymerase's ability to slide along the DNA template allows increased processivity. There is a dramatic increase in processivity at the replication fork. This increase is facilitated by the DNA polymerase's association with proteins known as the sliding DNA clamp. The clamps are multiple protein subunits associated in the shape of a ring. Using the hydrolysis of ATP, a class of proteins known as the sliding clamp loading proteins open up the ring structure of the sliding DNA clamps allowing binding to and release from the DNA strand. Protein–protein interaction with the clamp prevents DNA polymerase from diffusing from the DNA template, thereby ensuring that the enzyme binds the same primer/template junction and continues replication.: 207–208 DNA polymerase changes conformation, increasing affinity to the clamp when associated with it and decreasing affinity when it completes the replication of a stretch of DNA to allow release from the clamp.
DNA polymerase processivity has been studied with in vitro single-molecule experiments (namely, optical tweezers and magnetic tweezers) have revealed the synergies between DNA polymerases and other molecules of the replisome (helicases and SSBs) and with the DNA replication fork. These results have led to the development of synergetic kinetic models for DNA replication describing the resulting DNA polymerase processivity increase.
== Variation across species ==
Based on sequence homology, DNA polymerases can be further subdivided into seven different families: A, B, C, D, X, Y, and RT.
Some viruses also encode special DNA polymerases, such as Hepatitis B virus DNA polymerase. These may selectively replicate viral DNA through a variety of mechanisms. Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp). It polymerizes DNA from a template of RNA.
=== Prokaryotic polymerase ===
Prokaryotic polymerases exist in two forms: core polymerase and holoenzyme. Core polymerase synthesizes DNA from the DNA template but it cannot initiate the synthesis alone or accurately. Holoenzyme accurately initiates synthesis.
==== Pol I ====
Prokaryotic family A polymerases include the DNA polymerase I (Pol I) enzyme, which is encoded by the polA gene and ubiquitous among prokaryotes. This repair polymerase is involved in excision repair with both 3'–5' and 5'–3' exonuclease activity and processing of Okazaki fragments generated during lagging strand synthesis. Pol I is the most abundant polymerase, accounting for >95% of polymerase activity in E. coli; yet cells lacking Pol I have been found suggesting Pol I activity can be replaced by the other four polymerases. Pol I adds ~15-20 nucleotides per second, thus showing poor processivity. Instead, Pol I starts adding nucleotides at the RNA primer:template junction known as the origin of replication (ori). Approximately 400 bp downstream from the origin, the Pol III holoenzyme is assembled and takes over replication at a highly processive speed and nature.
Taq polymerase is a heat-stable enzyme of this family that lacks proofreading ability.
==== Pol II ====
DNA polymerase II is a family B polymerase encoded by the polB gene. Pol II has 3'–5' exonuclease activity and participates in DNA repair, replication restart to bypass lesions, and its cell presence can jump from ~30-50 copies per cell to ~200–300 during SOS induction. Pol II is also thought to be a backup to Pol III as it can interact with holoenzyme proteins and assume a high level of processivity. The main role of Pol II is thought to be the ability to direct polymerase activity at the replication fork and help stalled Pol III bypass terminal mismatches.
Pfu DNA polymerase is a heat-stable enzyme of this family found in the hyperthermophilic archaeon Pyrococcus furiosus. Detailed classification divides family B in archaea into B1, B2, B3, in which B2 is a group of pseudoenzymes. Pfu belongs to family B3. Others PolBs found in archaea are part of "Casposons", Cas1-dependent transposons. Some viruses (including Φ29 DNA polymerase) and mitochondrial plasmids carry polB as well.
==== Pol III ====
DNA polymerase III holoenzyme is the primary enzyme involved in DNA replication in E. coli and belongs to family C polymerases. It consists of three assemblies: the pol III core, the beta sliding clamp processivity factor, and the clamp-loading complex. The core consists of three subunits: α, the polymerase activity hub, ɛ, exonucleolytic proofreader, and θ, which may act as a stabilizer for ɛ. The beta sliding clamp processivity factor is also present in duplicate, one for each core, to create a clamp that encloses DNA allowing for high processivity. The third assembly is a seven-subunit (τ2γδδ′χψ) clamp loader complex.
The old textbook "trombone model" depicts an elongation complex with two equivalents of the core enzyme at each replication fork (RF), one for each strand, the lagging and leading. However, recent evidence from single-molecule studies indicates an average of three stoichiometric equivalents of core enzyme at each RF for both Pol III and its counterpart in B. subtilis, PolC. In-cell fluorescent microscopy has revealed that leading strand synthesis may not be completely continuous, and Pol III* (i.e., the holoenzyme α, ε, τ, δ and χ subunits without the ß2 sliding clamp) has a high frequency of dissociation from active RFs. In these studies, the replication fork turnover rate was about 10s for Pol III*, 47s for the ß2 sliding clamp, and 15m for the DnaB helicase. This suggests that the DnaB helicase may remain stably associated at RFs and serve as a nucleation point for the competent holoenzyme. In vitro single-molecule studies have shown that Pol III* has a high rate of RF turnover when in excess, but remains stably associated with replication forks when concentration is limiting. Another single-molecule study showed that DnaB helicase activity and strand elongation can proceed with decoupled, stochastic kinetics.
==== Pol IV ====
In E. coli, DNA polymerase IV (Pol IV) is an error-prone DNA polymerase involved in non-targeted mutagenesis. Pol IV is a Family Y polymerase expressed by the dinB gene that is switched on via SOS induction caused by stalled polymerases at the replication fork. During SOS induction, Pol IV production is increased tenfold and one of the functions during this time is to interfere with Pol III holoenzyme processivity. This creates a checkpoint, stops replication, and allows time to repair DNA lesions via the appropriate repair pathway. Another function of Pol IV is to perform translesion synthesis at the stalled replication fork like, for example, bypassing N2-deoxyguanine adducts at a faster rate than transversing undamaged DNA. Cells lacking the dinB gene have a higher rate of mutagenesis caused by DNA damaging agents.
==== Pol V ====
DNA polymerase V (Pol V) is a Y-family DNA polymerase that is involved in SOS response and translesion synthesis DNA repair mechanisms. Transcription of Pol V via the umuDC genes is highly regulated to produce only Pol V when damaged DNA is present in the cell generating an SOS response. Stalled polymerases causes RecA to bind to the ssDNA, which causes the LexA protein to autodigest. LexA then loses its ability to repress the transcription of the umuDC operon. The same RecA-ssDNA nucleoprotein posttranslationally modifies the UmuD protein into UmuD' protein. UmuD and UmuD' form a heterodimer that interacts with UmuC, which in turn activates umuC's polymerase catalytic activity on damaged DNA. In E. coli, a polymerase "tool belt" model for switching pol III with pol IV at a stalled replication fork, where both polymerases bind simultaneously to the β-clamp, has been proposed. However, the involvement of more than one TLS polymerase working in succession to bypass a lesion has not yet been shown in E. coli. Moreover, Pol IV can catalyze both insertion and extension with high efficiency, whereas pol V is considered the major SOS TLS polymerase. One example is the bypass of intra strand guanine thymine cross-link where it was shown on the basis of the difference in the mutational signatures of the two polymerases, that pol IV and pol V compete for TLS of the intra-strand crosslink.
==== Family D ====
In 1998, the family D of DNA polymerase was discovered in Pyrococcus furiosus and Methanococcus jannaschii. The PolD complex is a heterodimer of two chains, each encoded by DP1 (small proofreading) and DP2 (large catalytic). Unlike other DNA polymerases, the structure and mechanism of the DP2 catalytic core resemble that of multi-subunit RNA polymerases. The DP1-DP2 interface resembles that of Eukaryotic Class B polymerase zinc finger and its small subunit. DP1, a Mre11-like exonuclease, is likely the precursor of small subunit of Pol α and ε, providing proofreading capabilities now lost in Eukaryotes. Its N-terminal HSH domain is similar to AAA proteins, especially Pol III subunit δ and RuvB, in structure. DP2 has a Class II KH domain. Pyrococcus abyssi polD is more heat-stable and more accurate than Taq polymerase, but has not yet been commercialized. It has been proposed that family D DNA polymerase was the first to evolve in cellular organisms and that the replicative polymerase of the Last Universal Cellular Ancestor (LUCA) belonged to family D.
=== Eukaryotic DNA polymerase ===
==== Polymerases β, λ, σ, μ (beta, lambda, sigma, mu) and TdT ====
Family X polymerases contain the well-known eukaryotic polymerase pol β (beta), as well as other eukaryotic polymerases such as Pol σ (sigma), Pol λ (lambda), Pol μ (mu), and Terminal deoxynucleotidyl transferase (TdT). Family X polymerases are found mainly in vertebrates, and a few are found in plants and fungi. These polymerases have highly conserved regions that include two helix-hairpin-helix motifs that are imperative in the DNA-polymerase interactions. One motif is located in the 8 kDa domain that interacts with downstream DNA and one motif is located in the thumb domain that interacts with the primer strand. Pol β, encoded by POLB gene, is required for short-patch base excision repair, a DNA repair pathway that is essential for repairing alkylated or oxidized bases as well as abasic sites. Pol λ and Pol μ, encoded by the POLL and POLM genes respectively, are involved in non-homologous end-joining, a mechanism for rejoining DNA double-strand breaks due to hydrogen peroxide and ionizing radiation, respectively. TdT is expressed only in lymphoid tissue, and adds "n nucleotides" to double-strand breaks formed during V(D)J recombination to promote immunological diversity.
==== Polymerases α, δ and ε (alpha, delta, and epsilon) ====
Pol α (alpha), Pol δ (delta), and Pol ε (epsilon) are members of Family B Polymerases and are the main polymerases involved with nuclear DNA replication. Pol α complex (pol α-DNA primase complex) consists of four subunits: the catalytic subunit POLA1, the regulatory subunit POLA2, and the small and the large primase subunits PRIM1 and PRIM2 respectively. Once primase has created the RNA primer, Pol α starts replication elongating the primer with ~20 nucleotides. Due to its high processivity, Pol δ takes over the leading and lagging strand synthesis from Pol α.: 218–219 Pol δ is expressed by genes POLD1, creating the catalytic subunit, POLD2, POLD3, and POLD4 creating the other subunits that interact with Proliferating Cell Nuclear Antigen (PCNA), which is a DNA clamp that allows Pol δ to possess processivity. Pol ε is encoded by the POLE1, the catalytic subunit, POLE2, and POLE3 gene. It has been reported that the function of Pol ε is to extend the leading strand during replication, while Pol δ primarily replicates the lagging strand; however, recent evidence suggested that Pol δ might have a role in replicating the leading strand of DNA as well. Pol ε's C-terminus "polymerase relic" region, despite being unnecessary for polymerase activity, is thought to be essential to cell vitality. The C-terminus region is thought to provide a checkpoint before entering anaphase, provide stability to the holoenzyme, and add proteins to the holoenzyme necessary for initiation of replication. Pol ε has a larger "palm" domain that provides high processivity independently of PCNA.
Compared to other Family B polymerases, the DEDD exonuclease family responsible for proofreading is inactivated in Pol α. Pol ε is unique in that it has two zinc finger domains and an inactive copy of another family B polymerase in its C-terminal. The presence of this zinc finger has implications in the origins of Eukaryota, which in this case is placed into the Asgard group with archaeal B3 polymerase.
==== Polymerases η, ι and κ (eta, iota, and kappa) ====
Pol η (eta), Pol ι (iota), and Pol κ (kappa), are Family Y DNA polymerases involved in the DNA repair by translation synthesis and encoded by genes POLH, POLI, and POLK respectively. Members of Family Y have five common motifs to aid in binding the substrate and primer terminus and they all include the typical right hand thumb, palm and finger domains with added domains like little finger (LF), polymerase-associated domain (PAD), or wrist. The active site, however, differs between family members due to the different lesions being repaired. Polymerases in Family Y are low-fidelity polymerases, but have been proven to do more good than harm as mutations that affect the polymerase can cause various diseases, such as skin cancer and Xeroderma Pigmentosum Variant (XPS). The importance of these polymerases is evidenced by the fact that gene encoding DNA polymerase η is referred as XPV, because loss of this gene results in the disease Xeroderma Pigmentosum Variant. Pol η is particularly important for allowing accurate translesion synthesis of DNA damage resulting from ultraviolet radiation. The functionality of Pol κ is not completely understood, but researchers have found two probable functions. Pol κ is thought to act as an extender or an inserter of a specific base at certain DNA lesions. All three translesion synthesis polymerases, along with Rev1, are recruited to damaged lesions via stalled replicative DNA polymerases. There are two pathways of damage repair leading researchers to conclude that the chosen pathway depends on which strand contains the damage, the leading or lagging strand.
==== Polymerases Rev1 and ζ (zeta) ====
Pol ζ, another B family polymerase, is made of two subunits: Rev3 – the catalytic subunit; and Rev7 (MAD2L2) – which increases the catalytic function of the polymerase, and is involved in translation synthesis. Pol ζ lacks 3' to 5' exonuclease activity, and is unique in that it can extend primers with terminal mismatches. Rev1 has three regions of interest in the BRCT domain, ubiquitin-binding domain, and C-terminal domain and has dCMP transferase ability, which adds deoxycytidine opposite lesions that would stall replicative polymerases Pol δ and Pol ε. These stalled polymerases activate ubiquitin complexes that, in turn, disassociate replication polymerases and recruit Pol ζ and Rev1. Together, Pol ζ and Rev1 add deoxycytidine, and Pol ζ extends past the lesion. Through a yet undetermined process, Pol ζ disassociates, and replication polymerases reassociate and continue replication. Pol ζ and Rev1 are not required for replication, but loss of REV3 gene in budding yeast can cause increased sensitivity to DNA-damaging agents due to collapse of replication forks where replication polymerases have stalled.
==== Telomerase ====
Telomerase is a ribonucleoprotein which functions to replicate ends of linear chromosomes since normal DNA polymerase cannot replicate the ends, or telomeres. The single-strand 3' overhang of the double-strand chromosome with the sequence 5'-TTAGGG-3' recruits telomerase. Telomerase acts like other DNA polymerases by extending the 3' end, but, unlike other DNA polymerases, telomerase does not require a template. The TERT subunit, an example of a reverse transcriptase, uses the RNA subunit to form the primer–template junction that allows telomerase to extend the 3' end of chromosome ends. The gradual decrease in size of telomeres as the result of many replications over a lifetime are thought to be associated with the effects of aging.: 248–249
==== Polymerases γ, θ and ν (gamma, theta and nu) ====
Pol γ (gamma), Pol θ (theta), and Pol ν (nu) are Family A polymerases. Pol γ, encoded by the POLG gene, was long thought to be the only mitochondrial polymerase. However, recent research shows that at least Pol β (beta), a Family X polymerase, is also present in mitochondria. Any mutation that leads to limited or non-functioning Pol γ has a significant effect on mtDNA and is the most common cause of autosomal inherited mitochondrial disorders. Pol γ contains a C-terminus polymerase domain and an N-terminus 3'–5' exonuclease domain that are connected via the linker region, which binds the accessory subunit. The accessory subunit binds DNA and is required for processivity of Pol γ. Point mutation A467T in the linker region is responsible for more than one-third of all Pol γ-associated mitochondrial disorders. While many homologs of Pol θ, encoded by the POLQ gene, are found in eukaryotes, its function is not clearly understood. The sequence of amino acids in the C-terminus is what classifies Pol θ as Family A polymerase, although the error rate for Pol θ is more closely related to Family Y polymerases. Pol θ extends mismatched primer termini and can bypass abasic sites by adding a nucleotide. It also has Deoxyribophosphodiesterase (dRPase) activity in the polymerase domain and can show ATPase activity in close proximity to ssDNA. Pol ν (nu) is considered to be the least effective of the polymerase enzymes. However, DNA polymerase nu plays an active role in homology repair during cellular responses to crosslinks, fulfilling its role in a complex with helicase.
Plants use two Family A polymerases to copy both the mitochondrial and plastid genomes. They are more similar to bacterial Pol I than they are to mammalian Pol γ.
==== Reverse transcriptase ====
Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp) that synthesizes DNA from a template of RNA. The reverse transcriptase family contain both DNA polymerase functionality and RNase H functionality, which degrades RNA base-paired to DNA. An example of a retrovirus is HIV. Reverse transcriptase is commonly employed in amplification of RNA for research purposes. Using an RNA template, PCR can utilize reverse transcriptase, creating a DNA template. This new DNA template can then be used for typical PCR amplification. The products of such an experiment are thus amplified PCR products from RNA.
Each HIV retrovirus particle contains two RNA genomes, but, after an infection, each virus generates only one provirus. After infection, reverse transcription is accompanied by template switching between the two genome copies (copy choice recombination). From 5 to 14 recombination events per genome occur at each replication cycle. Template switching (recombination) appears to be necessary for maintaining genome integrity and as a repair mechanism for salvaging damaged genomes.
=== Bacteriophage T4 DNA polymerase ===
Bacteriophage (phage) T4 encodes a DNA polymerase that catalyzes DNA synthesis in a 5' to 3' direction. The phage polymerase also has an exonuclease activity that acts in a 3' to 5' direction, and this activity is employed in the proofreading and editing of newly inserted bases. A phage mutant with a temperature sensitive DNA polymerase, when grown at permissive temperatures, was observed to undergo recombination at frequencies that are about two-fold higher than that of wild-type phage.
It was proposed that a mutational alteration in the phage DNA polymerase can stimulate template strand switching (copy choice recombination) during replication.
== See also ==
Biological machines
DNA sequencing
Enzyme catalysis
Genetic recombination
Molecular cloning
Polymerase chain reaction
Protein domain dynamics
Reverse transcription
RNA polymerase
Taq DNA polymerase
== References ==
== Further reading ==
== External links ==
DNA+polymerases at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
PDB Molecule of the Month DNA polymerase
Unusual repair mechanism in DNA polymerase lambda, Ohio State University, July 25, 2006.
A great animation of DNA Polymerase from WEHI at 1:45 minutes in Archived 2014-12-05 at the Wayback Machine
3D macromolecular structures of DNA polymerase from the EM Data Bank(EMDB) | Wikipedia/Eukaryotic_DNA_polymerase |
A DNA virus is a virus that has a genome made of deoxyribonucleic acid (DNA) that is replicated by a DNA polymerase. They can be divided between those that have two strands of DNA in their genome, called double-stranded DNA (dsDNA) viruses, and those that have one strand of DNA in their genome, called single-stranded DNA (ssDNA) viruses. dsDNA viruses primarily belong to two realms: Duplodnaviria and Varidnaviria, and ssDNA viruses are almost exclusively assigned to the realm Monodnaviria, which also includes some dsDNA viruses. Additionally, many DNA viruses are unassigned to higher taxa. Reverse transcribing viruses, which have a DNA genome that is replicated through an RNA intermediate by a reverse transcriptase, are classified into the kingdom Pararnavirae in the realm Riboviria.
DNA viruses are ubiquitous worldwide, especially in marine environments where they form an important part of marine ecosystems, and infect both prokaryotes and eukaryotes. They appear to have multiple origins, as viruses in Monodnaviria appear to have emerged from archaeal and bacterial plasmids on multiple occasions, though the origins of Duplodnaviria and Varidnaviria are less clear.
Prominent disease-causing DNA viruses include herpesviruses, papillomaviruses, and poxviruses.
== Baltimore classification ==
The Baltimore classification system is used to group viruses together based on their manner of messenger RNA (mRNA) synthesis and is often used alongside standard virus taxonomy, which is based on evolutionary history. DNA viruses constitute two Baltimore groups: Group I: double-stranded DNA viruses, and Group II: single-stranded DNA viruses. While Baltimore classification is chiefly based on transcription of mRNA, viruses in each Baltimore group also typically share their manner of replication. Viruses in a Baltimore group do not necessarily share genetic relation or morphology.
=== Double-stranded DNA viruses ===
The first Baltimore group of DNA viruses are those that have a double-stranded DNA genome. All dsDNA viruses have their mRNA synthesized in a three-step process. First, a transcription preinitiation complex binds to the DNA upstream of the site where transcription begins, allowing for the recruitment of a host RNA polymerase. Second, once the RNA polymerase is recruited, it uses the negative strand as a template for synthesizing mRNA strands. Third, the RNA polymerase terminates transcription upon reaching a specific signal, such as a polyadenylation site.
dsDNA viruses make use of several mechanisms to replicate their genome. Bidirectional replication, in which two replication forks are established at a replication origin site and move in opposite directions of each other, is widely used. A rolling circle mechanism that produces linear strands while progressing in a loop around the circular genome is also common. Some dsDNA viruses use a strand displacement method whereby one strand is synthesized from a template strand, and a complementary strand is then synthesized from the prior synthesized strand, forming a dsDNA genome. Lastly, some dsDNA viruses are replicated as part of a process called replicative transposition whereby a viral genome in a host cell's DNA is replicated to another part of a host genome.
dsDNA viruses can be subdivided between those that replicate in the cell nucleus, and as such are relatively dependent on host cell machinery for transcription and replication, and those that replicate in the cytoplasm, in which case they have evolved or acquired their own means of executing transcription and replication. dsDNA viruses are also commonly divided between tailed dsDNA viruses, referring to members of the realm Duplodnaviria, usually the tailed bacteriophages of the order Caudovirales, and tailless or non-tailed dsDNA viruses of the realm Varidnaviria.
=== Single-stranded DNA viruses ===
The second Baltimore group of DNA viruses are those that have a single-stranded DNA genome. ssDNA viruses have the same manner of transcription as dsDNA viruses. However, because the genome is single-stranded, it is first made into a double-stranded form by a DNA polymerase upon entering a host cell. mRNA is then synthesized from the double-stranded form. The double-stranded form of ssDNA viruses may be produced either directly after entry into a cell or as a consequence of replication of the viral genome. Eukaryotic ssDNA viruses are replicated in the nucleus.
Most ssDNA viruses contain circular genomes that are replicated via rolling circle replication (RCR). ssDNA RCR is initiated by an endonuclease that bonds to and cleaves the positive strand, allowing a DNA polymerase to use the negative strand as a template for replication. Replication progresses in a loop around the genome by means of extending the 3'-end of the positive strand, displacing the prior positive strand, and the endonuclease cleaves the positive strand again to create a standalone genome that is ligated into a circular loop. The new ssDNA may be packaged into virions or replicated by a DNA polymerase to form a double-stranded form for transcription or continuation of the replication cycle.
Parvoviruses contain linear ssDNA genomes that are replicated via rolling hairpin replication (RHR), which is similar to RCR. Parvovirus genomes have hairpin loops at each end of the genome that repeatedly unfold and refold during replication to change the direction of DNA synthesis to move back and forth along the genome, producing numerous copies of the genome in a continuous process. Individual genomes are then excised from this molecule by the viral endonuclease. For parvoviruses, either the positive or negative sense strand may be packaged into capsids, varying from virus to virus.
Nearly all ssDNA viruses have positive sense genomes, but a few exceptions and peculiarities exist. The family Anelloviridae is the only ssDNA family whose members have negative sense genomes, which are circular. Parvoviruses, as previously mentioned, may package either the positive or negative sense strand into virions. Lastly, bidnaviruses package both the positive and negative linear strands.
== ICTV classification ==
The International Committee on Taxonomy of Viruses (ICTV) oversees virus taxonomy and organizes viruses at the basal level at the rank of realm. Virus realms correspond to the rank of domain used for cellular life but differ in that viruses within a realm do not necessarily share common ancestry, nor do the realms share common ancestry with each other. As such, each virus realm represents at least one instance of viruses coming into existence. Within each realm, viruses are grouped together based on shared characteristics that are highly conserved over time. Three DNA virus realms are recognized: Duplodnaviria, Monodnaviria, and Varidnaviria.
=== Duplodnaviria ===
Duplodnaviria contains dsDNA viruses that encode a major capsid protein (MCP) that has the HK97 fold. Viruses in the realm also share a number of other characteristics involving the capsid and capsid assembly, including an icosahedral capsid shape and a terminase enzyme that packages viral DNA into the capsid during assembly. Two groups of viruses are included in the realm: tailed bacteriophages, which infect prokaryotes and are assigned to the order Caudovirales, and herpesviruses, which infect animals and are assigned to the order Herpesvirales.
Duplodnaviria is a very ancient realm, perhaps predating the last universal common ancestor (LUCA) of cellular life. Its origins not known, nor whether it is monophyletic or polyphyletic. A characteristic feature is the HK97-fold found in the MCP of all members, which is found outside the realm only in encapsulins, a type of nanocompartment found in bacteria: this relation is not fully understood.
The relation between caudoviruses and herpesviruses is also uncertain: they may share a common ancestor or herpesviruses may be a divergent clade from the realm Caudovirales. A common trait among duplodnaviruses is that they cause latent infections without replication while still being able to replicate in the future. Tailed bacteriophages are ubiquitous worldwide, important in marine ecology, and the subject of much research. Herpesviruses are known to cause a variety of epithelial diseases, including herpes simplex, chickenpox and shingles, and Kaposi's sarcoma.
=== Monodnaviria ===
Monodnaviria contains ssDNA viruses that encode an endonuclease of the HUH superfamily that initiates rolling circle replication and all other viruses descended from such viruses. The prototypical members of the realm are called CRESS-DNA viruses and have circular ssDNA genomes. ssDNA viruses with linear genomes are descended from them, and in turn some dsDNA viruses with circular genomes are descended from linear ssDNA viruses.
Viruses in Monodnaviria appear to have emerged on multiple occasions from archaeal and bacterial plasmids, a type of extra-chromosomal DNA molecule that self-replicates inside its host. The kingdom Shotokuvirae in the realm likely emerged from recombination events that merged the DNA of these plasmids and complementary DNA encoding the capsid proteins of RNA viruses.
CRESS-DNA viruses include three kingdoms that infect prokaryotes: Loebvirae, Sangervirae, and Trapavirae. The kingdom Shotokuvirae contains eukaryotic CRESS-DNA viruses and the atypical members of Monodnaviria. Eukaryotic monodnaviruses are associated with many diseases, and they include papillomaviruses and polyomaviruses, which cause many cancers, and geminiviruses, which infect many economically important crops.
=== Varidnaviria ===
Varidnaviria contains DNA viruses that encode MCPs that have a jelly roll fold folded structure in which the jelly roll (JR) fold is perpendicular to the surface of the viral capsid. Many members also share a variety of other characteristics, including a minor capsid protein that has a single JR fold, an ATPase that packages the genome during capsid assembly, and a common DNA polymerase. Two kingdoms are recognized: Helvetiavirae, whose members have MCPs with a single vertical JR fold, and Bamfordvirae, whose members have MCPs with two vertical JR folds.
Varidnaviria is either monophyletic or polyphyletic and may predate the LUCA. The kingdom Bamfordvirae is likely derived from the other kingdom Helvetiavirae via fusion of two MCPs to have an MCP with two jelly roll folds instead of one. The single jelly roll (SJR) fold MCPs of Helvetiavirae show a relation to a group of proteins that contain SJR folds, including the Cupin superfamily and nucleoplasmins.
Marine viruses in Varidnaviria are ubiquitous worldwide and, like tailed bacteriophages, play an important role in marine ecology. Most identified eukaryotic DNA viruses belong to the realm. Notable disease-causing viruses in Varidnaviria include adenoviruses, poxviruses, and the African swine fever virus. Poxviruses have been highly prominent in the history of modern medicine, especially Variola virus, which caused smallpox. Many varidnaviruses can become endogenized in their host's genome; a peculiar example are virophages, which after infecting a host, can protect the host against giant viruses.
=== Baltimore classification ===
dsDNA viruses are classified into three realms and include many taxa that are unassigned to a realm:
All viruses in Duplodnaviria are dsDNA viruses.
In Monodnaviria, members of the class Papovaviricetes are dsDNA viruses.
All viruses in Varidnaviria are dsDNA viruses.
The following taxa that are unassigned to a realm exclusively contain dsDNA viruses:
Orders: Ligamenvirales
Families: Ampullaviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Halspiviridae, Hytrosaviridae, Nimaviridae, Nudiviridae, Ovaliviridae, Plasmaviridae, Polydnaviridae, Portogloboviridae, Thaspiviridae, Tristromaviridae
Genera: Dinodnavirus, Rhizidiovirus
ssDNA viruses are classified into one realm and include several families that are unassigned to a realm:
In Monodnaviria, all members except viruses in Papovaviricetes are ssDNA viruses.
The unassigned families Anelloviridae and Spiraviridae are ssDNA virus families.
Viruses in the family Finnlakeviridae contain ssDNA genomes. Finnlakeviridae is unassigned to a realm but is a proposed member of Varidnaviria.
== References ==
=== Bibliography === | Wikipedia/DNA_virus |
Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication to once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome.
DNA replication is the action of DNA polymerases synthesizing a DNA strand complementary to the original template strand. To synthesize DNA, the double-stranded DNA is unwound by DNA helicases ahead of polymerases, forming a replication fork containing two single-stranded templates. Replication processes permit copying a single DNA double helix into two DNA helices, which are divided into the daughter cells at mitosis. The major enzymatic functions carried out at the replication fork are well conserved from prokaryotes to eukaryotes, but the replication machinery in eukaryotic DNA replication is a much larger complex, coordinating many proteins at the site of replication, forming the replisome.
The replisome is responsible for copying the entirety of genomic DNA in each proliferative cell. This process allows for the high-fidelity passage of hereditary/genetic information from parental cell to daughter cell and is thus essential to all organisms. Much of the cell cycle is built around ensuring that DNA replication occurs without errors.
In G1 phase of the cell cycle, many of the DNA replication regulatory processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S phase of the cell cycle, and the entire genome must be unwound and duplicated to form two daughter copies. During G2, any damaged DNA or replication errors are corrected. Finally, one copy of the genomes is segregated into each daughter cell at the mitosis or M phase. These daughter copies each contains one strand from the parental duplex DNA and one nascent antiparallel strand.
This mechanism is conserved from prokaryotes to eukaryotes and is known as semiconservative DNA replication. The process of semiconservative replication for the site of DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is open, or unwound, exposing unpaired DNA nucleotides for recognition and base pairing for the incorporation
of free nucleotides into double-stranded DNA.
== Initiation ==
Initiation of eukaryotic DNA replication is the first stage of DNA synthesis where the DNA double helix is unwound and an initial priming event by DNA polymerase α occurs on the leading strand. The priming event on the lagging strand establishes a replication fork. Priming of the DNA helix consists of the synthesis of an RNA primer to allow DNA synthesis by DNA polymerase α. Priming occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand.
=== Origin of replication ===
Replication starts at origins of replication. DNA sequences containing these sites were initially isolated in the late 1970s on the basis of their ability to support replication of plasmids, hence the designation of autonomously replicating sequences (ARS). Origins vary widely in their efficiency, with some being used in almost every cell cycle while others may be used in only one in one thousand S phases. The total number of yeast ARSs is at least 1600, but may be more than 5000 if less active sites are counted, that is, there may be an ARS every 2000 to 8000 base pairs.
=== Pre-replicative complex ===
Multiple replicative proteins assemble on and dissociate from these replicative origins to initiate DNA replication. with the formation of the pre-replication complex (pre-RC) being a key intermediate in the replication initiation process.
Association of the origin recognition complex (ORC) with a replication origin recruits the cell division cycle 6 protein (Cdc6) to form a platform for the loading of the minichromosome maintenance (Mcm 2–7) complex proteins, facilitated by the chromatin licensing and DNA replication factor 1 protein (Cdt1). The ORC, Cdc6, and Cdt1 together are required for the stable association of the Mcm2-7 complex with replicative origins during the G1 phase of the cell cycle.
Eukaryotic origins of replication control the formation of several protein complexes that lead to the assembly of two bidirectional DNA replication forks. These events are initiated by the formation of the pre-replication complex (pre-RC) at the origins of replication. This process takes place in the G1 stage of the cell cycle. The pre-RC formation involves the ordered assembly of many replication factors including the origin recognition complex (ORC), Cdc6 protein, Cdt1 protein, and minichromosome maintenance proteins (Mcm2-7). Once the pre-RC is formed, activation of the complex is triggered by two kinases, cyclin-dependent kinase 2 (CDK) and Dbf4-dependent kinase (DDK) that help transition the pre-RC to the initiation complex before the initiation of DNA replication. This transition involves the ordered assembly of additional replication factors to unwind the DNA and accumulate the multiple eukaryotic DNA polymerases around the unwound DNA. Central to the question of how bidirectional replication forks are established at replication origins is the mechanism by which ORC recruits two head-to-head Mcm2-7 complexes to every replication origin to form the pre-replication complex.
==== Origin recognition complex ====
The first step in the assembly of the pre-replication complex (pre-RC) is the binding of the origin recognition complex (ORC) to the replication origin. In late mitosis, the Cdc6 protein joins the bound ORC followed by binding the Cdt1-Mcm2-7 complex. ORC, Cdc6, and Cdt1 are all required to load the six protein minichromosome maintenance (Mcm 2–7) complex onto the DNA. The ORC is a six-subunit, Orc1p-6, protein complex that selects the replicative origin sites on DNA for initiation of replication and ORC binding to chromatin is regulated through the cell cycle. Generally, the function and size of the ORC subunits are conserved throughout many eukaryotic genomes with the difference being their diverged DNA binding sites.
The most widely studied origin recognition complex is that of Saccharomyces cerevisiae or yeast which is known to bind to the autonomously replicating sequence (ARS). The S. cerevisiae ORC interacts specifically with both the A and B1 elements of yeast origins of replication, spanning a region of 30 base pairs. The binding to these sequences requires ATP.
The atomic structure of the S. cerevisiae ORC bound to ARS DNA has been determined. Orc1, Orc2, Orc3, Orc4, and Orc5 encircle the A element by means of two types of interactions, base non-specific and base-specific, that bend the DNA at the A element. All five subunits contact the sugar phosphate backbone at multiple points of the A element to form a tight grip without base specificity. Orc1 and Orc2 contact the minor groove of the A element while a winged helix domain of Orc4 contacts the methyl groups of the invariant Ts in the major groove of the A element via an insertion helix (IH). The absence of this IH in metazoans explains the lack of sequence specificity in human ORC. Removing the IH from the ScORC causes it to lose its specificity for the A element, and to bind promiscuously and preferentially (83%) to promoter regions. The ARS DNA is also bent at the B1 element through interactions with Orc2, Orc5 and Orc6. The bending of origin DNA by ORC appears to be evolutionarily conserved suggesting that it may be required for the Mcm2-7 complex loading mechanism.
When the ORC binds to DNA at replication origins, it serves as a scaffold for the assembly of other key initiation factors of the pre-replicative complex. This pre-replicative complex assembly during the G1 stage of the cell cycle is required prior to the activation of DNA replication during the S phase. The removal of at least part of the complex (Orc1) from the chromosome at metaphase is part of the regulation of mammalian ORC to ensure that the pre-replicative complex formation prior to the completion of metaphase is eliminated.
==== Cdc6 protein ====
Binding of the cell division cycle 6 (Cdc6) protein to the origin recognition complex (ORC) is an essential step in the assembly of the pre-replication complex (pre-RC) at the origins of replication. Cdc6 binds to the ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires Orc1 ATPase. Cdc6 requires ORC in order to associate with chromatin and is in turn required for the Cdt1-Mcm2-7 heptamer to bind to the chromatin. The ORC-Cdc6 complex forms a ring-shaped structure and is analogous to other ATP-dependent protein machines. The levels and activity of Cdc6 regulate the frequency with which the origins of replication are utilized during the cell cycle.
==== Cdt1 protein ====
The chromatin licensing and DNA replication factor 1 (Cdt1) protein is required for the licensing of chromatin for DNA replication. In S. cerevisiae, Cdt1 facilitates the loading of the Mcm2-7 complex one at a time onto the chromosome by stabilising the left-handed open-ring structure of the Mcm2-7 single hexamer. Cdt1 has been shown to associate with the C terminus of Cdc6 to cooperatively promote the association of Mcm proteins to the chromatin. The cryo-EM structure of the OCCM (ORC-Cdc6-Cdt1-MCM) complex shows that the Cdt1-CTD interacts with the Mcm6-WHD. In metazoans, Cdt1 activity during the cell cycle is tightly regulated by its association with the protein geminin, which both inhibits Cdt1 activity during S phase in order to prevent re-replication of DNA and prevents it from ubiquitination and subsequent proteolysis.
==== Minichromosome maintenance protein complex ====
The minichromosome maintenance (Mcm) proteins were named after a genetic screen for DNA replication initiation mutants in S. cerevisiae that affect plasmid stability in an ARS-specific manner. Mcm2, Mcm3, Mcm4, Mcm5, Mcm6 and Mcm7 form a hexameric complex that has an open-ring structure with a gap between Mcm2 and Mcm5. The assembly of the Mcm proteins onto chromatin requires the coordinated function of the origin recognition complex (ORC), Cdc6, and Cdt1. Once the Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from the chromatin without preventing subsequent DNA replication. This observation suggests that the primary role of the pre-replication complex is to correctly load the Mcm proteins.
The Mcm proteins on chromatin form a head-to-head double hexamer with the two rings slightly tilted, twisted and off-centred to create a kink in the central channel where the bound DNA is captured at the interface of the two rings. Each hexameric Mcm2-7 ring first serves as the scaffold for the assembly of the replisome and then as the core of the catalytic CMG (Cdc45-MCM-GINS) helicase, which is a main component of the replisome. Each Mcm protein is highly related to all others, but unique sequences distinguishing each of the subunit types are conserved across eukaryotes. All eukaryotes have exactly six Mcm protein analogs that each fall into one of the existing classes (Mcm2-7), indicating that each Mcm protein has a unique and important function.
Minichromosome maintenance proteins are required for DNA helicase activity. Inactivation of any of the six Mcm proteins during S phase irreversibly prevents further progression of the replication fork suggesting that the helicase cannot be recycled and must be assembled at replication origins. Along with the minichromosome maintenance protein complex helicase activity, the complex also has associated ATPase activity. Studies have shown that within the Mcm protein complex are specific catalytic pairs of Mcm proteins that function together to coordinate ATP hydrolysis. These studies, confirmed by cryo-EM structures of the Mcm2-7 complexes, showed that the Mcm complex is a hexamer with subunits arranged in a ring in the order of Mcm2-Mcm6-Mcm4-Mcm7-Mcm3-Mcm5-. Both members of each catalytic pair contribute to the conformation that allows ATP binding and hydrolysis and the mixture of active and inactive subunits presumably allows the Mcm hexameric complex to complete ATP binding and hydrolysis as a whole to create a coordinated ATPase activity.
The nuclear localization of the minichromosome maintenance proteins is regulated in budding yeast cells. The Mcm proteins are present in the nucleus in G1 stage and S phase of the cell cycle, but are exported to the cytoplasm during the G2 stage and M phase. A complete and intact six subunit Mcm complex is required to enter into the cell nucleus. In S. cerevisiae, nuclear export is promoted by cyclin-dependent kinase (CDK) activity. Mcm proteins that are associated with chromatin are protected from CDK export machinery due to the lack of accessibility to CDK.
=== Initiation complex ===
During the G1 stage of the cell cycle, the replication initiation factors, origin recognition complex (ORC), Cdc6, Cdt1, and minichromosome maintenance (Mcm) protein complex, bind sequentially to DNA to form a head-to-head dimer of the MCM ring complex, known as the pre-replication complex (pre-RC). While the yeast pre-RC forms a closed DNA complex, the human pre-RC forms an open complex. At the transition of the G1 stage to the S phase of the cell cycle, S phase–specific cyclin-dependent protein kinase (CDK) and Cdc7/Dbf4 kinase (DDK) transform the inert pre-RC into an active complex capable of assembling two bidirectional replisomes. CryoEM structures showed that two DDKs independently dock onto the interface of the MCM double hexamer straddling across the two rings. The sequential phosphorylation of multiple substrates on the NTEs of Mcm4, Mcm2 and Mcm6 is achieved by a wobble mechanism whereby Dbf4 assumes different wobble states to position Cdc7 over its multiple substrates. Phosphorylation of the MCM double hexamer, the Mcm4-NSD in particular, by DDK is essential for viability in yeast. The recruitment of Cdc45 and GINS follows after the activation of the MCMs by DDK and CDK.
==== Cdc45 protein ====
Cell division cycle 45 (Cdc45) protein is a critical component for the conversion of the pre-replicative complex to the initiation complex. The Cdc45 protein assembles at replication origins before initiation and is required for replication to begin in Saccharomyces cerevisiae, and has an essential role during elongation. Thus, Cdc45 has central roles in both initiation and elongation phases of chromosomal DNA replication.
Cdc45 associates with chromatin after the beginning of initiation in late G1 stage and during the S phase of the cell cycle. Cdc45 physically associates with Mcm5 and displays genetic interactions with five of the six members of the Mcm gene family and the ORC2 gene. The loading of Cdc45 onto chromatin is critical for loading other various replication proteins, including DNA polymerase α, DNA polymerase ε, replication protein A (RPA) and proliferating cell nuclear antigen (PCNA) onto chromatin.
Within a Xenopus nucleus-free system, it has been demonstrated that Cdc45 is required for the unwinding of plasmid DNA. The Xenopus nucleus-free system also demonstrates that DNA unwinding and tight RPA binding to chromatin occurs only in the presence of Cdc45.
Binding of Cdc45 to chromatin depends on Clb-Cdc28 kinase activity as well as functional Cdc6 and Mcm2, which suggests that Cdc45 associates with the pre-RC after activation of S-phase cyclin-dependent kinases (CDKs). As indicated by the timing and the CDK dependence, binding of Cdc45 to chromatin is crucial for commitment to initiation of DNA replication. During S phase, Cdc45 physically interacts with Mcm proteins on chromatin; however, dissociation of Cdc45 from chromatin is slower than that of the Mcm, which indicates that the proteins are released by different mechanisms.
==== GINS ====
The six minichromosome maintenance proteins and Cdc45 are essential during initiation and elongation for the movement of replication forks and for unwinding of the DNA. GINS are essential for the interaction of Mcm and Cdc45 at the origins of replication during initiation and then at DNA replication forks as the replisome progresses. The GINS complex is composed of four small proteins Sld5 (Cdc105), Psf1 (Cdc101), Psf2 (Cdc102) and Psf3 (Cdc103), GINS represents 'go, ichi, ni, san' which means '5, 1, 2, 3' in Japanese. Cdc45, Mcm2-7 and GINS together form the CMG helicase, the replicative helicase of the replisome. Although the Mcm2-7 complex alone has weak helicase activity Cdc45 and GINS are required for robust helicase activity
==== Mcm10 ====
Mcm10 is essential for chromosome replication and interacts with the minichromosome maintenance 2-7 helicase that is loaded in an inactive form at origins of DNA replication. Mcm10 also chaperones the catalytic DNA polymerase α and helps stabilize the polymerase at replication forks.
==== DDK and CDK kinases ====
At the onset of S phase, the pre-replicative complex must be activated by two S phase-specific kinases in order to form an initiation complex at an origin of replication. One kinase is the Cdc7-Dbf4 kinase called Dbf4-dependent kinase (DDK) and the other is cyclin-dependent kinase (CDK). Chromatin-binding assays of Cdc45 in yeast and Xenopus have shown that a downstream event of CDK action is loading of Cdc45 onto chromatin. Cdc6 has been speculated to be a target of CDK action, because of the association between Cdc6 and CDK, and the CDK-dependent phosphorylation of Cdc6. The CDK-dependent phosphorylation of Cdc6 has been considered to be required for entry into the S phase.
Both the catalytic subunits of DDK, Cdc7, and the activator protein, Dbf4, are conserved in eukaryotes and are required for the onset of S phase of the cell cycle. Both Dbf4 and Cdc7 are required for the loading of Cdc45 onto chromatin origins of replication. The target for binding of the DDK kinase is the chromatin-bound form of the Mcm complex. High resolution cryoEM structures showed that the Dbf4 subunit of DDK straddles across the hexamer interface of the DNA-bound MCM-DH, contacting Mcm2 of one hexamer and Mcm4/6 of the opposite hexamer. Mcm2, Mcm4 and Mcm6 are all substrates of phosphorylation by DDK but only the N-terminal serine/threonine-rich domain (NSD) of Mcm4 is an essential DDK target. Phosphorylation of the NSD leads to the activation of Mcm helicase activity.
==== Dpb11, Sld3, and Sld2 proteins ====
Sld3, Sld2, and Dpb11 interact with many replication proteins. Sld3 and Cdc45 form a complex that associated with the pre-RC at the early origins of replication even in the G11 phase and with the later origins of replication in the S phase in a mutually Mcm-dependent manner. Dpb11 and Sld2 interact with Polymerase ɛ and cross-linking experiments have indicated that Dpb11 and Polymerase ɛ coprecipitate in the S phase and associate with replication origins.
Sld3 and Sld2 are phosphorylated by CDK, which enables the two replicative proteins to bind to Dpb11. Dpb11 had two pairs of BRCA1 C Terminus (BRCT) domains which are known as a phosphopeptide-binding domains. The N-terminal pair of the BRCT domains binds to phosphorylated Sld3, and the C-terminal pair binds to phosphorylated Sld2. Both of these interactions are essential for CDK-dependent activation of DNA budding in yeast.
Dpb11 also interacts with GINS and participates in the initiation and elongation steps of chromosomal DNA replication. GINS are one of the replication proteins found at the replication forks and forms a complex with Cdc45 and Mcm.
These phosphorylation-dependent interactions between Dpb11, Sld2, and Sld3 are essential for CDK-dependent activation of DNA replication, and by using cross-linking reagents within some experiments, a fragile complex was identified called the pre-loading complex (pre-LC). This complex contains Pol ɛ, GINS, Sld2, and Dpb11. The pre-LC is found to form before any association with the origins in a CDK-dependent and DDK-dependent manner and CDK activity regulates the initiation of DNA replication through the formation of the pre-LC.
== Elongation ==
The formation of the pre-replicative complex (pre-RC) marks the potential sites for the initiation of DNA replication. Consistent with the minichromosome maintenance complex encircling double stranded DNA, formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead, the pre-RC that is formed during the G1 of the cell cycle is only activated to unwind the DNA and initiate replication after the cells pass from the G1 to the S phase of the cell cycle.
Once the initiation complex is formed and the cells pass into the S phase, the complex then becomes a replisome. The eukaryotic replisome complex is responsible for coordinating DNA replication. Replication on the leading and lagging strands is performed by DNA polymerase ε and DNA polymerase δ. Many replisome factors including Claspin, And1, replication factor C clamp loader and the fork protection complex are responsible for regulating polymerase functions and coordinating DNA synthesis with the unwinding of the template strand by Cdc45-Mcm-GINS complex. As the DNA is unwound the twist number decreases. To compensate for this the writhe number increases, introducing positive supercoils in the DNA. These supercoils would cause DNA replication to halt if they were not removed. Topoisomerases are responsible for removing these supercoils ahead of the replication fork.
The replisome is responsible for copying the entire genomic DNA in each proliferative cell. The base pairing and chain formation reactions, which form the daughter helix, are catalyzed by DNA polymerases. These enzymes move along single-stranded DNA and allow for the extension of the nascent DNA strand by "reading" the template strand and allowing for incorporation of the proper purine nucleobases, adenine and guanine, and pyrimidine nucleobases, thymine and cytosine. Activated free deoxyribonucleotides exist in the cell as deoxyribonucleotide triphosphates (dNTPs). These free nucleotides are added to an exposed 3'-hydroxyl group on the last incorporated nucleotide. In this reaction, a pyrophosphate is released from the free dNTP, generating energy for the polymerization reaction and exposing the 5' monophosphate, which is then covalently bonded to the 3' oxygen. Additionally, incorrectly inserted nucleotides can be removed and replaced by the correct nucleotides in an energetically favorable reaction. This property is vital to proper proofreading and repair of errors that occur during DNA replication.
=== Replication fork ===
The replication fork is the junction between the newly separated template strands, known as the leading and lagging strands, and the double stranded DNA. Since duplex DNA is antiparallel, DNA replication occurs in opposite directions
between the two new strands at the replication fork, but all DNA polymerases synthesize DNA in the 5' to 3' direction with respect to the newly synthesized strand. Further coordination is required during DNA replication. Two replicative polymerases synthesize DNA in opposite orientations. Polymerase ε synthesizes DNA on the "leading" DNA strand continuously as it is pointing in the same direction as DNA unwinding by the replisome. In contrast, polymerase δ synthesizes DNA on the "lagging" strand, which is the opposite DNA template strand, in a fragmented or discontinuous manner.
The discontinuous stretches of DNA replication products on the lagging strand are known as Okazaki fragments and are about 100 to 200 bases in length at eukaryotic replication forks. The lagging strand usually contains longer stretches of single-stranded DNA that is coated with single-stranded binding proteins, which help stabilize the single-stranded templates by preventing a secondary structure formation. In eukaryotes, these single-stranded binding proteins are a heterotrimeric complex known as replication protein A (RPA).
Each Okazaki fragment is preceded by an RNA primer, which is displaced by the procession of the next Okazaki fragment during synthesis. RNase H recognizes the DNA:RNA hybrids that are created by the use of RNA primers and is responsible for removing these from the replicated strand, leaving behind a primer:template junction. DNA polymerase α, recognizes these sites and elongates the breaks left by primer removal. In eukaryotic cells, a small amount of the DNA segment immediately upstream of the RNA primer is also displaced, creating a flap structure. This flap is then cleaved by endonucleases. At the replication fork, the gap in DNA after removal of the flap is sealed by DNA ligase I, which repairs the nicks that are left between the 3'-OH and 5'phosphate of the newly synthesized strand. Owing to the relatively short nature of the eukaryotic Okazaki fragment, DNA replication synthesis occurring discontinuously on the lagging strand is less efficient and more time-consuming than leading-strand synthesis. DNA synthesis is complete once all RNA primers are removed and nicks are repaired.
==== Leading strand ====
During DNA replication, the replisome will unwind the parental duplex DNA into a two single-stranded DNA template replication fork in a 5' to 3' direction. The leading strand is the template strand that is being replicated in the same direction as the movement of the replication fork. This allows the newly synthesized strand complementary to the original strand to be synthesized 5' to 3' in the same direction as the movement of the replication fork.
Once an RNA primer has been added by a primase to the 3' end of the leading strand, DNA synthesis will continue in a 3' to 5' direction with respect to the leading strand uninterrupted. DNA Polymerase ε will continuously add nucleotides to the template strand therefore making leading strand synthesis require only one primer and has uninterrupted DNA polymerase activity.
==== Lagging strand ====
DNA replication on the lagging strand is discontinuous. In lagging strand synthesis, the movement of DNA polymerase in the opposite direction of the replication fork requires the use of multiple RNA primers. DNA polymerase will synthesize short fragments of DNA called Okazaki fragments which are added to the 3' end of the primer. These fragments can be anywhere between 100 and 400 nucleotides long in eukaryotes.
At the end of Okazaki fragment synthesis, DNA polymerase δ runs into the previous Okazaki fragment and displaces its 5' end containing the RNA primer and a small segment of DNA. This generates an RNA-DNA single strand flap, which must be cleaved, and the nick between the two Okazaki fragments must be sealed by DNA ligase I. This process is known as Okazaki fragment maturation and can be handled in two ways: one mechanism processes short flaps, while the other deals with long flaps. DNA polymerase δ is able to displace up to 2 to 3 nucleotides of DNA or RNA ahead of its polymerization, generating a short "flap" substrate for Fen1, which can remove nucleotides from the flap, one nucleotide at a time.
By repeating cycles of this process, DNA polymerase δ and Fen1 can coordinate the removal of RNA primers and leave a DNA nick at the lagging strand. It has been proposed that this iterative process is preferable to the cell because it is tightly
regulated and does not generate large flaps that need to be excised. In the event of deregulated Fen1/DNA polymerase δ activity, the cell uses an alternative mechanism to generate and process long flaps by using Dna2, which has both helicase and nuclease activities. The nuclease activity of Dna2 is required for removing these long flaps, leaving a shorter flap to be processed by Fen1. Electron microscopy studies indicate that nucleosome loading on the lagging strand occurs very close to the site of synthesis. Thus, Okazaki fragment maturation is an efficient process that occurs immediately after the nascent DNA is synthesized.
=== Replicative DNA polymerases ===
After the replicative helicase has unwound the parental DNA duplex, exposing two single-stranded DNA templates, replicative polymerases are needed to generate two copies of the parental genome. DNA polymerase function is highly specialized and accomplish replication on specific templates and in narrow localizations. At the eukaryotic replication fork, there are three distinct replicative polymerase complexes that contribute to DNA replication: Polymerase α, Polymerase δ, and Polymerase ε. These three polymerases are essential for viability of the cell.
Because DNA polymerases require a primer on which to begin DNA synthesis, polymerase α (Pol α) acts as a replicative primase. Pol α is associated with an RNA primase and this complex accomplishes the priming task by synthesizing a primer that contains a short 10 nucleotide stretch of RNA followed by 10 to 20 DNA bases. Importantly, this priming action occurs at replication initiation at origins to begin leading-strand synthesis and also at the 5' end of each Okazaki fragment on the lagging strand.
However, Pol α is not able to continue DNA replication and must be replaced with another polymerase to continue DNA synthesis. Polymerase switching requires clamp loaders and it has been proven that normal DNA replication requires the coordinated actions of all three DNA polymerases: Pol α for priming synthesis, Pol ε for leading-strand replication, and the Pol δ, which is constantly loaded, for generating Okazaki fragments during lagging-strand synthesis.
Polymerase α (Pol α): Forms a complex with a small catalytic subunit (PriS) and a large noncatalytic (PriL) subunit. First, synthesis of an RNA primer allows DNA synthesis by DNA polymerase alpha. Occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand. Pri subunits act as a primase, synthesizing an RNA primer. DNA Pol α elongates the newly formed primer with DNA nucleotides. After around 20 nucleotides, elongation is taken over by Pol ε on the leading strand and Pol δ on the lagging strand.
Polymerase δ (Pol δ): Highly processive and has proofreading, 3'->5' exonuclease activity. In vivo, it is the main polymerase involved in both lagging strand and leading strand synthesis.
Polymerase ε (Pol ε): Highly processive and has proofreading, 3'->5' exonuclease activity. Highly related to pol δ, in vivo it functions mainly in error checking of pol δ.
=== Cdc45–Mcm–GINS helicase complex ===
The DNA helicases and polymerases must remain in close contact at the replication fork. If unwinding occurs too far in advance of synthesis, large tracts of single-stranded DNA are exposed. This can activate DNA damage signaling or induce DNA repair processes. To thwart these problems, the eukaryotic replisome contains specialized proteins that are designed to regulate the helicase activity ahead of the replication fork. These proteins also provide docking sites for physical interaction between helicases and polymerases, thereby ensuring that duplex unwinding is coupled with DNA synthesis.
For DNA polymerases to function, the double-stranded DNA helix has to be unwound to expose two single-stranded DNA templates for replication. DNA helicases are responsible for unwinding the double-stranded DNA during chromosome replication. Helicases in eukaryotic cells are remarkably complex. The catalytic core of the helicase is composed of six minichromosome maintenance (Mcm2-7) proteins, forming a hexameric ring. Away from DNA, the Mcm2-7 proteins form a single heterohexamer and are loaded in an inactive form at origins of DNA replication as a head-to-head double hexamers around double-stranded DNA. The Mcm proteins are recruited to replication origins then redistributed throughout the genomic DNA during S phase, indicative of their localization to the replication fork.
Loading of Mcm proteins can only occur during the G1 of the cell cycle, and the loaded complex is then activated during S phase by recruitment of the Cdc45 protein and the GINS complex to form the active Cdc45–Mcm–GINS (CMG) helicase at DNA replication forks. Mcm activity is required throughout the S phase for DNA replication. A variety of regulatory factors assemble around the CMG helicase to produce the ‘Replisome Progression Complex’ which associates with DNA polymerases to form the eukaryotic replisome, the structure of which is still quite poorly defined in comparison with its bacterial counterpart.
The isolated CMG helicase and Replisome Progression Complex contain a single Mcm protein ring complex suggesting that the loaded double hexamer of the Mcm proteins at origins might be broken into two single hexameric rings as part of the initiation process, with each Mcm protein complex ring forming the core of a CMG helicase at the two replication forks established from each origin. The full CMG complex is required for DNA unwinding, and the complex of CDC45-Mcm-GINS is the functional DNA helicase in eukaryotic cells.
==== Ctf4 and And1 proteins ====
The CMG complex interacts with the replisome through the interaction with Ctf4 and And1 proteins. Ctf4/And1 proteins interact with both the CMG complex and DNA polymerase α. Ctf4 is a polymerase α accessory factor, which is required for the recruitment of polymerase α to replication origins.
==== Mrc1 and Claspin proteins ====
Mrc1/Claspin proteins couple leading-strand synthesis with the CMG complex helicase activity. Mrc1 interacts with polymerase ε as well as Mcm proteins. The importance of this direct link between the helicase and the leading-strand polymerase is underscored by results in cultured human cells, where Mrc1/Claspin is required for efficient replication fork progression. These results suggest that efficient DNA replication also requires the coupling of helicases and leading-strand synthesis...
=== Proliferating cell nuclear antigen ===
DNA polymerases require additional factors to support DNA replication. DNA polymerases have a semiclosed 'hand' structure, which allows the polymerase to load onto the DNA and begin translocating. This structure permits DNA polymerase to hold the single-stranded DNA template, incorporate dNTPs at the active site, and release the newly formed double-stranded DNA. However, the structure of DNA polymerases does not allow a continuous stable interaction with the template DNA.
To strengthen the interaction between the polymerase and the template DNA, DNA sliding clamps associate with the polymerase to promote the processivity of the replicative polymerase. In eukaryotes, the sliding clamp is a homotrimer ring structure known as the proliferating cell nuclear antigen (PCNA). The PCNA ring has polarity with surfaces that interact with DNA polymerases and tethers them securely to the DNA template. PCNA-dependent stabilization of DNA polymerases has a significant effect on DNA replication because PCNAs are able to enhance the polymerase processivity up to 1,000-fold. PCNA is an essential cofactor and has the distinction of being one of the most common interaction platforms in the replisome to accommodate multiple processes at the replication fork, and so PCNA is also viewed as a regulatory cofactor for DNA polymerases.
=== Replication factor C ===
PCNA fully encircles the DNA template strand and must be loaded onto DNA at the replication fork. At the leading strand, loading of the PCNA is an infrequent process, because DNA replication on the leading strand is continuous until replication is terminated. However, at the lagging strand, DNA polymerase δ needs to be continually loaded at the start of each Okazaki fragment. This constant initiation of Okazaki fragment synthesis requires repeated PCNA loading for efficient DNA replication.
PCNA loading is accomplished by the replication factor C (RFC) complex. The RFC complex is composed of five ATPases: Rfc1, Rfc2, Rfc3, Rfc4 and Rfc5. RFC recognizes primer-template junctions and loads PCNA at these sites. The PCNA homotrimer is opened by RFC by ATP hydrolysis and is then loaded onto DNA in the proper orientation to facilitate its association with the polymerase. Clamp loaders can also unload PCNA from DNA; a mechanism needed when replication must be terminated.
=== Stalled replication fork ===
DNA replication at the replication fork can be halted by a shortage of deoxynucleotide triphosphates (dNTPs) or by DNA damage, resulting in replication stress. This halting of replication is described as a stalled replication fork. A fork protection complex of proteins stabilizes the replication fork until DNA damage or other replication problems can be fixed. Prolonged replication fork stalling can lead to further DNA damage. Stalling signals are deactivated if the problems causing the replication fork are resolved.
== Termination ==
Termination of eukaryotic DNA replication requires different processes depending on whether the chromosomes are circular or linear. Unlike linear molecules, circular chromosomes are able to replicate the entire molecule. However, the two DNA molecules will remain linked together. This issue is handled by decatenation of the two DNA molecules by a type II topoisomerase. Type II topoisomerases are also used to separate linear strands as they are intricately folded into a nucleosome within the cell.
As previously mentioned, linear chromosomes face another issue that is not seen in circular DNA replication. Due to the fact that an RNA primer is required for initiation of DNA synthesis, the lagging strand is at a disadvantage in replicating the entire chromosome. While the leading strand can use a single RNA primer to extend the 5' terminus of the replicating DNA strand, multiple RNA primers are responsible for lagging strand synthesis, creating Okazaki fragments. This leads to an issue due to the fact that DNA polymerase is only able to add to the 3' end of the DNA strand. The 3'-5' action of DNA polymerase along the parent strand leaves a short single-stranded DNA (ssDNA) region at the 3' end of the parent strand when the Okazaki fragments have been repaired. Since replication occurs in opposite directions at opposite ends of parent chromosomes, each strand is a lagging strand at one end. Over time this would result in progressive shortening of both daughter chromosomes. This is known as the end replication problem.
The end replication problem is handled in eukaryotic cells by telomere regions and telomerase. Telomeres extend the 3' end of the parental chromosome beyond the 5' end of the daughter strand. This single-stranded DNA structure can act as an origin of replication that recruits telomerase. Telomerase is a specialized DNA polymerase that consists of multiple protein subunits and an RNA component. The RNA component of telomerase anneals to the single stranded 3' end of the template DNA and contains 1.5 copies of the telomeric sequence. Telomerase contains a protein subunit that is a reverse transcriptase called telomerase reverse transcriptase or TERT. TERT synthesizes DNA until the end of the template telomerase RNA and then disengages. This process can be repeated as many times as needed with the extension of the 3' end of the parental DNA molecule. This 3' addition provides a template for extension of the 5' end of the daughter strand by lagging strand DNA synthesis. Regulation of telomerase activity is handled by telomere-binding proteins.
=== Replication fork barriers ===
Prokaryotic DNA replication is bidirectional; within a replicative origin, replisome complexes are created at each end of the replication origin and replisomes move away from each other from the initial starting point. In prokaryotes, bidirectional replication initiates at one replicative origin on the circular chromosome and terminates at a site opposed from the initial start of the origin. These termination regions have DNA sequences known as Ter sites. These Ter sites are bound by the Tus protein. The Ter-Tus complex is able to stop helicase activity, terminating replication.
In eukaryotic cells, termination of replication usually occurs through the collision of the two replicative forks between two active replication origins. The location of the collision varies on the timing of origin firing. In this way, if a replication fork becomes stalled or collapses at a certain site, replication of the site can be rescued when a replisome traveling in the opposite direction completes copying the region. There are programmed replication fork barriers (RFBs) bound by RFB proteins in various locations, throughout the genome, which are able to terminate or pause replication forks, stopping progression of the replisome.
== Replication factories ==
It has been found that replication happens in a localised way in the cell nucleus. Contrary to the traditional view of moving replication forks along stagnant DNA, a concept of replication factories emerged, which means replication forks are concentrated towards some immobilised 'factory' regions through which the template DNA strands pass like conveyor belts.
== Cell cycle regulation ==
DNA replication is a tightly orchestrated process that is controlled within the context of the cell cycle. Progress through the cell cycle and in turn DNA replication is tightly regulated by the formation and activation of pre-replicative complexes (pre-RCs) which is achieved through the activation and inactivation of cyclin-dependent kinases (Cdks, CDKs). Specifically it is the interactions of cyclins and cyclin dependent kinases that are responsible for the transition from G1 into S-phase.
During the G1 phase of the cell cycle there are low levels of CDK activity. This low level of CDK activity allows for the formation of new pre-RC complexes but is not sufficient for DNA replication to be initiated by the newly formed pre-RCs. During the remaining phases of the cell cycle there are elevated levels of CDK activity. This high level of CDK activity is responsible for initiating DNA replication as well as inhibiting new pre-RC complex formation. Once DNA replication has been initiated the pre-RC complex is broken down. Due to the fact that CDK levels remain high during the S phase, G2, and M phases of the cell cycle no new pre-RC complexes can be formed. This all helps to ensure that no initiation can occur until the cell division is complete.
In addition to cyclin dependent kinases a new round of replication is thought to be prevented through the downregulation of Cdt1. This is achieved via degradation of Cdt1 as well as through the inhibitory actions of a protein known as geminin. Geminin binds tightly to Cdt1 and is thought to be the major inhibitor of re-replication. Geminin first appears in S-phase and is degraded at the metaphase-anaphase transition, possibly through ubiquination by anaphase promoting complex (APC).
Various cell cycle checkpoints are present throughout the course of the cell cycle that determine whether a cell will progress through division entirely. Importantly in replication the G1, or restriction, checkpoint makes the determination of whether or not initiation of replication will begin or whether the cell will be placed in a resting stage known as G0. Cells in the G0 stage of the cell cycle are prevented from initiating a round of replication because the minichromosome maintenance proteins are not expressed. Transition into the S-phase indicates replication has begun.
=== Replication checkpoint proteins ===
In order to preserve genetic information during cell division, DNA replication must be completed with high fidelity. In order to achieve this task, eukaryotic cells have proteins in place during certain points in the replication process that are able to detect any errors during DNA replication and are able to preserve genomic integrity. These checkpoint proteins are able to stop the cell cycle from entering mitosis in order to allow time for DNA repair. Checkpoint proteins are also involved in some DNA repair pathways, while they stabilize the structure of the replication fork to prevent further damage. These checkpoint proteins are essential to avoid passing down mutations or other chromosomal aberrations to offspring.
Eukaryotic checkpoint proteins are well conserved and involve two phosphatidylinositol 3-kinase-related kinases (PIKKs), ATR and ATM. Both ATR and ATM share a target phosphorylation sequence, the SQ/TQ motif, but their individual roles in cells differ.
ATR is involved in arresting the cell cycle in response to DNA double-stranded breaks. ATR has an obligate checkpoint partner, ATR-interacting-protein (ATRIP), and together these two proteins are responsive to stretches of single-stranded DNA that are coated by replication protein A (RPA). The formation of single-stranded DNA occurs frequently, more often during replication stress. ATR-ATRIP is able to arrest the cell cycle to preserve genome integrity. ATR is found on chromatin during S phase, similar to RPA and claspin.
The generation of single-stranded DNA tracts is important in initiating the checkpoint pathways downstream of replication damage. Once single-stranded DNA becomes sufficiently long, single-stranded DNA coated with RPA are able to recruit ATR-ATRIP. In order to become fully active, the ATR kinase rely on sensor proteins that sense whether the checkpoint proteins are localized to a valid site of DNA replication stress. The RAD9-HUS1-Rad1 (9-1-1) heterotrimeric clamp and its clamp loader RFCRad17 are able to recognize gapped or nicked DNA. The RFCRad17 clamp loader loads 9-1-1 onto the damaged DNA. The presence of 9-1-1 on DNA is enough to facilitate the interaction between ATR-ATRIP and a group of proteins termed checkpoint mediators, such as TOPBP1 and Mrc1/claspin. TOPBP1 interacts with and recruits the phosphorylated Rad9 component of 9-1-1 and binds ATR-ATRIP, which phosphorylates Chk1. Mrc1/Claspin is also required for
the complete activation of ATR-ATRIP that phosphorylates Chk1, the major downstream checkpoint effector kinase. Claspin is a component of the replisome and contains a domain for docking with Chk1, revealing a specific function of Claspin during DNA replication: the promotion of
checkpoint signaling at the replisome.
Chk1 signaling is vital for arresting the cell cycle and preventing cells from entering mitosis with incomplete DNA replication or DNA damage. The Chk1-dependent Cdk inhibition is important for the function of the ATR-Chk1 checkpoint and to arrest the cell cycle and allow sufficient time for completion of DNA repair mechanisms, which in turn prevents the inheritance of damaged DNA. In addition, Chk1-dependent Cdk inhibition plays a critical role in inhibiting origin firing during S phase. This mechanism prevents continued DNA synthesis and is required for the protection of the genome in the
presence of replication stress and potential genotoxic conditions. Thus, ATR-Chk1 activity further prevents potential replication problems at the level of single replication origins by inhibiting initiation of replication throughout the genome, until the signaling cascade maintaining cell-cycle arrest is turned off.
== Replication through nucleosomes ==
Eukaryotic DNA must be tightly compacted in order to fit within the confined space of the nucleus. Chromosomes are packaged by wrapping 147 nucleotides around an octamer of histone proteins, forming a nucleosome. The nucleosome octamer includes two copies of each histone H2A, H2B, H3, and H4. Due to the tight association of histone proteins to DNA, eukaryotic cells have proteins that are designed to remodel histones ahead of the replication fork, in order to allow smooth progression of the replisome. There are also proteins involved in reassembling histones behind the replication fork to reestablish the nucleosome conformation.
There are several histone chaperones that are known to be involved in nucleosome assembly after replication. The FACT complex has been found to interact with DNA polymerase α-primase complex, and the subunits of the FACT complex interacted genetically with replication factors. The FACT complex is a heterodimer that does not hydrolyze ATP, but is able to facilitate "loosening" of histones in nucleosomes, but how the FACT complex is able to relieve the tight association of histones for DNA removal remains unanswered.
Another histone chaperone that associates with the replisome is Asf1, which interacts with the Mcm complex dependent on histone dimers H3-H4. Asf1 is able to pass newly synthesized H3-H4 dimer to deposition factors behind the replication fork and this activity makes the H3-H4 histone dimers available at the site of histone deposition just after replication. Asf1 (and its partner Rtt109) has also been implicated in inhibiting gene expression from replicated genes during S-phase.
The heterotrimeric chaperone chromatin assembly factor 1 (CAF-1) is a chromatin formation protein that is involved in depositing histones onto both newly replicated DNA strands to form chromatin. CAF-1 contains a PCNA-binding motif, called a PIP-box, that allows CAF-1 to associate with the replisome through PCNA and is able to deposit histone H3-H4 dimers onto newly synthesized DNA. The Rtt106 chaperone is also involved in this process, and associated with CAF-1 and H3-H4 dimers during chromatin formation. These processes load newly synthesized histones onto DNA.
After the deposition of histones H3-H4, nucleosomes form by the association of histone H2A-H2B. This process is thought to occur through the FACT complex, since it already associated with the replisome and is able to bind free H2A-H2B, or there is the possibility of another H2A-H2B chaperone, Nap1. Electron microscopy studies show that this occurs very quickly, as nucleosomes can be observed forming just a few hundred base pairs after the replication fork. Therefore, the entire process of forming new
nucleosomes takes place just after replication due to the coupling of histone chaperones to the replisome.
== Mitotic DNA Synthesis ==
Mitotic DNA synthesis (MiDAS) is a process of irregular DNA replication where DNA synthesis, naturally occurring in the S phase, takes place in the M phase of the cell cycle. Mitotic DNA synthesis is known to occur when cells are experiencing stress related to DNA replication. Certain loci in the genome, considered common fragile sites (CFS) or ALT-associated replication defects can induce replication stress that may lead to MiDAS. Mitotic DNA synthesis is enabled by a protein known as RAD52, which then recruits enzymes, including MUS81 and POLD3. These enzymes work to promote MiDAS, operating outside of ATR, BRCA2, and RAD51 which are necessary to prevent replication stress at CFS loci throughout S phase. MiDAS has been recorded in mammals and yeast, however, its occurrence in other eukaryotic organisms is yet to be discovered.
== Comparisons between prokaryotic and eukaryotic DNA replication ==
When compared to prokaryotic DNA replication, namely in bacteria, the completion of eukaryotic DNA replication is more complex and involves multiple origins of replication and replicative proteins to accomplish. Prokaryotic DNA is arranged in a circular shape, and has only one replication origin when replication starts. By contrast, eukaryotic DNA is linear. When replicated, there are as many as one thousand origins of replication.
Eukaryotic DNA is bidirectional. Here the meaning of the word bidirectional is different. Eukaryotic linear DNA has many origins (called O) and termini (called T). "T" is present to the right of "O". One "O" and one "T" together form one replicon. After the formation of pre-initiation complex, when one replicon starts elongation, initiation starts in second replicon. Now, if the first replicon moves in clockwise direction, the second replicon moves in anticlockwise direction, until "T" of first replicon is reached. At "T", both the replicons merge to complete the process of replication. Meanwhile, the second replicon is moving in forward direction also, to meet with the third replicon. This clockwise and counter-clockwise movement of two replicons is termed as bidirectional replication.
Eukaryotic DNA replication requires precise coordination of all DNA polymerases and associated proteins to replicate the entire genome each time a cell divides. This process is achieved through a series of steps of protein assemblies at origins of replication, mainly focusing the regulation of DNA replication on the association of the MCM helicase with the DNA. These origins of replication direct the number of protein complexes that will form to initiate replication. In bacterial DNA replication, regulation focuses on the binding of the DnaA initiator protein to the DNA, with initiation of replication occurring multiple times during one cell cycle. Both prokaryotic and eukaryotic DNA use ATP binding and hydrolysis to direct helicase loading and in both cases the helicase is loaded in the inactive form. However, eukaryotic helicases are double hexamers that are loaded onto double stranded DNA whereas bacterial helicases are single hexamers loaded onto single stranded DNA.
Segregation of chromosomes is another difference between prokaryotic and eukaryotic cells. Rapidly dividing cells, such as bacteria, will often begin to segregate chromosomes that are still in the process of replication. In eukaryotic cells chromosome segregation into the daughter cells is not initiated until replication is complete in all chromosomes. Despite these differences, however, the underlying process of replication is similar for both prokaryotic and eukaryotic DNA.
== Eukaryotic DNA replication protein list ==
List of major proteins involved in eukaryotic DNA replication:
== See also ==
DNA replication
Prokaryotic DNA replication
Processivity
== References == | Wikipedia/Eukaryotic_DNA_replication |
DNA polymerase alpha catalytic subunit is an enzyme that in humans is encoded by the POLA1 gene.
== Function ==
This gene encodes the p180 catalytic subunit of DNA polymerase α-primase. Pol α has limited processivity and lacks 3′ exonuclease activity for proofreading errors. Thus it is not well suited to efficiently and accurately copy long templates (unlike Pol Delta and Epsilon). Instead it plays a more limited role in replication. Pol α is responsible for the initiation of DNA replication at origins of replication (on both the leading and lagging strands) and during synthesis of Okazaki fragments on the lagging strand. The Pol α complex (pol α-DNA primase complex) consists of four subunits: the catalytic subunit POLA1, the regulatory subunit POLA2, and the small and the large primase subunits PRIM1 and PRIM2 respectively. Once primase has created the RNA primer, Pol α starts replication elongating the primer with ~20 nucleotides.
== Clinical significance ==
In addition to its role during DNA replication, POLA1 plays a role in type I interferon activation. The POLA1 gene was found to be the site of a mutation resulting in X-linked reticulate pigmentary disorder (XLPDR), OMIM 301220). This leads to altered mRNA splicing and decreased expression of POLA1 protein to a level that does not impair DNA replication. The reduction in POLA1 expression is accompanied by marked reduction in cytosolic RNA:DNA hybrid molecules and a concomitant hyperactivation of the IRF3 pathway, with consequent overproduction of type I interferons.
Moreover, POLA1 deficiency, typical for XLPDR, also impair direct cytotoxicity of NK cells. POLA1 inhibition or a natural deficiency (XLPDR) affects the way the lytic granules secreted toward target cells. As a result, NK cells in XLPDR patients display functional deficiency. Interestingly, the POLA1 deficiency typical for XLPDR is not associated with any genomic damages or cell cycle arrest.
While the XLPDR mutation is resided in intron 13th, other somatic mutations in POLA1 were also described. Somatic mutation are associated with more profound deficiency of POLA1, with develops into X-linked intellectual disability (XLID). In a case of non-XLPDR mutations, beside of type I interferon signature patients also display mild to medium signs of intellectual disability, cell cycle arrest, proportionate short stature, microcephaly and hypogonadism.
== Interactions ==
DNA dependent polymerase alpha (Pol α) has been shown to interact with MCM4 and GINS1, Retinoblastoma protein, PARP1 and RBMS1.
== See also ==
DNA Polymerase
DNA polymerase alpha subunit 2
== References ==
== External links ==
PDBe-KB provides an overview of all the structure information available in the PDB for Human DNA polymerase alpha catalytic subunit
== Further reading == | Wikipedia/DNA_polymerase_alpha_catalytic_subunit |
Proteinoids, or thermal proteins, are protein-like, often cross-linked molecules formed abiotically from amino acids. Sidney W. Fox initially proposed that they may have been precursors to the first living cells (protocells). The term was also used in the 1960s to describe peptides that are shorter than twenty amino acids found in hydrolysed protein, but this term is no longer commonly used.
== History ==
In trying to uncover the intermediate stages of abiogenesis, scientist Sidney W. Fox in the 1950s and 1960s, studied the spontaneous formation of peptide structures under conditions that might plausibly have existed early in Earth's history.: 199–201 He demonstrated that amino acids could spontaneously form small chains called peptides. In one of his experiments, he allowed amino acids to dry out as if puddled in a warm, dry spot in prebiotic conditions. He found that, as they dried, the amino acids formed long, often cross-linked, thread-like microscopic polypeptide globules, he named "proteinoid microspheres".
== Polymerization ==
The abiotic polymerization of amino acids into proteins through the formation of peptide bonds was thought to occur only at temperatures over 140 °C. However, the biochemist Sidney Walter Fox and his co-workers discovered that phosphoric acid acted as a catalyst for this reaction. They were able to form protein-like chains from a mixture of 18 common amino acids at 70 °C in the presence of phosphoric acid, and dubbed these protein-like chains proteinoids. Fox later found naturally occurring proteinoids similar to those he had created in his laboratory in lava and cinders from Hawaiian volcanic vents and determined that the amino acids present polymerized due to the heat of escaping gases and lava. Other catalysts have since been found; one of them, amidinium carbodiimide, is formed in primitive Earth experiments and is effective in dilute aqueous solutions.
When present in certain concentrations in aqueous solutions, proteinoids form small microspheres. This is because some of the amino acids incorporated into proteinoid chains are more hydrophobic than others, and so proteinoids cluster together like droplets of oil in water. These structures exhibit a few characteristics of living cells:
An outer wall.
Osmotic swelling and shrinking.
Budding.
Binary fission (dividing into two daughter microspheres).
Streaming movement of internal particles.
Fox thought that the microspheres may have provided a cell compartment within which organic molecules could have become concentrated and protected from the outside environment during the process of chemical evolution.
Proteinoid microspheres are today being considered for use in pharmaceuticals, providing microscopic biodegradable capsules in which to package and deliver oral drugs.
In another experiment using a similar method to set suitable conditions for life to form, Fox collected volcanic material from a cinder cone in Hawaii. He discovered that the temperature was over 100 °C (212 °F) just 4 inches (100 mm) beneath the surface of the cinder cone, and suggested that this might have been the environment in which life was created—molecules could have formed and then been washed through the loose volcanic ash and into the sea. He placed lumps of lava over amino acids derived from methane, ammonia and water, sterilized all materials, and baked the lava over the amino acids for a few hours in a glass oven. A brown, sticky substance formed over the surface and when the lava was drenched in sterilized water a thick, brown liquid leached out. It turned out that the amino acids had combined to form proteinoids, and the proteinoids had combined to form small spheres. Fox called these "microspheres". His protobionts were not cells, although they formed clumps and chains reminiscent of bacteria. Based upon such experiments, Colin Pittendrigh stated in December 1967 that "laboratories will be creating a living cell within ten years," a remark that reflected the typical contemporary levels of ignorance of the complexity of cell structures.
== Legacy ==
Fox has likened the amino acid globules to cells, and proposed it bridged the macromolecule to cell transition. However, his hypothesis was later dismissed as proteinoids are not proteins, they feature mostly non-peptide bonds and amino acid cross-linkages not present in living organisms. Furthermore, they have no compartmentalization and there is no information content in the molecules.
Although their role as an evolutionary precursor has been superseded, the hypothesis was a catalyst to further investigate other mechanisms that could have brought about abiogenesis,: 201 such as the RNA world, PAH world, Iron–sulfur world, and protocell hypotheses.
== See also ==
Abiogenesis
Jeewanu
Protocell
Proto-mitochondrion
== References ==
== Further reading == | Wikipedia/Proteinoid |
Single-strand DNA-binding protein (SSB) is a protein found in Escherichia coli (E. coli) bacteria, that binds to single-stranded regions of deoxyribonucleic acid (DNA). Single-stranded DNA is produced during all aspects of DNA metabolism: replication, recombination, and repair. As well as stabilizing this single-stranded DNA, SSB proteins bind to and modulate the function of numerous proteins involved in all of these processes.
Active E. coli SSB is composed of four identical 19 kDa subunits. Binding of single-stranded DNA to the tetramer can occur in different "modes", with SSB occupying different numbers of DNA bases depending on a number of factors, including salt concentration. For example, the (SSB)65 binding mode, in which approximately 65 nucleotides of DNA wrap around the SSB tetramer and contact all four of its subunits, is favoured at high salt concentrations in vitro. At lower salt concentrations, the (SSB)35 binding mode, in which about 35 nucleotides bind to only two of the SSB subunits, tends to form. Further work is required to elucidate the functions of the various binding modes in vivo.
== Bacterial SSB ==
SSB protein domains in bacteria are important in its function of maintaining DNA metabolism, more specifically DNA replication, repair, and recombination. It has a structure of three beta-strands to a single six-stranded beta-sheet to form a protein dimer.
== See also ==
DNA-binding protein
Single-stranded binding protein
Comparison of nucleic acid simulation software
== References ==
== External links ==
Single-Stranded+DNA+Binding+Proteins at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
SSB in PFAM | Wikipedia/Single-strand_binding_protein |
Deoxyribonucleic acid ( ; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.
The two DNA strands are known as polynucleotides as they are composed of simpler monomeric units called nucleotides. Each nucleotide is composed of one of four nitrogen-containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine [T]), a sugar called deoxyribose, and a phosphate group. The nucleotides are joined to one another in a chain by covalent bonds (known as the phosphodiester linkage) between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The nitrogenous bases of the two separate polynucleotide strands are bound together, according to base pairing rules (A with T and C with G), with hydrogen bonds to make double-stranded DNA. The complementary nitrogenous bases are divided into two groups, the single-ringed pyrimidines and the double-ringed purines. In DNA, the pyrimidines are thymine and cytosine; the purines are adenine and guanine.
Both strands of double-stranded DNA store the same biological information. This information is replicated when the two strands separate. A large part of DNA (more than 98% for humans) is non-coding, meaning that these sections do not serve as patterns for protein sequences. The two strands of DNA run in opposite directions to each other and are thus antiparallel. Attached to each sugar is one of four types of nucleobases (or bases). It is the sequence of these four nucleobases along the backbone that encodes genetic information. RNA strands are created using DNA strands as a template in a process called transcription, where DNA bases are exchanged for their corresponding bases except in the case of thymine (T), for which RNA substitutes uracil (U). Under the genetic code, these RNA strands specify the sequence of amino acids within proteins in a process called translation.
Within eukaryotic cells, DNA is organized into long structures called chromosomes. Before typical cell division, these chromosomes are duplicated in the process of DNA replication, providing a complete set of chromosomes for each daughter cell. Eukaryotic organisms (animals, plants, fungi and protists) store most of their DNA inside the cell nucleus as nuclear DNA, and some in the mitochondria as mitochondrial DNA or in chloroplasts as chloroplast DNA. In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm, in circular chromosomes. Within eukaryotic chromosomes, chromatin proteins, such as histones, compact and organize DNA. These compacting structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.
== Properties ==
DNA is a long polymer made from repeating units called nucleotides. The structure of DNA is dynamic along its length, being capable of coiling into tight loops and other shapes. In all species it is composed of two helical chains, bound to each other by hydrogen bonds. Both chains are coiled around the same axis, and have the same pitch of 34 ångströms (3.4 nm). The pair of chains have a radius of 10 Å (1.0 nm). According to another study, when measured in a different solution, the DNA chain measured 22–26 Å (2.2–2.6 nm) wide, and one nucleotide unit measured 3.3 Å (0.33 nm) long. The buoyant density of most DNA is 1.7g/cm3.
DNA does not usually exist as a single strand, but instead as a pair of strands that are held tightly together. These two long strands coil around each other, in the shape of a double helix. The nucleotide contains both a segment of the backbone of the molecule (which holds the chain together) and a nucleobase (which interacts with the other DNA strand in the helix). A nucleobase linked to a sugar is called a nucleoside, and a base linked to a sugar and to one or more phosphate groups is called a nucleotide. A biopolymer comprising multiple linked nucleotides (as in DNA) is called a polynucleotide.
The backbone of the DNA strand is made from alternating phosphate and sugar groups. The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These are known as the 3′-end (three prime end), and 5′-end (five prime end) carbons, the prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond.
Therefore, any DNA strand normally has one end at which there is a phosphate group attached to the 5′ carbon of a ribose (the 5′ phosphoryl) and another end at which there is a free hydroxyl group attached to the 3′ carbon of a ribose (the 3′ hydroxyl). The orientation of the 3′ and 5′ carbons along the sugar-phosphate backbone confers directionality (sometimes called polarity) to each DNA strand. In a nucleic acid double helix, the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are said to have a directionality of five prime end (5′ ), and three prime end (3′), with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the related pentose sugar ribose in RNA.
The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases. The four bases found in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar-phosphate to form the complete nucleotide, as shown for adenosine monophosphate. Adenine pairs with thymine and guanine pairs with cytosine, forming A-T and G-C base pairs.
=== Nucleobase classification ===
The nucleobases are classified into two types: the purines, A and G, which are fused five- and six-membered heterocyclic compounds, and the pyrimidines, the six-membered rings C and T. A fifth pyrimidine nucleobase, uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. In addition to RNA and DNA, many artificial nucleic acid analogues have been created to study the properties of nucleic acids, or for use in biotechnology.
=== Non-canonical bases ===
Modified bases occur in DNA. The first of these recognized was 5-methylcytosine, which was found in the genome of Mycobacterium tuberculosis in 1925. The reason for the presence of these noncanonical bases in bacterial viruses (bacteriophages) is to avoid the restriction enzymes present in bacteria. This enzyme system acts at least in part as a molecular immune system protecting bacteria from infection by viruses. Modifications of the bases cytosine and adenine, the more common and modified DNA bases, play vital roles in the epigenetic control of gene expression in plants and animals.
A number of noncanonical bases are known to occur in DNA. Most of these are modifications of the canonical bases plus uracil.
Modified Adenine
N6-carbamoyl-methyladenine
N6-methyadenine
Modified Guanine
7-Deazaguanine
7-Methylguanine
Modified Cytosine
N4-Methylcytosine
5-Carboxylcytosine
5-Formylcytosine
5-Glycosylhydroxymethylcytosine
5-Hydroxycytosine
5-Methylcytosine
Modified Thymidine
α-Glutamythymidine
α-Putrescinylthymine
Uracil and modifications
Base J
Uracil
5-Dihydroxypentauracil
5-Hydroxymethyldeoxyuracil
Others
Deoxyarchaeosine
2,6-Diaminopurine (2-Aminoadenine)
=== Grooves ===
Twin helical strands form the DNA backbone. Another double helix may be found tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site. As the strands are not symmetrically located with respect to each other, the grooves are unequally sized. The major groove is 22 ångströms (2.2 nm) wide, while the minor groove is 12 Å (1.2 nm) in width. Due to the larger width of the major groove, the edges of the bases are more accessible in the major groove than in the minor groove. As a result, proteins such as transcription factors that can bind to specific sequences in double-stranded DNA usually make contact with the sides of the bases exposed in the major groove. This situation varies in unusual conformations of DNA within the cell (see below), but the major and minor grooves are always named to reflect the differences in width that would be seen if the DNA was twisted back into the ordinary B form.
=== Base pairing ===
In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other strand. This is called complementary base pairing. Purines form hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds. This arrangement of two nucleotides binding together across the double helix (from six-carbon ring to six-carbon ring) is called a Watson-Crick base pair. DNA with high GC-content is more stable than DNA with low GC-content. A Hoogsteen base pair (hydrogen bonding the 6-carbon ring to the 5-carbon ring) is a rare variation of base-pairing. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can thus be pulled apart like a zipper, either by a mechanical force or high temperature. As a result of this base pair complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. This reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in organisms.
==== ssDNA vs. dsDNA ====
Most DNA molecules are actually two polymer strands, bound together in a helical fashion by noncovalent bonds; this double-stranded (dsDNA) structure is maintained largely by the intrastrand base stacking interactions, which are strongest for G,C stacks. The two strands can come apart—a process known as melting—to form two single-stranded DNA (ssDNA) molecules. Melting occurs at high temperatures, low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH is rarely used).
The stability of the dsDNA form depends not only on the GC-content (% G,C basepairs) but also on sequence (since stacking is sequence specific) and also length (longer molecules are more stable). The stability can be measured in various ways; a common way is the melting temperature (also called Tm value), which is the temperature at which 50% of the double-strand molecules are converted to single-strand molecules; melting temperature is dependent on ionic strength and the concentration of DNA. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determines the strength of the association between the two strands of DNA. Long DNA helices with a high GC-content have more strongly interacting strands, while short helices with high AT content have more weakly interacting strands. In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters, tend to have a high AT content, making the strands easier to pull apart.
In the laboratory, the strength of this interaction can be measured by finding the melting temperature Tm necessary to break half of the hydrogen bonds. When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others.
=== Amount ===
In humans, the total female diploid nuclear genome per cell extends for 6.37 Gigabase pairs (Gbp), is 208.23 cm long and weighs 6.51 picograms (pg). Male values are 6.27 Gbp, 205.00 cm, 6.41 pg. Each DNA polymer can contain hundreds of millions of nucleotides, such as in chromosome 1. Chromosome 1 is the largest human chromosome with approximately 220 million base pairs, and would be 85 mm long if straightened.
In eukaryotes, in addition to nuclear DNA, there is also mitochondrial DNA (mtDNA) which encodes certain proteins used by the mitochondria. The mtDNA is usually relatively small in comparison to the nuclear DNA. For example, the human mitochondrial DNA forms closed circular molecules, each of which contains 16,569 DNA base pairs, with each such molecule normally containing a full set of the mitochondrial genes. Each human mitochondrion contains, on average, approximately 5 such mtDNA molecules. Each human cell contains approximately 100 mitochondria, giving a total number of mtDNA molecules per human cell of approximately 500. However, the amount of mitochondria per cell also varies by cell type, and an egg cell can contain 100,000 mitochondria, corresponding to up to 1,500,000 copies of the mitochondrial genome (constituting up to 90% of the DNA of the cell).
=== Sense and antisense ===
A DNA sequence is called a "sense" sequence if it is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands can contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear. One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.
A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes. In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription, while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome.
=== Supercoiling ===
DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound. If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases. These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.
=== Alternative DNA structures ===
DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although only B-DNA and Z-DNA have been directly observed in functional organisms. The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, and the presence of polyamines in solution.
The first published reports of A-DNA X-ray diffraction patterns—and also B-DNA—used analyses based on Patterson functions that provided only a limited amount of structural information for oriented fibers of DNA. An alternative analysis was proposed by Wilkins et al. in 1953 for the in vivo B-DNA X-ray diffraction-scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions. In the same journal, James Watson and Francis Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double helix.
Although the B-DNA form is most common under the conditions found in cells, it is not a well-defined conformation but a family of related DNA conformations that occur at the high hydration levels present in cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder.
Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a narrower, deeper major groove. The A form occurs under non-physiological conditions in partly dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, and in enzyme-DNA complexes. Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form. Here, the strands turn about the helical axis in a left-handed spiral, the opposite of the more common B form. These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription.
=== Alternative DNA chemistry ===
For many years, exobiologists have proposed the existence of a shadow biosphere, a postulated microbial biosphere of Earth that uses radically different biochemical and molecular processes than currently known life. One of the proposals was the existence of lifeforms that use arsenic instead of phosphorus in DNA. A report in 2010 of the possibility in the bacterium GFAJ-1 was announced, though the research was disputed, and evidence suggests the bacterium actively prevents the incorporation of arsenic into the DNA backbone and other biomolecules.
=== Quadruplex structures ===
At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes. These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected. In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence.
These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases, known as a guanine tetrad, form a flat plate. These flat four-base units then stack on top of each other to form a stable G-quadruplex structure. These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit. Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure.
In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins. At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop or D-loop.
=== Branched DNA ===
In DNA, fraying occurs when non-complementary regions exist at the end of an otherwise complementary double-strand of DNA. However, branched DNA can occur if a third strand of DNA is introduced and contains adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Although the simplest example of branched DNA involves only three strands of DNA, complexes involving additional strands and multiple branches are also possible. Branched DNA can be used in nanotechnology to construct geometric shapes, see the section on uses in technology below.
=== Artificial bases ===
Several artificial nucleobases have been synthesized, and successfully incorporated in the eight-base DNA analogue named Hachimoji DNA. Dubbed S, B, P, and Z, these artificial bases are capable of bonding with each other in a predictable way (S–B and P–Z), maintain the double helix structure of DNA, and be transcribed to RNA. Their existence could be seen as an indication that there is nothing special about the four natural nucleobases that evolved on Earth. On the other hand, DNA is tightly related to RNA which does not only act as a transcript of DNA but also performs as molecular machines many tasks in cells. For this purpose it has to fold into a structure. It has been shown that to allow to create all possible structures at least four bases are required for the corresponding RNA, while a higher number is also possible but this would be against the natural principle of least effort.
=== Acidity ===
The phosphate groups of DNA give it similar acidic properties to phosphoric acid and it can be considered as a strong acid. It will be fully ionized at a normal cellular pH, releasing protons which leave behind negative charges on the phosphate groups. These negative charges protect DNA from breakdown by hydrolysis by repelling nucleophiles which could hydrolyze it.
=== Macroscopic appearance ===
Pure DNA extracted from cells forms white, stringy clumps.
== Chemical modifications and altered DNA packaging ==
=== Base modifications and DNA packaging ===
The expression of genes is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin. Base modifications can be involved in packaging, with regions that have low or no gene expression usually containing high levels of methylation of cytosine bases. DNA packaging and its influence on gene expression can also occur by covalent modifications of the histone protein core around which DNA is wrapped in the chromatin structure or else by remodeling carried out by chromatin remodeling complexes (see Chromatin remodeling). There is, further, crosstalk between DNA methylation and histone modification, so they can coordinately affect chromatin and gene expression.
For one example, cytosine methylation produces 5-methylcytosine, which is important for X-inactivation of chromosomes. The average level of methylation varies between organisms—the worm Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing 5-methylcytosine. Despite the importance of 5-methylcytosine, it can deaminate to leave a thymine base, so methylated cytosines are particularly prone to mutations. Other base modifications include adenine methylation in bacteria, the presence of 5-hydroxymethylcytosine in the brain, and the glycosylation of uracil to produce the "J-base" in kinetoplastids.
=== Damage ===
DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Mutagens include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers, which are cross-links between pyrimidine bases. On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, and double-strand breaks. A typical human cell contains about 150,000 bases that have suffered oxidative damage. Of these oxidative lesions, the most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations, insertions, deletions from the DNA sequence, and chromosomal translocations. These mutations can cause cancer. Because of inherent limits in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer. DNA damages that are naturally occurring, due to normal cellular processes that produce reactive oxygen species, the hydrolytic activities of cellular water, etc., also occur frequently. Although most of these damages are repaired, in any cell some DNA damage may remain despite the action of repair processes. These remaining DNA damages accumulate with age in mammalian postmitotic tissues. This accumulation appears to be an important underlying cause of aging.
Many mutagens fit into the space between two adjacent base pairs, this is called intercalation. Most intercalators are aromatic and planar molecules; examples include ethidium bromide, acridines, daunomycin, and doxorubicin. For an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen. Others such as benzo[a]pyrene diol epoxide and aflatoxin form DNA adducts that induce errors in replication. Nevertheless, due to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to inhibit rapidly growing cancer cells.
== Biological functions ==
DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation, which depends on the same interaction between RNA nucleotides. In an alternative fashion, a cell may copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles; here the focus is on the interactions between DNA and other molecules that mediate the function of the genome.
=== Genes and genomes ===
Genomic DNA is tightly and orderly packed in the process called DNA condensation, to fit the small available volumes of the cell. In eukaryotes, DNA is located in the cell nucleus, with small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid. The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, and regulatory sequences such as promoters and enhancers, which control transcription of the open reading frame.
In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences. The reasons for the presence of so much noncoding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species, represent a long-standing puzzle known as the "C-value enigma". However, some DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.
Some noncoding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes but are important for the function and stability of chromosomes. An abundant form of noncoding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation. These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence.
=== Transcription and translation ===
A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).
In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (43 combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAG, TAA, and TGA codons, (UAG, UAA, and UGA on the mRNA).
=== Replication ===
Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Here, the two strands are separated and then each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix. In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.
=== Extracellular nucleic acids ===
Naked extracellular DNA (eDNA), most of it released by cell death, is nearly ubiquitous in the environment. Its concentration in soil may be as high as 2 μg/L, and its concentration in natural aquatic environments may be as high at 88 μg/L. Various possible functions have been proposed for eDNA: it may be involved in horizontal gene transfer; it may provide nutrients; and it may act as a buffer to recruit or titrate ions or antibiotics. Extracellular DNA acts as a functional extracellular matrix component in the biofilms of several bacterial species. It may act as a recognition factor to regulate the attachment and dispersal of specific cell types in the biofilm; it may contribute to biofilm formation; and it may contribute to the biofilm's physical strength and resistance to biological stress.
Cell-free fetal DNA is found in the blood of the mother, and can be sequenced to determine a great deal of information about the developing fetus.
Under the name of environmental DNA eDNA has seen increased use in the natural sciences as a survey tool for ecology, monitoring the movements and presence of species in water, air, or on land, and assessing an area's biodiversity.
=== Neutrophil extracellular traps ===
Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of DNA, which allow neutrophils, a type of white blood cell, to kill extracellular pathogens while minimizing damage to the host cells.
== Interactions with proteins ==
All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.
=== DNA-binding proteins ===
Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes, this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones, making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are thus largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation, phosphorylation, and acetylation. These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA. These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.
A distinct group of DNA-binding proteins is the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination, and DNA repair. These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.
In contrast, other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription. Alternatively, transcription factors can bind enzymes that modify the histones at the promoter. This changes the accessibility of the DNA template to the polymerase.
As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes. Consequently, these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.
=== DNA-modifying enzymes ===
==== Nucleases and ligases ====
Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GATATC-3′ and makes a cut at the horizontal line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system. In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.
Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly important in lagging strand DNA replication, as they join the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.
==== Topoisomerases and helicases ====
Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the DNA break. Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix. Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.
Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly adenosine triphosphate (ATP), to break hydrogen bonds between bases and unwind the DNA double helix into single strands. These enzymes are essential for most processes where enzymes need to access the DNA bases.
==== Polymerases ====
Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products is created based on existing polynucleotide chains—which are called templates. These enzymes function by repeatedly adding a nucleotide to the 3′ hydroxyl group at the end of the growing polynucleotide chain. As a consequence, all polymerases work in a 5′ to 3′ direction. In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.
In DNA replication, DNA-dependent DNA polymerases make copies of DNA polynucleotide chains. To preserve biological information, it is essential that the sequence of bases in each copy are precisely complementary to the sequence of bases in the template strand. Many DNA polymerases have a proofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed. In most organisms, DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases.
RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres. For example, HIV reverse transcriptase is an enzyme for AIDS virus replication. Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure. It synthesizes telomeres at the ends of chromosomes. Telomeres prevent fusion of the ends of neighboring chromosomes and protect chromosome ends from damage.
Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits.
== Genetic recombination ==
A DNA helix usually does not interact with other segments of DNA, and in human cells, the different chromosomes even occupy separate areas in the nucleus called "chromosome territories". This physical separation of different chromosomes is important for the ability of DNA to function as a stable repository for information, as one of the few times chromosomes interact is in chromosomal crossover which occurs during sexual reproduction, when genetic recombination occurs. Chromosomal crossover is when two DNA helices break, swap a section and then rejoin.
Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of new proteins. Genetic recombination can also be involved in DNA repair, particularly in the cell's response to double-strand breaks.
The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51. The first step in recombination is a double-stranded break caused by either an endonuclease or damage to the DNA. A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA. Only strands of like polarity exchange DNA during recombination. There are two types of cleavage: east-west cleavage and north–south cleavage. The north–south cleavage nicks both strands of DNA, while the east–west cleavage has one strand of DNA intact. The formation of a Holliday junction during recombination makes it possible for genetic diversity, genes to exchange on chromosomes, and expression of wild-type viral genomes.
== Evolution ==
DNA contains the genetic information that allows all forms of life to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material. RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes. This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes. However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible because DNA survives in the environment for less than one million years, and slowly degrades into short fragments in solution. Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old, but these claims are controversial.
Building blocks of DNA (adenine, guanine, and related organic molecules) may have been formed extraterrestrially in outer space. Complex DNA and RNA organic compounds of life, including uracil, cytosine, and thymine, have also been formed in the laboratory under conditions mimicking those found in outer space, using starting chemicals, such as pyrimidine, found in meteorites. Pyrimidine, like polycyclic aromatic hydrocarbons (PAHs), the most carbon-rich chemical found in the universe, may have been formed in red giants or in interstellar cosmic dust and gas clouds.
Ancient DNA has been recovered from ancient organisms at a timescale where genome evolution can be directly observed, including from extinct organisms up to millions of years old, such as the woolly mammoth.
== Uses in technology ==
=== Genetic engineering ===
Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction, and to manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant DNA technology. Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector. The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research, or be grown in agriculture.
=== DNA profiling ===
Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual, such as a perpetrator. This process is formally termed DNA profiling, also called DNA fingerprinting. In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a matching DNA. However, identification can be complicated if the scene is contaminated with DNA from several people. DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.
The development of forensic science and the ability to now obtain genetic matching on minute samples of blood, skin, saliva, or hair has led to re-examining many cases. Evidence can now be uncovered that was scientifically impossible at the time of the original examination. Combined with the removal of the double jeopardy law in some places, this can allow cases to be reopened where prior trials have failed to produce sufficient evidence to convince a jury. People charged with serious crimes may be required to provide a sample of DNA for matching purposes. The most obvious defense to DNA matches obtained forensically is to claim that cross-contamination of evidence has occurred. This has resulted in meticulous strict handling procedures with new cases of serious crime.
DNA profiling is also used successfully to positively identify victims of mass casualty incidents, bodies or body parts in serious accidents, and individual victims in mass war graves, via matching to family members.
DNA profiling is also used in DNA paternity testing to determine if someone is the biological parent or grandparent of a child with the probability of parentage is typically 99.99% when the alleged parent is biologically related to the child. Normal DNA sequencing methods happen after birth, but there are new methods to test paternity while a mother is still pregnant.
=== DNA enzymes or catalytic DNA ===
Deoxyribozymes, also called DNAzymes or catalytic DNA, were first discovered in 1994. They are mostly single stranded DNA sequences isolated from a large pool of random DNA sequences through a combinatorial approach called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). DNAzymes catalyze variety of chemical reactions including RNA-DNA cleavage, RNA-DNA ligation, amino acids phosphorylation-dephosphorylation, carbon-carbon bond formation, etc. DNAzymes can enhance catalytic rate of chemical reactions up to 100,000,000,000-fold over the uncatalyzed reaction. The most extensively studied class of DNAzymes is RNA-cleaving types which have been used to detect different metal ions and designing therapeutic agents. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific), the CA1-3 DNAzymes (copper-specific), the 39E DNAzyme (uranyl-specific) and the NaA43 DNAzyme (sodium-specific). The NaA43 DNAzyme, which is reported to be more than 10,000-fold selective for sodium over other metal ions, was used to make a real-time sodium sensor in cells.
=== Bioinformatics ===
Bioinformatics involves the development of techniques to store, data mine, search and manipulate biological data, including DNA nucleic acid sequence data. These have led to widely applied advances in computer science, especially string searching algorithms, machine learning, and database theory. String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides. The DNA sequence may be aligned with other DNA sequences to identify homologous sequences and locate the specific mutations that make them distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function. Data sets representing entire genomes' worth of DNA sequences, such as those produced by the Human Genome Project, are difficult to use without the annotations that identify the locations of genes and regulatory elements on each chromosome. Regions of DNA sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products and their possible functions in an organism even before they have been isolated experimentally. Entire genomes may also be compared, which can shed light on the evolutionary history of particular organism and permit the examination of complex evolutionary events.
=== DNA nanotechnology ===
DNA nanotechnology uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties. DNA is thus used as a structural material rather than as a carrier of biological information. This has led to the creation of two-dimensional periodic lattices (both tile-based and using the DNA origami method) and three-dimensional structures in the shapes of polyhedra. Nanomechanical devices and algorithmic self-assembly have also been demonstrated, and these DNA structures have been used to template the arrangement of other molecules such as gold nanoparticles and streptavidin proteins. DNA and other nucleic acids are the basis of aptamers, synthetic oligonucleotide ligands for specific target molecules used in a range of biotechnology and biomedical applications.
=== History and anthropology ===
Because DNA collects mutations over time, which are then inherited, it contains historical information, and, by comparing DNA sequences, geneticists can infer the evolutionary history of organisms, their phylogeny. This field of phylogenetics is a powerful tool in evolutionary biology. If DNA sequences within a species are compared, population geneticists can learn the history of particular populations. This can be used in studies ranging from ecological genetics to anthropology.
=== Information storage ===
DNA as a storage device for information has enormous potential since it has much higher storage density compared to electronic devices. However, high costs, slow read and write times (memory latency), and insufficient reliability has prevented its practical use.
== History ==
DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein". In 1878, Albrecht Kossel isolated the non-protein component of "nuclein", nucleic acid, and later isolated its five primary nucleobases.
In 1909, Phoebus Levene identified the base, sugar, and phosphate nucleotide unit of RNA (then named "yeast nucleic acid"). In 1929, Levene identified deoxyribose sugar in "thymus nucleic acid" (DNA). Levene suggested that DNA consisted of a string of four nucleotide units linked together through the phosphate groups ("tetranucleotide hypothesis"). Levene thought the chain was short and the bases repeated in a fixed order. In 1927, Nikolai Koltsov proposed that inherited traits would be inherited via a "giant hereditary molecule" made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template". In 1928, Frederick Griffith in his experiment discovered that traits of the "smooth" form of Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form. This system provided the first clear suggestion that DNA carries genetic information.
In 1933, while studying virgin sea urchin eggs, Jean Brachet suggested that DNA is found in the cell nucleus and that RNA is present exclusively in the cytoplasm. At the time, "yeast nucleic acid" (RNA) was thought to occur only in plants, while "thymus nucleic acid" (DNA) only in animals. The latter was thought to be a tetramer, with the function of buffering cellular pH.
In 1937, William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure.
In 1943, Oswald Avery, along with co-workers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle, supporting Griffith's suggestion (Avery–MacLeod–McCarty experiment). Erwin Chargaff developed and published observations now known as Chargaff's rules, stating that in DNA from any species of any organism, the amount of guanine should be equal to cytosine and the amount of adenine should be equal to thymine.
Late in 1951, Francis Crick started working with James Watson at the Cavendish Laboratory within the University of Cambridge. DNA's role in heredity was confirmed in 1952 when Alfred Hershey and Martha Chase in the Hershey–Chase experiment showed that DNA is the genetic material of the enterobacteria phage T2.
In May 1952, Raymond Gosling, a graduate student working under the supervision of Rosalind Franklin, took an X-ray diffraction image, labeled as "Photo 51", at high hydration levels of DNA. This photo was given to Watson and Crick by Maurice Wilkins and was critical to their obtaining the correct structure of DNA. Franklin told Crick and Watson that the backbones had to be on the outside. Before then, Linus Pauling, and Watson and Crick, had erroneous models with the chains inside and the bases pointing outwards. Franklin's identification of the space group for DNA crystals revealed to Crick that the two DNA strands were antiparallel. In February 1953, Linus Pauling and Robert Corey proposed a model for nucleic acids containing three intertwined chains, with the phosphates near the axis, and the bases on the outside. Watson and Crick completed their model, which is now accepted as the first correct model of the double helix of DNA. On 28 February 1953 Crick interrupted patrons' lunchtime at The Eagle pub in Cambridge, England to announce that he and Watson had "discovered the secret of life".
The 25 April 1953 issue of the journal Nature published a series of five articles giving the Watson and Crick double-helix structure DNA and evidence supporting it. The structure was reported in a letter titled "MOLECULAR STRUCTURE OF NUCLEIC ACIDS A Structure for Deoxyribose Nucleic Acid", in which they said, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." This letter was followed by a letter from Franklin and Gosling, which was the first publication of their own X-ray diffraction data and of their original analysis method. Then followed a letter by Wilkins and two of his colleagues, which contained an analysis of in vivo B-DNA X-ray patterns, and which supported the presence in vivo of the Watson and Crick structure.
In April 2023, scientists, based on new evidence, concluded that Rosalind Franklin was a contributor and "equal player" in the discovery process of DNA, rather than otherwise, as may have been presented subsequently after the time of the discovery.
In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine. Nobel Prizes are awarded only to living recipients. A debate continues about who should receive credit for the discovery.
In an influential presentation in 1957, Crick laid out the central dogma of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis". Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson–Stahl experiment. Further work by Crick and co-workers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley, and Marshall Warren Nirenberg to decipher the genetic code. These findings represent the birth of molecular biology.
In 1986, DNA analysis was first used in a criminal investigation when police in the UK requested Alec Jeffreys of the University of Leicester to prove or disprove the involvement in a particular case of a suspect who claimed innocence in the matter. Although the suspect had already confessed to committing a recent rape-murder, he was denying any involvement in a similar crime committed three years earlier. Yet the details of the two cases were so alike that the police concluded both crimes had been committed by the same person. However, all charges against the suspect were dropped when Jeffreys' DNA testing exonerated the suspect — from both the earlier murder and the one to which he'd confessed. Further such DNA profiling led to positive identification of another suspect who, in 1988, was found guilty of both rape-murders.
== See also ==
== References ==
== Further reading ==
== External links ==
DNA binding site prediction on protein
DNA the Double Helix Game From the official Nobel Prize web site
DNA under electron microscope
Dolan DNA Learning Center
Double Helix: 50 years of DNA, Nature
Proteopedia DNA
Proteopedia Forms_of_DNA
ENCODE threads explorer ENCODE home page at Nature
Double Helix 1953–2003 National Centre for Biotechnology Education
Genetic Education Modules for Teachers – DNA from the Beginning Study Guide
PDB Molecule of the Month DNA
"Clue to chemistry of heredity found". The New York Times, June 1953. First American newspaper coverage of the discovery of the DNA structure
DNA from the Beginning Another DNA Learning Center site on DNA, genes, and heredity from Mendel to the human genome project.
The Register of Francis Crick Personal Papers 1938 – 2007 at Mandeville Special Collections Library, University of California, San Diego
Seven-page, handwritten letter that Crick sent to his 12-year-old son Michael in 1953 describing the structure of DNA. See Crick's medal goes under the hammer, Nature, 5 April 2013. | Wikipedia/DNA_strand |
A single-molecule experiment is an experiment that investigates the properties of individual molecules. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior of molecules cannot be distinguished, and only average characteristics can be measured. Since many measurement techniques in biology, chemistry, and physics are not sensitive enough to observe single molecules, single-molecule fluorescence techniques (that have emerged since the 1990s for probing various processes on the level of individual molecules) caused a lot of excitement, since these supplied many new details on the measured processes that were not accessible in the past. Indeed, since the 1990s, many techniques for probing individual molecules have been developed.
The first single-molecule experiments were patch clamp experiments performed in the 1970s, but these were limited to studying ion channels. Today, systems investigated using single-molecule techniques include the movement of myosin on actin filaments in muscle tissue and the spectroscopic details of individual local environments in solids. Biological polymers' conformations have been measured using atomic force microscopy (AFM). Using force spectroscopy, single molecules (or pairs of interacting molecules), usually polymers, can be mechanically stretched, and their elastic response recorded in real time.
== History ==
In the gas phase at ultralow pressures, single-molecule experiments have been around for decades, but in the condensed phase only since 1989 with the work by W. E. Moerner and Lothar Kador. One year later, Michel Orrit and Jacky Bernard were able to show also the detection of the absorption of single molecules by their fluorescence.
Many techniques have the ability to observe one molecule at a time, most notably mass spectrometry, where single ions are detected. In addition, one of the earliest means of detecting single molecules, came about in the field of ion channels with the development of the patch clamp technique by Erwin Neher and Bert Sakmann (who later went on to win the Nobel prize for their seminal contributions). However, the idea of measuring conductance to look at single molecules placed a serious limitation on the kind of systems which could be observed.
Fluorescence is a convenient means of observing one molecule at a time, mostly due to the sensitivity of commercial optical detectors, capable of counting single photons. However, spectroscopically, the observation of one molecule requires that the molecule is in an isolated environment and that it emits photons upon excitation, which owing to the technology to detect single photons by use of photomultiplier tubes (PMT) or avalanche photodiodes (APD), enables one to record photon emission events with great sensitivity and time resolution.
More recently, single-molecule fluorescence is the subject of intense interest for biological imaging, through the labeling of biomolecules such as proteins and nucleotides to study enzymatic function which cannot easily be studied on the bulk scale, due to subtle time-dependent movements in catalysis and structural reorganization. The most studied protein has been the class of myosin/actin enzymes found in muscle tissues. Through single-molecule techniques the step mechanism has been observed and characterized in many of these proteins.
In 1997, single-molecule detection was demonstrated with surface-enhanced Raman spectroscopy (SERS) by Katrin Kneipp, H. Kneipp, Y. Wang, L.T. Perelman and others at MIT and independently by S. Nie and S. R. Emory at Indiana University. The MIT team used non-resonance Raman excitation and surface enhancement with silver nanoclusters to detect single cresyl violet molecules, while the team at Indiana University used resonance Raman excitation and surface enhancement with silver nanoparticles to detect single rhodamine 6G molecules.
Nanomanipulators such as the atomic force microscope are also suited to single-molecule experiments of biological significance, since they work on the same length scale of most biological polymers. Besides, atomic force microscopy (AFM) is appropriate for the studies of synthetic polymer molecules. AFM provides a unique possibility of 3D visualization of polymer chains. For instance, AFM tapping mode is gentle enough for the recording of adsorbed polyelectrolyte molecules (for example, 0.4 nm thick chains of poly(2-vinylpyridine)) under liquid medium. The location of two-chain-superposition correspond in these experiments to twice the thickness of single chain (0.8 nm in the case of the mentioned example). At the application of proper scanning parameters, conformation of such molecules remain unchanged for hours that allows the performance of experiments under liquid media having various properties. Furthermore, by controlling the force between the tip and the sample high resolution images can be obtained. Optical tweezers have also been used to study and quantify DNA-protein interactions.
== About the experiments ==
=== Concept ===
Single-molecule fluorescence spectroscopy uses the fluorescence of a molecule for obtaining information on its environment, structure, and position. The technique affords the ability of obtaining information otherwise not available due to ensemble averaging (that is, a signal obtained when recording many molecules at the same time represents an average property of the molecules' dynamics). The results in many experiments of individual molecules are two-state trajectories.
=== Single-channel recording ===
As in the case of single molecule fluorescence spectroscopy, the technique known as single channel recording can be used to obtain specific kinetic information—in this case about ion channel function—that is not available when ensemble recording, such as whole-cell recording, is performed. Specifically, ion channels alternate between conducting and non-conducting classes, which differ in conformation. Therefore, the functional state of ion channels can be directly measured with sufficiently sensitive electronics, provided that proper precautions are taken to minimize noise. In turn, each of these classes may be divided into one or more kinetic states with direct bearing on the underlying function of the ion channel. Performing these types of single molecule studies under systematically varying conditions (e.g. agonist concentration and structure, permeant ion and/or channel blocker, mutations in the ion channel amino acids), can provide information regarding the interconversion of various kinetic states of the ion channel. In a minimal model for an ion channel, there are two states: open and closed. However, other states are often needed in order to accurately represent the data, including multiple closed states as well as inactive and/or desensitized states, which are non-conducting states that can occur even in the presence of stimulus.
=== Biomolecule labeling ===
Single fluorophores can be chemically attached to biomolecules, such as proteins or DNA, and the dynamics of individual molecules can be tracked by monitoring the fluorescent probe. Spatial movements within the Rayleigh limit can be tracked, along with changes in emission intensity and/or radiative lifetime, which often indicate changes in local environment. For instance, single-molecule labeling has yielded a vast quantity of information on how kinesin motor proteins move along microtubule strands in muscle cells. Single-molecule imaging in live cells reveals interesting information about protein dynamics under its physiological environment. Several biophysical parameters about protein dynamics can be quantified such as diffusion coefficient, mean squared displacements, residence time, the fraction of bound and unbound molecules, and target-search mechanism of protein binding to its target site in the live cell.
=== Single-molecule fluorescence resonance energy transfer (FRET) ===
Main article smFRET.
In single-molecule fluorescence resonance energy transfer, the molecule is labeled in (at least) two places. A laser beam is focused on the molecule exciting the first probe. When this probe relaxes and emits a photon, it has a chance of exciting the other probe. The efficiency of the absorption of the photon emitted from the first probe in the second probe depends on the distance between these probes. Since the distance changes with time, this experiment probes the internal dynamics of the molecule.
== Versus ensemble experiments ==
When looking at data related to individual molecules, one usually can construct propagators, and jumping time probability density functions, of the first order, the second order and so on, whereas from bulk experiments, one usually obtains the decay of a correlation function. From the information contained in these unique functions (obtained from individual molecules), one can extract a relatively clear picture on the way the system behaves; e.g. its kinetic scheme, or its potential of activity, or its reduced dimensions form. In particular, one can construct (many properties of) the reaction pathway of an enzyme when monitoring the activity of an individual enzyme. Additionally, significant aspects regarding the analysis of single molecule data—such as fitting methods and tests for homogeneous populations—have been described by several authors. On the other hand, there are several issues with the analysis of single molecule data including construction of a low noise environment and insulated pipet tips, filtering some of the remaining unwanted components (noise) found in recordings, and the length of time required for data analysis (pre-processing, unambiguous event detection, plotting data, fitting kinetic schemes, etc.).
== Impact ==
Single-molecule techniques impacted optics, electronics, biology, and chemistry. In the biological sciences, the study of proteins and other complex biological machinery was limited to ensemble experiments that nearly made impossible the direct observation of their kinetics. For example, it was only after single molecule fluorescence microscopy was used to study kinesin-myosin pairs in muscle tissue that direct observation of the walking mechanisms were understood. These experiments, however, have for the most part been limited to in vitro studies, as useful techniques for live cell imaging have yet to be fully realized. The promise of single molecule in vivo imaging, however, brings with it an enormous potential to directly observe bio-molecules in native processes. These techniques are often targeted for studies involving low-copy proteins, many of which are still being discovered. These techniques have also been extended to study areas of chemistry, including the mapping of heterogeneous surfaces.
== See also ==
Electron microscopy
Force spectroscopy
Magnetic tweezers
Optical tweezers
Raman spectroscopy
Scanning probe microscopy
Single molecule real time sequencing
Single-molecule magnet
Single-particle tracking
Super-resolution microscopy
Tethered particle motion (TPM)
Tunable resistive pulse sensing
Voltage clamp
== References == | Wikipedia/Single-molecule_experiment |
Replication protein A (RPA) is the major protein that binds to single-stranded DNA (ssDNA) in eukaryotic cells. In vitro, RPA shows a much higher affinity for ssDNA than RNA or double-stranded DNA. RPA is required in replication, recombination and repair processes such as nucleotide excision repair and homologous recombination. It also plays roles in responding to damaged DNA.
== Structure ==
RPA is a heterotrimer, composed of the subunits RPA1 (RPA70) (70kDa subunit), RPA2 (RPA32) (32kDa subunit) and RPA3 (RPA14) (14kDa subunit). The three RPA subunits contain six OB-folds (oligonucleotide/oligosaccharide binding), with DNA-binding domains (DBD) designated DBDs A-F, that bind RPA to single-stranded DNA.
DBDs A, B, C and F are located on RPA1, DBD D is located on RPA2, and DBD E is located on RPA3. DBDs C, D, and E make up the trimerization core of the protein with flexible linker regions connecting them all together. Due to these flexible linker regions RPA is considered highly flexible and this supports the dynamic binding that RPA is able to achieve. Because of this dynamic binding, RPA is also capable of different conformations that leads to varied numbers of nucleotides that it can engage.
DBDs A, B, C and D are the sites that are involved in ssDNA binding. Protein-protein interactions between RPA and other proteins happen at the N-terminal of RPA1, specifically DBD F, along with the C-terminal of RPA2. Phosphorylation of RPA takes place at the N-terminus of RPA2.
RPA shares many features with the CST complex heterotrimer, although RPA has a more uniform 1:1:1 stoichiometry.
== Functions ==
During DNA replication, RPA prevents single-stranded DNA (ssDNA) from winding back on itself or from forming secondary structures. It also helps protect the ssDNA from being attacked by endonucleases. This keeps DNA unwound for the polymerase to replicate it. RPA also binds to ssDNA during the initial phase of homologous recombination, an important process in DNA repair and prophase I of meiosis.
RPA has a key role in the maintenance of the recombination checkpoint during meiosis of the yeast Saccharomyces cerevisiae. RPA appears to act as a sensor of single-strand DNA for the activation of the meiotic DNA damage response.
Hypersensitivity to DNA damaging agents can be caused by mutations in the RPA gene. Like its role in DNA replication, this keeps ssDNA from binding to itself (self-complementizing) so that the resulting nucleoprotein filament can then be bound by Rad51 and its cofactors.
RPA also binds to DNA during the nucleotide excision repair process. This binding stabilizes the repair complex during the repair process. A bacterial homolog is called single-strand binding protein (SSB).
== See also ==
Single-stranded binding protein
Replication protein A1
Replication protein A2
Replication protein A3
== References == | Wikipedia/Replication_protein_A |
CDT1 (Chromatin licensing and DNA replication factor 1) is a protein that in humans is encoded by the CDT1 gene. It is a licensing factor that functions to limit DNA from replicating more than once per cell cycle.
== Role in pre-replication complexes ==
The protein encoded by this gene is a key licensing factor in the assembly of pre-replication complexes (pre-RC), which occurs during the G1 phase of the cell cycle. In the assembly of pre-RCs, origin recognition complexes (ORC1-6) recognize and bind to DNA replication origins. CDT1, along with the protein CDC6, are then recruited to the forming pre-RC, followed by minichromosome maintenance complexes (MCM2-7).
The activity of CDT1 during the cell cycle is tightly regulated during the S phase by the protein geminin, which inhibits it, and by SCFSKP2, which ubiquinates the protein to tag it for proteasomal degradation. This regulation is important in preventing relicensing, thus ensuring that DNA is only replicated once per cell cycle.
== Orthologs ==
CDT1 belongs to a family of replication proteins conserved from yeast to humans. Examples of orthologs in other species include:
S. pombe – CDT1 (CDC10-dependent transcript 1)
Drosophila melanogaster – 'double parked' or Dup
Xenopus laevis - CDT1
== Interactions ==
DNA replication factor CDT1 has been shown to interact with SKP2. Cdt1 is recruited by the origin recognition complex in origin licensing. Null-mutations for CDT1 are lethal in yeast; the spores undergo mitosis without DNA replication. The overexpression of CDT1 causes rereplication in H. sapiens, which activates the Chk1 pathway, preventing entry into mitosis.
== References ==
== Further reading ==
== External links ==
Overview of all the structural information available in the PDB for UniProt: Q9H211 (Human DNA replication factor Cdt1) at the PDBe-KB.
Overview of all the structural information available in the PDB for UniProt: Q8R4E9 (Mouse DNA replication factor Cdt1) at the PDBe-KB. | Wikipedia/DNA_replication_factor_CDT1 |
Thermostable DNA polymerases are DNA polymerases that originate from thermophiles, usually bacterial or archaeal species, and are therefore thermostable. They are used for the polymerase chain reaction and related methods for the amplification and modification of DNA.
== Properties ==
Several DNA polymerases have been described with distinct properties that define their specific utilisation in a PCR, in real-time PCR or in an isothermal amplification. Being DNA polymerases, the thermostable DNA polymerases all have a 5'→3' polymerase activity, and either a 5'→3' or a 3'→5' exonuclease activity.
== Structure ==
DNA polymerases are roughly shaped like a hand with a thumb, palm and fingers. The thumb is involved in binding and moving double-stranded DNA. The palm carries the polymerase active site, whereas the fingers bind substrates (template DNA and nucleoside triphosphates). The exonuclease activity is in a separate protein domain. Mg2+ is a cofactor.
The polymerase active site in the palm catalyses the prolongation of DNA, starting from a primer bound to a template DNA single strand:
deoxynucleoside triphosphate + DNAn ⇌ pyrophosphate + DNAn+1.
== Bacterial polymerases ==
Thermostable DNA polymerases of natural origin are found in thermophilic bacteria, archaea and their pathogens. Among the bacterial thermostable DNA polymerases, Taq polymerase, Tfl polymerase, Tma polymerase, Tne polymerase, Tth and Bst polymerase are used.
In addition to 5'→3' polymerase activity, the bacterial thermostable DNA polymerases (belonging to the A-type DNA polymerases) have 5'→3' exonuclease activity and generate an adenosine overhang (sticky ends) at the 3' end of the newly generated strand. The Klenow fragment of Bst (BF) has a strand displacement activity which allows for use in isothermal amplification without the necessity of denaturation of the DNA in a thermocycler, and its 5'→3' exonuclease activity is deleted for higher yield.
== Archaeal polymerases ==
Frequently used B-type DNA polymerases are the Pfu polymerase, the Pwo polymerase, the KOD polymerase, the Tli polymerase (also called Vent), which originates from various archaea, the Tag polymerase, the Tce polymerase, the Tgo polymerase, the TNA1 polymerase, the Tpe polymerase, the Tthi polymerase, the Neq polymerase and the Pab polymerase.
The archaeal variants (belonging to the B-type) produce blunt ends (the Tli polymerase produces an overhang in about 30% of the products) and instead of the 5'→3' exonuclease activity have an activity for correcting synthesis errors (proof-reading), the 3'→5' exonuclease activity. In archaeal polymerases, the error rate suffers when a Klenow fragment analogue is generated, as the correcting exonuclease activity is removed in the process. Some archaeal DNA polymerases are characterised less by their suitability for standard PCR than by their reduced inhibition in the amplification of A-DNA or DNA with modified bases.
== Modified polymerases ==
Various fusion proteins with the low error rate of archaeal and the high synthesis rate of bacterial thermostable DNA polymerases (Q5 polymerase) were generated from various thermostable polymerases and the DNA clamp of the thermostable DNA-binding protein SSo7d by protein design. A fusion protein of the PCNA homologue from Archaeoglobus fulgidus was also generated with archaeal thermostable DNA polymerases. Similarly, fusion proteins of thermostable DNA polymerases with the thermostable DNA-binding protein domain of a topoisomerase (type V, with helix-hairpin-helix motif, HhH) from Methanopyrus kandleri were generated (TopoTaq and PfuC2). A modified Pfu polymerase was also generated by protein design (Pfu Ultra). Similar effects are also achieved with mixtures of thermostable DNA polymerases of both types with a mixing ratio of the enzyme activities of type A and B polymerases of 30 to 1, e.g. Herculase and TaqPlus as a commercial mixture of Taq and Pfu polymerase, Expand as a commercial mixture of Taq and Pwo, Expand High Fidelity as a commercial mixture of Taq and Tgo, Platinum Taq High Fidelity as a commercial mixture of Taq and Tli (Vent), and Advantage HF 2 as a commercial mixture of Titanium Taq and an unnamed proof-reading polymerase. These mixtures can be used for long-range PCR to synthesize products of up to 35kb length. Other additives are used to help against difficult GC-rich sequences, avoid or neutralise the negative effects of PCR inhibitors (like blood components or detergents or dUTP), or alter the reaction kinetics.
== Speed & Processivity ==
The baseline synthesis rates (speed, productivity) of various polymerases have been compared. The synthesis rate of Taq polymerase is around 60 base pairs per second. Among the unmodified thermostable DNA polymerases, only the synthesis rate of KOD polymerase is above 100 base pairs per second (approx. 120 bp/s). Among the modified thermostable DNA polymerases, various mutations have been described that increase the synthesis rate. KOD polymerase and some modified thermostable DNA polymerases (iProof/Phusion, Pfu Ultra, Velocity or Z-Taq) are used as a PCR variant with shorter amplification cycles (fast PCR, high-speed PCR) due to their high synthesis rate. Processivity describes the average number of base pairs before a polymerase falls off the DNA template. The processivity of the polymerase limits the maximum distance between the primer and the probe in some forms of real-time quantitative PCR (qPCR).
== Fidelity ==
The error rates of various polymerases (fidelity) have been described. The error rate of Taq polymerase is 8 × 10−6 errors per base, that of Advantage HF 6.1 × 10−6 errors per base, that of Platinum Taq High Fidelity 5.8 × 10−6 errors per base and doubling, that of TaqPlus 4 × 10−6 errors per base and doubling, that of KOD polymerase 3.5 × 10−6 errors per base and doubling, that of Tli polymerase and Herculase 2.8 × 10−6 errors per base and doubling, that of Deep Vent 2.8 × 10−6 errors per base and doubling, that of Pfu, Phusion DNA Polymerase (identical with iProof DNA Polymerase) and Herculase II Fusion 1.3 × 10−6 errors per base and doubling and that of Pfu Ultra and Pfu Ultra II 4.3 × 10−7 errors per base and doubling. A newer analysis found slightly different error rates: Deep Vent (exo-) polymerase (5.0 × 10−4 errors per base and doubling), Taq polymerase (1.5 × 10−4 errors per base and doubling), Kapa HiFi HotStart ReadyMix (1.6 × 10−5 errors per base and doubling), KOD (1.2 × 10−5 errors per base and doubling), PrimeSTAR GXL (8.4 × 10−6 errors per base and doubling), Pfu (5.1 × 10−6 errors per base and doubling), Deep Vent DNA polymerase (4.0 × 10−6) errors per base and doubling, Phusion (3.9 × 10−6 errors per base and doubling), and Q5 DNA polymerase (5.3 × 10−7 errors per base and doubling). Yet another found error rates of 3–5.6 × 10−6 for Taq, 7.6 × 10−6 for KOD, 2.8 × 10−6 for Pfu, 2.6 × 10−6 for Phusion, and 2.4 × 10−6 for Pwo. To reduce the number of mutations in the PCR product (e.g. for molecular cloning), more template DNA and less cycles can be used in the PCR.
== Yield ==
Bacterial thermostable DNA polymerases generally produce higher product concentrations than archaeal, but with more copy errors. In the bacterial thermostable DNA polymerases, a Klenow fragment (Klen-Taq) or a Stoffel fragment can be generated by deleting the exonuclease domain in the course of protein design, analogous to the DNA polymerase from E. coli, which results in a higher product concentration. Two amino acids required for the exonuclease function of Taq polymerase were identified by mutagenesis as arginines at positions 25 and 74 (R25 and R74). A histidine to glutamic acid mutation at position 147 (short: H147E) in KOD polymerase lowers the relatively high exonuclease activity of KOD.
== Nucleotide specificity ==
The favouring of individual nucleotides by a thermostable DNA polymerase is referred to as nucleotide specificity (bias). In PCR-based DNA sequencing with chain termination substrates (dideoxy method), their uniform incorporation and thus unbiased generation of all chain termination products is often desired in order to enable higher sensitivity and easier analysis. For this purpose, a KlenTaq polymerase was generated by deletion and a phenylalanine at position 667 was exchanged for tyrosine by site-directed mutagenesis (short: F667Y) and named Thermo Sequenase. This polymerase can also be used for the incorporation of fluorescence-labelled dideoxynucleotides.
== Hot-start thermostable DNA polymerases ==
The template specificity of the polymerases is increased by using hot-start polymerases, to avoid binding of primers to unwanted DNA templates or to each other at low temperatures before the beginning of the PCR. Examples are the antibody-inhibited Pfu polymerase Pfu Turbo, the Platinum Pfx as a commercial KOD polymerase with an inhibiting antibody and the Platinum Taq as an antibody-inhibited Taq polymerase. Hot-start polymerases are either inhibited by inactivation with formaldehyde (or maleic anhydride, exo-cis-3,6-endoxo-Δ4-tetrahydropthalic anhydride, citraconic anhydride, 3,4,5,6-tetrahydrophthalic anhydride, cis-aconitic anhydride, or 2,3-dimethylmaleic anhydride), by complexing the magnesium with phosphates or by binding an antibody to their active site. Upon heating to 95 °C, the formaldehyde dissociates from proteins, or the magnesium ions are released, or the antibody is denatured and released in the process. Furthermore, polymerases can be inhibited with aptamers that denature upon heating. A fifth variant is a polymerase adsorbed on latex beads via hydrophobic effects, which dissolves with increasing temperature. In the sixth and oldest variant, the reaction mixture without polymerase is coated with wax and the polymerase is added on top of the cooled wax. When heated, the wax layer melts and the polymerase mixes with the reaction mixture.
== Other DNA polymerases ==
Some DNA polymerases used in isothermal DNA amplification, e.g. in loop-mediated isothermal amplification, multidisplacement amplification, recombinase polymerase amplification or isothermal assembly, for the amplification of entire genomes (e.g. the φ29 DNA polymerase from the bacteriophage phi29, B35DNAP from the phage Bam35) are not thermostable, while others like the Bst Klenow fragment are thermostable. The T4, T6 and T7 DNA polymerases are also not thermostable.
== RNA-dependent DNA polymerases ==
The standard reverse transcriptases (RNA-dependent DNA polymerases) of retroviral origin used for RT-PCR, like the AMV- and the MoMuLV-Reverse-Transcriptase, are not thermostable at 95 °C. At the lower temperatures of a reverse transcription unspecific hybridisation of primers to wrong sequences can occur, as well as unwanted secondary structures in the DNA template, which can lead to unwanted PCR products and less desired PCR products. The AMV reverse transcriptase may be used up to 70 °C. Also, some thermostable DNA-dependent DNA polymerases can be used as RNA-dependent DNA polymerases by exchanging Mg2+ as cofactors with Mn2+, so that they may be used for an RT-PCR. But since the synthesis rate of Taq with Mn2+ is relatively low, Tth was increasingly used for this approach. The use of Mn2+ also increases the error rate and the necessary amount of template, so that this method is rarely used. These problems can be avoided with the thermostable 3173-Polymerase from a thermophilic bacteriophage, which can withstand the high temperatures of a PCR and prefers RNA as a template.
== Applications ==
In addition to the choice of thermostable DNA polymerase, other parameters of a PCR are specifically changed in the course of PCR optimisation.
In addition to PCR, thermostable DNA polymerases are also used for RT-PCR variants, qPCR in different variants, site-specific mutagenesis and DNA sequencing. They are also used to produce hybridisation probes for Southern blot and Northern blot by random priming. The 5'→3' exonuclease activity is used for nick translation and TaqMan, among other things, without DNA replication (amplification).
== History ==
Alice Chien and colleagues were the first to characterise the thermostable Taq polymerase in 1976. The first use of a thermostable DNA polymerase was by Randall K. Saiki and colleagues in 1988, introducing Taq polymerase for PCR. The thermostability of Taq polymerase obliviated the need to add a non-thermostable DNA polymerase to the reaction after every melting phase of the PCR, because the Taq polymerase is not denatured by heating to 95 °C during the melting phase of each cyle. In 1989, the Taq polymerase gene was cloned and the Taq polymerase was produced in Escherichia coli as a recombinant protein. DNA of up to 35,000 basepairs was synthesized by Wayne M. Barnes by using different mixtures of A and B type polymerases, thereby creating the long-range PCR. The high synthesis rate of KOD polymerase was published in 1997 by Masahiro Takagi and colleagues, thereby creating the fundamentals of high speed PCR. Other optimisations to the PCR were developed in the following years, e.g. circumventing PCR inhibitors and amplifying difficult GC-rich DNA sequences, as well as modifying thermostable DNA polymerases by protein design. In 1998 the loop-mediated isothermal amplification was developed by Tsugunori Notomi and colleagues at Eiken Chemical Company, using Bst polymerase at 65 °C.
== Further reading ==
J. Sambrook, T. Maniatis, D. W. Russel: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press; 3rd edition (2001), ISBN 0-87969-577-3.
F. Akram, F. I. Shah, R. Ibrar, T. Fatima, I. U. Haq, W. Naseem, M. A. Gul, L. Tehreem, G. Haider: Bacterial thermophilic DNA polymerases: A focus on prominent biotechnological applications. In: Analytical biochemistry. Volume 671, June 2023, p. 115150, doi:10.1016/j.ab.2023.115150, PMID 37054862.
K. Terpe: Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems. In: Applied Microbiology and Biotechnology. Volume 97, issue 24, December 2013, p. 10243–10254, doi:10.1007/s00253-013-5290-2, PMID 24177730.
== External links ==
NEB Polbase. Accessed September 27, 2012.
== References == | Wikipedia/Thermostable_DNA_polymerase |
In molecular biology the SeqA protein is found in bacteria and archaea. The function of this protein is highly important in DNA replication. The protein negatively regulates the initiation of DNA replication at the origin of replication, in Escherichia coli, OriC. Additionally the protein plays a further role in sequestration. The importance of this protein is vital, without its help in DNA replication, cell division and other crucial processes could not occur. This protein domain is thought to be part of a much larger protein complex which includes other proteins such as SeqB.
== Function ==
DNA replication is an energy consuming process and hence in bacteria the process only occurs at a specific checkpoint in the cell cycle.
The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation.
SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Apart from its function in the control of DNA replication, SeqA may also be a specific transcription factor.
Additionally, SeqA is also thought to have a role in chromosome organisation and gene regulation.
== Localisation ==
Most of the SeqA in the cell is found bound to new DNA, at the replication fork.
== Structure ==
=== N terminal domain ===
The N-terminal domain folds into two alpha-helices and one beta-strand. This protein domain is vital in assisting multimerisation.
=== C terminal domain ===
The C-terminal protein domain has an important role in binding to DNA. It binds to fully methylated and hemimethylated GATC sequences at oriC. The structure of the C-terminal domain consists of seven alpha-helices and a three-stranded beta-sheet.
== References == | Wikipedia/SeqA_protein_domain |
DnaG is a bacterial DNA primase and is encoded by the dnaG gene. The enzyme DnaG, and any other DNA primase, synthesizes short strands of RNA known as oligonucleotides during DNA replication. These oligonucleotides are known as primers because they act as a starting point for DNA synthesis. DnaG catalyzes the synthesis of oligonucleotides that are 10 to 60 nucleotides (the fundamental unit of DNA and RNA) long, however most of the oligonucleotides synthesized are 11 nucleotides. These RNA oligonucleotides serve as primers, or starting points, for DNA synthesis by bacterial DNA polymerase III (Pol III). DnaG is important in bacterial DNA replication because DNA polymerase cannot initiate the synthesis of a DNA strand, but can only add nucleotides to a preexisting strand. DnaG synthesizes a single RNA primer at the origin of replication. This primer serves to prime leading strand DNA synthesis. For the other parental strand, the lagging strand, DnaG synthesizes an RNA primer every few kilobases (kb). These primers serve as substrates for the synthesis of Okazaki fragments.
In E. coli DnaG associates through noncovalent interactions with bacterial replicative helicase DnaB to perform its primase activity, with three DnaG primase proteins associating with each DnaB helicase to form the primosome. Primases tend to initiate synthesis at specific three nucleotide sequences on single-stranded DNA (ssDNA) templates and for E. coli DnaG the sequence is 5'-CTG-3'.
DnaG contains three separate protein domains: a zinc binding domain, an RNA polymerase domain, and a DnaB helicase binding domain. There are several bacteria that use the DNA primase DnaG. A few organisms that have DnaG as their DNA primase are Escherichia coli (E. coli), Bacillus stearothermophilus, and Mycobacterium tuberculosis (MTB). E. coli DnaG has a molecular weight of 60 kilodaltons (kDa) and contains 581 amino acids.
== Function ==
DnaG catalyzes the synthesis of oligonucleotides in five discrete steps: template binding, nucleoside triphosphate (NTP) binding, initiation, extension to form a primer, and primer transfer to DNA polymerase III. DnaG performs this catalysis near the replication fork that is formed by DnaB helicase during DNA replication. DnaG must be complexed with DnaB in order for it to catalyze the formation of the oligonucleotide primers.
The mechanism for primer synthesis by primases involves two NTP binding sites on the primase protein (DnaG). Prior to the binding of any NTPs to form the RNA primer, the ssDNA template sequence binds to DnaG. The ssDNA contains a three nucleotide recognition sequence that recruits NTPs based on Watson-Crick base pairing. After binding DNA, DnaG must bind two NTPs in order to generate an enzyme-DNA-NTP-NTP quaternary complex. The Michaelis constant's (km) for the NTPs vary depending on the primase and templates. The two NTP binding sites on DnaG are referred to as the initiation site and elongation site. The initiation site is the site at which the NTP to be incorporated at the 5' end of the primer binds. The elongation site binds the NTP that is added to the 3' end of the primer.
Once two nucleotides are bound to the primase, DnaG catalyzes the formation of a dinucleotide by forming a phosphodiester bond via dehydration synthesis between the 3' hydroxyl of the nucleotide in the initiation site and the α-phosphate of the nucleotide in the elongation site. This reaction results in a dinucleotide and breaking of the bond between the α and β phosphorus, releasing pyrophosphate. This reaction is irreversible because the pyrophosphate that is formed is hydrolyzed into two inorganic phosphate molecules by the enzyme inorganic pyrophosphatase. This dinucleotide synthesis reaction is the same reaction as any other enzyme that catalyzes the formation of DNA or RNA (DNA Polymerase, RNA Polymerase), therefore DnaG must always synthesize oligonucleotides in the 5' to 3' direction. In E. coli, primers begin with a triphosphate adenine-guanine (pppAG) dinucleotide at the 5' end.
In order for further elongation of the dinucleotide to occur, oligonucleotide must be moved so that the 3' NTP is transferred from the elongation site to the initiation site, allowing for another NTP to bind to the elongation site and attach to the 3' hydroxyl of the oligonucleotide. Once an oligonucleotide of appropriate length has been synthesized from the elongation step of primer synthesis, DnaG transfers the newly synthesized primer to DNA polymerase III for it to synthesize the DNA leading strand or Okazaki fragments for the lagging strand. The rate limiting step of the primer synthesis occurs after NTP binding but before or during dinucleotide synthesis.
== Structure ==
The E. Coli DnaG primase is a 581 residue monomeric protein with three functional domains, according to proteolysis studies. There is an N-terminal Zinc-binding domain (residues 1–110) where a zinc ion is tetrahedrally coordinated between one histidine and three cysteine residues, which plays a role in recognizing sequence specific DNA binding sites. The central domain (residues 111–433) displays RNA polymerase activities, and is the site of RNA primer synthesis. The C-terminal domain (residues 434–581) is responsible for the noncovalent binding of DnaG to the DnaB helicase protein.
=== Zinc-Binding Domain ===
The zinc-binding domain, the domain responsible for recognizing sequence specific DNA binding sites, is conserved across all viral, bacteriophage, prokaryotic and eukaryotic DNA primases. The primase zinc-binding domain is part of the subfamily of zinc-binding domains known as the zinc ribbon. Zinc ribbon domains are characterized by two β-hairpin loops which form the zinc-binding domain. Typically, zinc ribbon domains are thought to lack α-helices, distinguishing them from other zinc-binding domains. However, in 2000 DnaG's zinc-binding domain was crystallized from Bacillus stearothermophilus revealing that the domain consisted of a five stranded antiparallel β sheet adjacent to four α helices and a 310 helix on the c-terminal end of the domain.
The zinc-binding site of B. stearothermophilus consists of three cysteine residues, Cys40, Cys61, and Cys64, and one histidine residue, His43. Cys40 and His43 are located on the β-hairpin between the second and third β sheet. Cys61 is located on the fifth β sheet, and Cys64 is on the β-hairpin between the fourth and fifth β sheet. These four residues coordinate the zinc ion tetrahedrally. The zinc ion is thought to stabilize the loops between the second and third β sheet as well as the fourth and fifth β sheet. The domain is further stabilized by a number of hydrophobic interactions between the hydrophobic inner surface of the β sheet which is packed against the second and third α helices. The outer surface of the β sheet also has many conserved hydrophobic and basic residues. These residues are Lys30, Arg34, Lys46, Pro48, Lys56, Ile58, His60 and Phe62.
==== DNA Binding ====
It is thought that the function of the zinc binding domain is for sequence specific DNA recognition. DNA primases make RNA primers which are then used for DNA synthesis. The placement of the RNA primers is not random, suggesting that they are placed on specific DNA sequences. Indeed, other DNA primases have been shown to recognize triplet sequences; the specific sequence recognized by B. stearothermophilus has not yet been identified. It has been shown that if the cystine residues that coordinate the zinc ion are mutated, the DNA primase stops functioning. This indicates that the zinc-binding domain does play a role in sequence recognition. In addition, the hydrophobic surface of the β sheet, as well as the basic residues which are clustered primarily on one edge of the sheet, serve to attract single stranded DNA, further facilitating DNA binding.
Based on previous studies of DNA binding by DNA Primases, it is thought that DNA binds to the zinc-binding domain across the surface of the β sheet, with the three nucleotides binding across three strands of the β sheet. The positively charged residues in the sheet would be able to form contacts with the phosphates and the aromatic residues would form stacking interactions with the bases. This is the model of DNA binding by the ssDNA-binding domain of replication protein A (RPA). It is logical to assume that B. stearothermophilus’ zinc-binding domain binds DNA in a similar manner, as the residues important for binding DNA in RPA occur in structurally equivalent positions in B. stearothermophilus.
=== RNA Polymerase Domain ===
As its name suggests, the RNA polymerase domain (RNAP) of DnaG is responsible for synthesizing the RNA primers on the single stranded DNA. In-vivo, DnaG is able to synthesis primer fragments of up to 60 nucleotides, but in-vivo primer fragments are limited to approximately 11 nucleotides. During the synthesis of the lagging strand DnaG synthesizes between 2000 and 3000 primers at a rate of one primer per-second.
RNAP domain of DnaG has three subdomains, the N-terminal domain, which has a mixed α and β fold, the central domain consisting of a 5 stranded β sheet and 6 α helices, and finally the C-terminal domain which is made up of a helical bundle consisting of 3 antiparallel α helices. The central domain is made up in part of the toprim fold, a fold that has been observed in many metal-binding phosphotransfer proteins. The central domain and the N-terminal domain form a shallow cleft, which makes up the active site of the RNA chain elongation in DnaG. The opening of the cleft is lined by several highly conserved basic residues: Arg146, Arg221, and Lys229. These residues are part of the electrostatically positive ridge of the N-terminal subdomain. It is this ridge that interacts with the ssDNA and helps guide it into the cleft, which consists of the metal binding center of the toprim motif on the central subdomain, and the conserved primase motifs of the N-terminal domain. The metal binding site of the toprim domain is where the primer is synthesized. The RNA:DNA duplex then exits through another basic depression.
=== C-Terminal Domain ===
Unlike both the zinc-binding domains, and the RNA polymerase domains, the C-terminal domains of DNA primases are not conserved. In prokaryotic primases, the only known function of this domain is to interact with the helicase, DnaB. Thus, this domain is called the helicase binding domain (HBD). The HBD of DnaG consists of two subdomains: a helical bundle, the C1 subdomain, and a helical hairpin, the C2 subdomain. For each of the two to three DnaG molecules that bind the DnaB hexamer, the C1 subdomains of the HBDs interact with DnaB at its N-terminal domains on the inner surface of the hexamer ring, while the C2 subdomains interact with the N-terminal domains on the outer surface of the hexamer.
Three residues in B. stearothermophilus DnaB have been identified as important for formation of the DnaB, DnaG interface. Those residues include Tyr88, Ile119, and Ile125. Tyr88 is close in proximity to, but does not make contact with, the HBD of DnaG. Mutation of Tyr88 inhibits the formation of the N-terminal domain helical bundle of DnaB, interrupting the contacts with the HBD of DnaG. The hexameric structure of DnaB is really a trimer of dimers. Both Ile119 and Ile125 are buried in the N-terminal domain dimer interface of DnaB and mutation of these residues inhibits formation of the hexameric structure and thus the interaction with DnaG. One other residue that has been identified as playing a crucial role in the interaction of DnaB and DnaG is Glu15. Mutation of Glu15 does not disrupt the formation of the DnaB, DnaG complex, but instead plays a role in modulating the length of primers synthesized by DnaG.
== Inhibition of DnaG ==
Inhibitors of DNA primases are valuable compounds for the elucidation of biochemical pathways and key interactions, but they are also of interest as lead compounds to design drugs against bacterial diseases. Most of the compounds known to inhibit primases are nucleotide analogs such as AraATP (see Vidarabine) and 2-fluoro-AraATP. These compounds will often be used as substrates by the primase, but once incorporated synthesis or elongation can no longer occur. For example, E. coli DnaG will use 2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs) as substrates, which act as chain terminators due to the lack of a 3' hydroxyl to form a phosphodiester bond with the next nucleotide.
The relatively small number of primase inhibitors likely reflects the inherent difficulty of primase assays rather than a lack of potential binding sites on the enzyme. The short length of products synthesized and the generally slow rate of the enzyme compared to other replication enzymes make developing high-throughput screening (HTS) approaches more difficult. Despite the difficulties, there are several known inhibitors of DnaG that are not NTP analogues. Doxorubicin and suramin are both DNA and NTP competitive inhibitors of Mycobacterium Tuberculosis DnaG. Suramin is also known to inhibit eukaryotic DNA primase by competing with GTP, so suramin is likely to inhibit DnaG via a similar mechanism.
== External links ==
dnaG+protein,+E+coli at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
DnaG+(Primase) at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
== References == | Wikipedia/DnaG |
dnaC is a prokaryotic loading factor found in Escherichia coli that complexes with the C-terminus of helicase dnaB during the initial stages of prokaryotic DNA replication, loading dnaB onto DNA and inhibiting it from unwinding double stranded DNA (dsDNA) at a replication fork. Both dnaB and dnaC associate near the dnaA bound origin for each of the single stranded DNA molecules (ssDNA). Since DNA is antiparallel, one dnaB-dnaC complex is oriented in the opposite direction to the other dnaB-dnaC complex. After the assembly of dnaG, a primase, onto the N-terminus of dnaB, dnaC is released and dnaB will be allowed to begin unwinding dsDNA to make room for DNA polymerase to begin synthesizing the daughter strands.
This interaction of dnaC with dnaB requires the hydrolysis of ATP.
== dnaC Function ==
=== Association of dnaB-dnaC complex ===
Since dnaC functions as a helicase loader, dnaB helicase is needed. Specifically, for dnaC function a complex with dnaB is formed. dnaB is a hexameric protein with helicase properties that allow it to unwind DNA at the origin site, oriC. When dnaC associates with dnaB and ATP, dnaB and dnaC form dimers with six dnaC polypeptides. This is due to a conformational change of dnaC. These dimers a specific structure, containing a small lobe and a large lobe. The small lobe attaches to one monomer of the dnaB, while the large lobe associates with subunits of neighboring dnaB. dnaB transforms from a closed ring structure to an open ring structure with the addition of dnaC and ATP. The binding of the two proteins is because of interactions regarding their amino acids. Amino acids on the N terminus of dnaC associate with the carboxyl terminal domain of dnaB. When this happens, there is also a conformational change of the RecA fold on dnaB and the AAA+ domain of dnaC. The RecA fold is responsible for DNA binding and the AAA+ domain of dnaC is needed for ATP binding and hydrolysis. ATP hydrolysis is necessary for the function of dnaC later in replication. Additionally, dnaC impacts hairpins of the N-terminal domain of dnaB and the N-terminal domain of dnaB can be modified by dnaC to impact interactions with dnaG, a primase. The new dnaB-dnaC complex formed can now aid in loading dnaB to the origin of replication.
=== Binding of dnaB-dnaC complex to DNA ===
The dnaB-dnaC complex is able to open and close like a clamp due to its ring-like structure. To start binding, a region in the DNA is unwound slightly by the protein dnaA attached to dnaA boxes. The slight unwinding allows for the dnaB-dnaC complex to associate with the DNA replication fork. These interactions with the replication fork are impacted by the AAA+ domain on the C-terminal domain of dnaC. For single strand binding, ATP hydrolysis of dnaC is needed for the complex to bind to the template ssDNA with a high affinity. ATP is hydrolyzed to ADP and the complex is able to bind and close its ring-like structure around the DNA strand. When the dnaB-dnaC complex initially binds to the DNA, it is inactive. To activate dnaB, dnaC has to be released. When this occurs, dnaB is translocated and can begin unwinding the DNA for replication.
=== Dissociation of dnaC and activation of dnaB ===
For dnaB to complete helicase activity, dnaC is required to dissociate from the dnaB-dnaC complex. The release of dnaC from dnaB relies on multiple factors. First, a hydrolysis reaction that specifically requires ATP needs to occur. This reaction is the same one used to bind the complex to the ssDNA at the replication fork. In addition, interactions with dnaG on the N-terminal domain of dnaB are necessary to disrupt the dnaB-dnaC complex. This interaction and hydrolysis reaction releases dnaC from the C-terminal domain of dnaB. Once dnaC dissociates from the complex, dnaB is able to perform helicase activities for DNA replication. These allow for the ssDNA to be available to primase and other proteins necessary to create a complementary strand of the template DNA.
== Current Research ==
Current research is ongoing regarding dnaC and its role in prokaryotic DNA replication. Research groups are using a variety of physical and molecular methods to further knowledge. Topics include the role of single stranded binding proteins, potentially exploiting the dnaC-dnaB complex for peptide antibiotics, interactions with other proteins like dnaE, and others. Additionally, other prokaryotic helicase loaders, like DciA in bacteria, are being investigated due to their similar properties to dnaC.
== References == | Wikipedia/DnaC |
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones.
These enzymes catalyze the chemical reaction
deoxynucleoside triphosphate + DNAn ⇌ pyrophosphate + DNAn+1.
DNA polymerase adds nucleotides to the three prime (3')-end of a DNA strand, one nucleotide at a time. Every time a cell divides, DNA polymerases are required to duplicate the cell's DNA, so that a copy of the original DNA molecule can be passed to each daughter cell. In this way, genetic information is passed down from generation to generation.
Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form, in the process breaking the hydrogen bonds between the nucleotide bases. This opens up or "unzips" the double-stranded DNA to give two single strands of DNA that can be used as templates for replication in the above reaction.
== History ==
In 1956, Arthur Kornberg and colleagues discovered DNA polymerase I (Pol I), in Escherichia coli. They described the DNA replication process by which DNA polymerase copies the base sequence of a template DNA strand. Kornberg was later awarded the Nobel Prize in Physiology or Medicine in 1959 for this work. DNA polymerase II was discovered by Thomas Kornberg (the son of Arthur Kornberg) and Malcolm E. Gefter in 1970 while further elucidating the role of Pol I in E. coli DNA replication. Three more DNA polymerases have been found in E. coli, including DNA polymerase III (discovered in the 1970s) and DNA polymerases IV and V (discovered in 1999). From 1983 on, DNA polymerases have been used in the polymerase chain reaction (PCR), and from 1988 thermostable DNA polymerases were used instead, as they do not need to be added in every cycle of a PCR.
== Function ==
The main function of DNA polymerase is to synthesize DNA from deoxyribonucleotides, the building blocks of DNA. The DNA copies are created by the pairing of nucleotides to bases present on each strand of the original DNA molecule. This pairing always occurs in specific combinations, with cytosine along with guanine, and thymine along with adenine, forming two separate pairs, respectively. By contrast, RNA polymerases synthesize RNA from ribonucleotides from either RNA or DNA.
When synthesizing new DNA, DNA polymerase can add free nucleotides only to the 3' end of the newly forming strand. This results in elongation of the newly forming strand in a 5'–3' direction.
It is important to note that the directionality of the newly forming strand (the daughter strand) is opposite to the direction in which DNA polymerase moves along the template strand. Since DNA polymerase requires a free 3' OH group for initiation of synthesis, it can synthesize in only one direction by extending the 3' end of the preexisting nucleotide chain. Hence, DNA polymerase moves along the template strand in a 3'–5' direction, and the daughter strand is formed in a 5'–3' direction. This difference enables the resultant double-strand DNA formed to be composed of two DNA strands that are antiparallel to each other.
The function of DNA polymerase is not quite perfect, with the enzyme making about one mistake for every billion base pairs copied. Error correction is a property of some, but not all DNA polymerases. This process corrects mistakes in newly synthesized DNA. When an incorrect base pair is recognized, DNA polymerase moves backwards by one base pair of DNA. The 3'–5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue forwards. This preserves the integrity of the original DNA strand that is passed onto the daughter cells.
Fidelity is very important in DNA replication. Mismatches in DNA base pairing can potentially result in dysfunctional proteins and could lead to cancer. Many DNA polymerases contain an exonuclease domain, which acts in detecting base pair mismatches and further performs in the removal of the incorrect nucleotide to be replaced by the correct one. The shape and the interactions accommodating the Watson and Crick base pair are what primarily contribute to the detection or error. Hydrogen bonds play a key role in base pair binding and interaction. The loss of an interaction, which occurs at a mismatch, is said to trigger a shift in the balance, for the binding of the template-primer, from the polymerase, to the exonuclease domain. In addition, an incorporation of a wrong nucleotide causes a retard in DNA polymerization. This delay gives time for the DNA to be switched from the polymerase site to the exonuclease site. Different conformational changes and loss of interaction occur at different mismatches. In a purine:pyrimidine mismatch there is a displacement of the pyrimidine towards the major groove and the purine towards the minor groove. Relative to the shape of DNA polymerase's binding pocket, steric clashes occur between the purine and residues in the minor groove, and important van der Waals and electrostatic interactions are lost by the pyrimidine. Pyrimidine:pyrimidine and purine:purine mismatches present less notable changes since the bases are displaced towards the major groove, and less steric hindrance is experienced. However, although the different mismatches result in different steric properties, DNA polymerase is still able to detect and differentiate them so uniformly and maintain fidelity in DNA replication. DNA polymerization is also critical for many mutagenesis processes and is widely employed in biotechnologies.
=== Structure ===
The known DNA polymerases have highly conserved structure, which means that their overall catalytic subunits vary very little from species to species, independent of their domain structures. Conserved structures usually indicate important, irreplaceable functions of the cell, the maintenance of which provides evolutionary advantages. The shape can be described as resembling a right hand with thumb, finger, and palm domains. The palm domain appears to function in catalyzing the transfer of phosphoryl groups in the phosphoryl transfer reaction. DNA is bound to the palm when the enzyme is active. This reaction is believed to be catalyzed by a two-metal-ion mechanism. The finger domain functions to bind the nucleoside triphosphates with the template base. The thumb domain plays a potential role in the processivity, translocation, and positioning of the DNA.
=== Processivity ===
DNA polymerase's rapid catalysis due to its processive nature. Processivity is a characteristic of enzymes that function on polymeric substrates. In the case of DNA polymerase, the degree of processivity refers to the average number of nucleotides added each time the enzyme binds a template. The average DNA polymerase requires about one second locating and binding a primer/template junction. Once it is bound, a nonprocessive DNA polymerase adds nucleotides at a rate of one nucleotide per second.: 207–208 Processive DNA polymerases, however, add multiple nucleotides per second, drastically increasing the rate of DNA synthesis. The degree of processivity is directly proportional to the rate of DNA synthesis. The rate of DNA synthesis in a living cell was first determined as the rate of phage T4 DNA elongation in phage infected E. coli. During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second.
DNA polymerase's ability to slide along the DNA template allows increased processivity. There is a dramatic increase in processivity at the replication fork. This increase is facilitated by the DNA polymerase's association with proteins known as the sliding DNA clamp. The clamps are multiple protein subunits associated in the shape of a ring. Using the hydrolysis of ATP, a class of proteins known as the sliding clamp loading proteins open up the ring structure of the sliding DNA clamps allowing binding to and release from the DNA strand. Protein–protein interaction with the clamp prevents DNA polymerase from diffusing from the DNA template, thereby ensuring that the enzyme binds the same primer/template junction and continues replication.: 207–208 DNA polymerase changes conformation, increasing affinity to the clamp when associated with it and decreasing affinity when it completes the replication of a stretch of DNA to allow release from the clamp.
DNA polymerase processivity has been studied with in vitro single-molecule experiments (namely, optical tweezers and magnetic tweezers) have revealed the synergies between DNA polymerases and other molecules of the replisome (helicases and SSBs) and with the DNA replication fork. These results have led to the development of synergetic kinetic models for DNA replication describing the resulting DNA polymerase processivity increase.
== Variation across species ==
Based on sequence homology, DNA polymerases can be further subdivided into seven different families: A, B, C, D, X, Y, and RT.
Some viruses also encode special DNA polymerases, such as Hepatitis B virus DNA polymerase. These may selectively replicate viral DNA through a variety of mechanisms. Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp). It polymerizes DNA from a template of RNA.
=== Prokaryotic polymerase ===
Prokaryotic polymerases exist in two forms: core polymerase and holoenzyme. Core polymerase synthesizes DNA from the DNA template but it cannot initiate the synthesis alone or accurately. Holoenzyme accurately initiates synthesis.
==== Pol I ====
Prokaryotic family A polymerases include the DNA polymerase I (Pol I) enzyme, which is encoded by the polA gene and ubiquitous among prokaryotes. This repair polymerase is involved in excision repair with both 3'–5' and 5'–3' exonuclease activity and processing of Okazaki fragments generated during lagging strand synthesis. Pol I is the most abundant polymerase, accounting for >95% of polymerase activity in E. coli; yet cells lacking Pol I have been found suggesting Pol I activity can be replaced by the other four polymerases. Pol I adds ~15-20 nucleotides per second, thus showing poor processivity. Instead, Pol I starts adding nucleotides at the RNA primer:template junction known as the origin of replication (ori). Approximately 400 bp downstream from the origin, the Pol III holoenzyme is assembled and takes over replication at a highly processive speed and nature.
Taq polymerase is a heat-stable enzyme of this family that lacks proofreading ability.
==== Pol II ====
DNA polymerase II is a family B polymerase encoded by the polB gene. Pol II has 3'–5' exonuclease activity and participates in DNA repair, replication restart to bypass lesions, and its cell presence can jump from ~30-50 copies per cell to ~200–300 during SOS induction. Pol II is also thought to be a backup to Pol III as it can interact with holoenzyme proteins and assume a high level of processivity. The main role of Pol II is thought to be the ability to direct polymerase activity at the replication fork and help stalled Pol III bypass terminal mismatches.
Pfu DNA polymerase is a heat-stable enzyme of this family found in the hyperthermophilic archaeon Pyrococcus furiosus. Detailed classification divides family B in archaea into B1, B2, B3, in which B2 is a group of pseudoenzymes. Pfu belongs to family B3. Others PolBs found in archaea are part of "Casposons", Cas1-dependent transposons. Some viruses (including Φ29 DNA polymerase) and mitochondrial plasmids carry polB as well.
==== Pol III ====
DNA polymerase III holoenzyme is the primary enzyme involved in DNA replication in E. coli and belongs to family C polymerases. It consists of three assemblies: the pol III core, the beta sliding clamp processivity factor, and the clamp-loading complex. The core consists of three subunits: α, the polymerase activity hub, ɛ, exonucleolytic proofreader, and θ, which may act as a stabilizer for ɛ. The beta sliding clamp processivity factor is also present in duplicate, one for each core, to create a clamp that encloses DNA allowing for high processivity. The third assembly is a seven-subunit (τ2γδδ′χψ) clamp loader complex.
The old textbook "trombone model" depicts an elongation complex with two equivalents of the core enzyme at each replication fork (RF), one for each strand, the lagging and leading. However, recent evidence from single-molecule studies indicates an average of three stoichiometric equivalents of core enzyme at each RF for both Pol III and its counterpart in B. subtilis, PolC. In-cell fluorescent microscopy has revealed that leading strand synthesis may not be completely continuous, and Pol III* (i.e., the holoenzyme α, ε, τ, δ and χ subunits without the ß2 sliding clamp) has a high frequency of dissociation from active RFs. In these studies, the replication fork turnover rate was about 10s for Pol III*, 47s for the ß2 sliding clamp, and 15m for the DnaB helicase. This suggests that the DnaB helicase may remain stably associated at RFs and serve as a nucleation point for the competent holoenzyme. In vitro single-molecule studies have shown that Pol III* has a high rate of RF turnover when in excess, but remains stably associated with replication forks when concentration is limiting. Another single-molecule study showed that DnaB helicase activity and strand elongation can proceed with decoupled, stochastic kinetics.
==== Pol IV ====
In E. coli, DNA polymerase IV (Pol IV) is an error-prone DNA polymerase involved in non-targeted mutagenesis. Pol IV is a Family Y polymerase expressed by the dinB gene that is switched on via SOS induction caused by stalled polymerases at the replication fork. During SOS induction, Pol IV production is increased tenfold and one of the functions during this time is to interfere with Pol III holoenzyme processivity. This creates a checkpoint, stops replication, and allows time to repair DNA lesions via the appropriate repair pathway. Another function of Pol IV is to perform translesion synthesis at the stalled replication fork like, for example, bypassing N2-deoxyguanine adducts at a faster rate than transversing undamaged DNA. Cells lacking the dinB gene have a higher rate of mutagenesis caused by DNA damaging agents.
==== Pol V ====
DNA polymerase V (Pol V) is a Y-family DNA polymerase that is involved in SOS response and translesion synthesis DNA repair mechanisms. Transcription of Pol V via the umuDC genes is highly regulated to produce only Pol V when damaged DNA is present in the cell generating an SOS response. Stalled polymerases causes RecA to bind to the ssDNA, which causes the LexA protein to autodigest. LexA then loses its ability to repress the transcription of the umuDC operon. The same RecA-ssDNA nucleoprotein posttranslationally modifies the UmuD protein into UmuD' protein. UmuD and UmuD' form a heterodimer that interacts with UmuC, which in turn activates umuC's polymerase catalytic activity on damaged DNA. In E. coli, a polymerase "tool belt" model for switching pol III with pol IV at a stalled replication fork, where both polymerases bind simultaneously to the β-clamp, has been proposed. However, the involvement of more than one TLS polymerase working in succession to bypass a lesion has not yet been shown in E. coli. Moreover, Pol IV can catalyze both insertion and extension with high efficiency, whereas pol V is considered the major SOS TLS polymerase. One example is the bypass of intra strand guanine thymine cross-link where it was shown on the basis of the difference in the mutational signatures of the two polymerases, that pol IV and pol V compete for TLS of the intra-strand crosslink.
==== Family D ====
In 1998, the family D of DNA polymerase was discovered in Pyrococcus furiosus and Methanococcus jannaschii. The PolD complex is a heterodimer of two chains, each encoded by DP1 (small proofreading) and DP2 (large catalytic). Unlike other DNA polymerases, the structure and mechanism of the DP2 catalytic core resemble that of multi-subunit RNA polymerases. The DP1-DP2 interface resembles that of Eukaryotic Class B polymerase zinc finger and its small subunit. DP1, a Mre11-like exonuclease, is likely the precursor of small subunit of Pol α and ε, providing proofreading capabilities now lost in Eukaryotes. Its N-terminal HSH domain is similar to AAA proteins, especially Pol III subunit δ and RuvB, in structure. DP2 has a Class II KH domain. Pyrococcus abyssi polD is more heat-stable and more accurate than Taq polymerase, but has not yet been commercialized. It has been proposed that family D DNA polymerase was the first to evolve in cellular organisms and that the replicative polymerase of the Last Universal Cellular Ancestor (LUCA) belonged to family D.
=== Eukaryotic DNA polymerase ===
==== Polymerases β, λ, σ, μ (beta, lambda, sigma, mu) and TdT ====
Family X polymerases contain the well-known eukaryotic polymerase pol β (beta), as well as other eukaryotic polymerases such as Pol σ (sigma), Pol λ (lambda), Pol μ (mu), and Terminal deoxynucleotidyl transferase (TdT). Family X polymerases are found mainly in vertebrates, and a few are found in plants and fungi. These polymerases have highly conserved regions that include two helix-hairpin-helix motifs that are imperative in the DNA-polymerase interactions. One motif is located in the 8 kDa domain that interacts with downstream DNA and one motif is located in the thumb domain that interacts with the primer strand. Pol β, encoded by POLB gene, is required for short-patch base excision repair, a DNA repair pathway that is essential for repairing alkylated or oxidized bases as well as abasic sites. Pol λ and Pol μ, encoded by the POLL and POLM genes respectively, are involved in non-homologous end-joining, a mechanism for rejoining DNA double-strand breaks due to hydrogen peroxide and ionizing radiation, respectively. TdT is expressed only in lymphoid tissue, and adds "n nucleotides" to double-strand breaks formed during V(D)J recombination to promote immunological diversity.
==== Polymerases α, δ and ε (alpha, delta, and epsilon) ====
Pol α (alpha), Pol δ (delta), and Pol ε (epsilon) are members of Family B Polymerases and are the main polymerases involved with nuclear DNA replication. Pol α complex (pol α-DNA primase complex) consists of four subunits: the catalytic subunit POLA1, the regulatory subunit POLA2, and the small and the large primase subunits PRIM1 and PRIM2 respectively. Once primase has created the RNA primer, Pol α starts replication elongating the primer with ~20 nucleotides. Due to its high processivity, Pol δ takes over the leading and lagging strand synthesis from Pol α.: 218–219 Pol δ is expressed by genes POLD1, creating the catalytic subunit, POLD2, POLD3, and POLD4 creating the other subunits that interact with Proliferating Cell Nuclear Antigen (PCNA), which is a DNA clamp that allows Pol δ to possess processivity. Pol ε is encoded by the POLE1, the catalytic subunit, POLE2, and POLE3 gene. It has been reported that the function of Pol ε is to extend the leading strand during replication, while Pol δ primarily replicates the lagging strand; however, recent evidence suggested that Pol δ might have a role in replicating the leading strand of DNA as well. Pol ε's C-terminus "polymerase relic" region, despite being unnecessary for polymerase activity, is thought to be essential to cell vitality. The C-terminus region is thought to provide a checkpoint before entering anaphase, provide stability to the holoenzyme, and add proteins to the holoenzyme necessary for initiation of replication. Pol ε has a larger "palm" domain that provides high processivity independently of PCNA.
Compared to other Family B polymerases, the DEDD exonuclease family responsible for proofreading is inactivated in Pol α. Pol ε is unique in that it has two zinc finger domains and an inactive copy of another family B polymerase in its C-terminal. The presence of this zinc finger has implications in the origins of Eukaryota, which in this case is placed into the Asgard group with archaeal B3 polymerase.
==== Polymerases η, ι and κ (eta, iota, and kappa) ====
Pol η (eta), Pol ι (iota), and Pol κ (kappa), are Family Y DNA polymerases involved in the DNA repair by translation synthesis and encoded by genes POLH, POLI, and POLK respectively. Members of Family Y have five common motifs to aid in binding the substrate and primer terminus and they all include the typical right hand thumb, palm and finger domains with added domains like little finger (LF), polymerase-associated domain (PAD), or wrist. The active site, however, differs between family members due to the different lesions being repaired. Polymerases in Family Y are low-fidelity polymerases, but have been proven to do more good than harm as mutations that affect the polymerase can cause various diseases, such as skin cancer and Xeroderma Pigmentosum Variant (XPS). The importance of these polymerases is evidenced by the fact that gene encoding DNA polymerase η is referred as XPV, because loss of this gene results in the disease Xeroderma Pigmentosum Variant. Pol η is particularly important for allowing accurate translesion synthesis of DNA damage resulting from ultraviolet radiation. The functionality of Pol κ is not completely understood, but researchers have found two probable functions. Pol κ is thought to act as an extender or an inserter of a specific base at certain DNA lesions. All three translesion synthesis polymerases, along with Rev1, are recruited to damaged lesions via stalled replicative DNA polymerases. There are two pathways of damage repair leading researchers to conclude that the chosen pathway depends on which strand contains the damage, the leading or lagging strand.
==== Polymerases Rev1 and ζ (zeta) ====
Pol ζ, another B family polymerase, is made of two subunits: Rev3 – the catalytic subunit; and Rev7 (MAD2L2) – which increases the catalytic function of the polymerase, and is involved in translation synthesis. Pol ζ lacks 3' to 5' exonuclease activity, and is unique in that it can extend primers with terminal mismatches. Rev1 has three regions of interest in the BRCT domain, ubiquitin-binding domain, and C-terminal domain and has dCMP transferase ability, which adds deoxycytidine opposite lesions that would stall replicative polymerases Pol δ and Pol ε. These stalled polymerases activate ubiquitin complexes that, in turn, disassociate replication polymerases and recruit Pol ζ and Rev1. Together, Pol ζ and Rev1 add deoxycytidine, and Pol ζ extends past the lesion. Through a yet undetermined process, Pol ζ disassociates, and replication polymerases reassociate and continue replication. Pol ζ and Rev1 are not required for replication, but loss of REV3 gene in budding yeast can cause increased sensitivity to DNA-damaging agents due to collapse of replication forks where replication polymerases have stalled.
==== Telomerase ====
Telomerase is a ribonucleoprotein which functions to replicate ends of linear chromosomes since normal DNA polymerase cannot replicate the ends, or telomeres. The single-strand 3' overhang of the double-strand chromosome with the sequence 5'-TTAGGG-3' recruits telomerase. Telomerase acts like other DNA polymerases by extending the 3' end, but, unlike other DNA polymerases, telomerase does not require a template. The TERT subunit, an example of a reverse transcriptase, uses the RNA subunit to form the primer–template junction that allows telomerase to extend the 3' end of chromosome ends. The gradual decrease in size of telomeres as the result of many replications over a lifetime are thought to be associated with the effects of aging.: 248–249
==== Polymerases γ, θ and ν (gamma, theta and nu) ====
Pol γ (gamma), Pol θ (theta), and Pol ν (nu) are Family A polymerases. Pol γ, encoded by the POLG gene, was long thought to be the only mitochondrial polymerase. However, recent research shows that at least Pol β (beta), a Family X polymerase, is also present in mitochondria. Any mutation that leads to limited or non-functioning Pol γ has a significant effect on mtDNA and is the most common cause of autosomal inherited mitochondrial disorders. Pol γ contains a C-terminus polymerase domain and an N-terminus 3'–5' exonuclease domain that are connected via the linker region, which binds the accessory subunit. The accessory subunit binds DNA and is required for processivity of Pol γ. Point mutation A467T in the linker region is responsible for more than one-third of all Pol γ-associated mitochondrial disorders. While many homologs of Pol θ, encoded by the POLQ gene, are found in eukaryotes, its function is not clearly understood. The sequence of amino acids in the C-terminus is what classifies Pol θ as Family A polymerase, although the error rate for Pol θ is more closely related to Family Y polymerases. Pol θ extends mismatched primer termini and can bypass abasic sites by adding a nucleotide. It also has Deoxyribophosphodiesterase (dRPase) activity in the polymerase domain and can show ATPase activity in close proximity to ssDNA. Pol ν (nu) is considered to be the least effective of the polymerase enzymes. However, DNA polymerase nu plays an active role in homology repair during cellular responses to crosslinks, fulfilling its role in a complex with helicase.
Plants use two Family A polymerases to copy both the mitochondrial and plastid genomes. They are more similar to bacterial Pol I than they are to mammalian Pol γ.
==== Reverse transcriptase ====
Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp) that synthesizes DNA from a template of RNA. The reverse transcriptase family contain both DNA polymerase functionality and RNase H functionality, which degrades RNA base-paired to DNA. An example of a retrovirus is HIV. Reverse transcriptase is commonly employed in amplification of RNA for research purposes. Using an RNA template, PCR can utilize reverse transcriptase, creating a DNA template. This new DNA template can then be used for typical PCR amplification. The products of such an experiment are thus amplified PCR products from RNA.
Each HIV retrovirus particle contains two RNA genomes, but, after an infection, each virus generates only one provirus. After infection, reverse transcription is accompanied by template switching between the two genome copies (copy choice recombination). From 5 to 14 recombination events per genome occur at each replication cycle. Template switching (recombination) appears to be necessary for maintaining genome integrity and as a repair mechanism for salvaging damaged genomes.
=== Bacteriophage T4 DNA polymerase ===
Bacteriophage (phage) T4 encodes a DNA polymerase that catalyzes DNA synthesis in a 5' to 3' direction. The phage polymerase also has an exonuclease activity that acts in a 3' to 5' direction, and this activity is employed in the proofreading and editing of newly inserted bases. A phage mutant with a temperature sensitive DNA polymerase, when grown at permissive temperatures, was observed to undergo recombination at frequencies that are about two-fold higher than that of wild-type phage.
It was proposed that a mutational alteration in the phage DNA polymerase can stimulate template strand switching (copy choice recombination) during replication.
== See also ==
Biological machines
DNA sequencing
Enzyme catalysis
Genetic recombination
Molecular cloning
Polymerase chain reaction
Protein domain dynamics
Reverse transcription
RNA polymerase
Taq DNA polymerase
== References ==
== Further reading ==
== External links ==
DNA+polymerases at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
PDB Molecule of the Month DNA polymerase
Unusual repair mechanism in DNA polymerase lambda, Ohio State University, July 25, 2006.
A great animation of DNA Polymerase from WEHI at 1:45 minutes in Archived 2014-12-05 at the Wayback Machine
3D macromolecular structures of DNA polymerase from the EM Data Bank(EMDB) | Wikipedia/Prokaryotic_DNA_polymerase |
T7 DNA helicase (gp4) is a hexameric motor protein encoded by T7 phages that uses energy from dTTP hydrolysis to process unidirectionally along single stranded DNA, separating (helicase) the two strands as it progresses. It is also a primase, making short stretches of RNA that initiates DNA synthesis. It forms a complex with T7 DNA polymerase. Its homologs are found in mitochondria (as Twinkle) and chloroplasts.
== Crystal structure ==
The crystal structure was solved to 3.0 Å resolution in 2000, as shown in the figure in the reference. In (A), notice that the separate subunits appear to be anchored through interactions between an alpha helix and an adjacent subunit. In (B), there are six sets of three loops. The red loop, known as loop II, contains three lysine residues and is thought to be involved in binding the ssDNA that is fed through the center of the enzyme.
== Mechanism of sequential dTTP hydrolysis ==
Crampton et al. have proposed a mechanism for the ssDNA-dependent hydrolysis of dTTP by T7 DNA helicase as shown in the figure below. In their model, protein loops located on each hexameric subunit, each of which contain three lysine residues, sequentially interact with the negatively charged phosphate backbone of ssDNA. This interaction presumably causes a conformational change in the actively bound subunit, providing for the efficient release of dTDP from its dTTP binding site. In the process of dTDP release, the ssDNA is transferred to the neighboring subunit, which undergoes a similar process. Previous studies have already suggested that ssDNA is able to bind to two hexameric subunits simultaneously.
== See also ==
Helicase
== References ==
== External links ==
T7+DNA+Primase-Helicase+Protein at the U.S. National Library of Medicine Medical Subject Headings (MeSH) | Wikipedia/T7_DNA_Helicase |
DNA gyrase, or simply gyrase, is an enzyme within the class of topoisomerase and is a subclass of Type II topoisomerases that reduces topological strain in an ATP dependent manner while double-stranded DNA is being unwound by elongating RNA-polymerase or by helicase in front of the progressing replication fork. It is the only known enzyme to actively contribute negative supercoiling to DNA, while it also is capable of relaxing positive supercoils. It does so by looping the template to form a crossing, then cutting one of the double helices and passing the other through it before releasing the break, changing the linking number by two in each enzymatic step. This process occurs in bacteria, whose single circular DNA is cut by DNA gyrase and the two ends are then twisted around each other to form supercoils. Gyrase is also found in eukaryotic plastids: it has been found in the apicoplast of the malarial parasite Plasmodium falciparum and in chloroplasts of several plants. Bacterial DNA gyrase is the target of many antibiotics, including nalidixic acid, novobiocin, albicidin, and ciprofloxacin.
The unique ability of gyrase to introduce negative supercoils into DNA at the expense of ATP hydrolysis is what allows bacterial DNA to have free negative supercoils. The ability of gyrase to relax positive supercoils comes into play during DNA replication and prokaryotic transcription. The helical nature of the DNA causes positive supercoils to accumulate ahead of a translocating enzyme, in the case of DNA replication, a DNA polymerase. The ability of gyrase (and topoisomerase IV) to relax positive supercoils allows superhelical tension ahead of the polymerase to be released so that replication can continue.
== Gyrase structure ==
DNA gyrase is a tetrameric enzyme that consists of 2 GyrA ("A") and 2 GyrB ("B") subunits. Structurally the complex is formed by 3 pairs of "gates", sequential opening and closing of which results into the direct transfer of DNA segment and introduction of 2 negative supercoils. N-gates are formed by ATPase domains of GyrB subunits. Binding of 2 ATP molecules leads to dimerization and, therefore, closing of the gates. Hydrolysis, on the contrary, opens them. DNA cleavage and reunion is performed by a catalytic center located in DNA-gates build by all gyrase subunits. C-gates are formed by GyrA subunits.
== Mechanochemical model of gyrase activity ==
A single molecule study has characterized gyrase activity as a function of DNA tension (applied force) and ATP, and proposed a mechanochemical model. Upon binding to DNA (the "Gyrase-DNA" state), there is a competition between DNA wrapping and dissociation, where increasing DNA tension increases the probability of dissociation. According to the catalytic cycle proposed, binding of 2 ATP molecules causes dimerization of ATPase domains of GyrB subunits and capturing of a T-segment of DNA (T- from transferring) in a cavity between GyrB subunits. On a next step the enzyme cleaves a G-segment of DNA (G- from gate) making a double-strand break. Then the T-segment is transferred through the break, which is accompanied by the hydrolysis of the first ATP molecule. DNA-gyrase ligates the break in a G-segment back and T-segment finally leaves the enzyme complex. Hydrolysis of the second ATP returns the system to the initial step of a cycle.
As the result of a catalytic cycle two ATP molecules are hydrolyzed and two negative supercoils are introduced into the DNA template. The number of superhelical turns introduced into an initially relaxed circular DNA has been calculated to be approximately equal to the number of ATP molecules hydrolyzed by gyrase. Therefore, it can be suggested that two ATP molecules are hydrolyzed per cycle of reaction by gyrase, leading to the introduction of a linking difference of -2.
== Gyrase specificity ==
Gyrase has a pronounced specificity to DNA substrates. Strong gyrase binding sites (SGS) were found in some phages (bacteriophage Mu group) and plasmids (pSC101, pBR322). Recently, high throughput mapping of DNA gyrase sites in the Escherichia coli genome using Topo-Seq approach revealed a long (≈130 bp) and degenerate binding motif that can explain the existence of SGSs. The gyrase motif reflects wrapping of DNA around the enzyme complex and DNA flexibility. It contains two periodic regions in which GC-rich islands are alternated with AT-rich patches by a period close to the period of DNA double helix (≈10.5 bp). The two regions correspond to DNA binding by C-terminal domains of GyrA subunits and resemble eukaryotic nucleosome binding motif.
== Inhibition by antibiotics ==
Gyrase is present in prokaryotes and some eukaryotes, but the enzymes are not entirely similar in structure or sequence, and have different affinities for different molecules. This makes gyrase a good target for antibiotics. Two classes of antibiotics that inhibit gyrase are:
The aminocoumarins (including novobiocin and Coumermycin A1), which work by competitive inhibition of energy transduction of DNA gyrase by binding to the ATPase active site on the GyrB subunit.
The quinolones (including nalidixic acid and ciprofloxacin) are known as topoisomerase poisons. By binding to the enzyme they trap it in a transient step of the catalytic cycle, preventing the reunion of a G-segment. This results in an accumulation of double-strand breaks, stalling of replication forks and cell death. Quinolone-resistant bacteria frequently harbor mutated topoisomerases that resist quinolone binding.
The subunit A is selectively inactivated by antibiotics such as oxolinic and nalidixic acids. The subunit B is selectively inactivated by antibiotics such as coumermycin A1 and novobiocin. Inhibition of either subunit blocks supertwisting activity.
== Phage T4 ==
Phage T4 genes 39, 52 and 60 encode proteins that form a DNA gyrase that is employed in phage DNA replication during infection of the E. coli bacterial host. The phage gene 52 protein shares homology with the bacterial gyrase gyrA subunit and the phage gene 39 protein shares homology with the gyrB subunit. Since the host E. coli DNA gyrase can partially compensate for the loss of the phage gene products, mutants defective in either genes 39, 52 or 60 do not completely abolish phage DNA replication, but rather delay its initiation. Mutants defective in genes 39, 52 or 60 show increased genetic recombination as well as increased base-substitution and deletion mutation suggesting that the host compensated DNA synthesis is less accurate than that directed by wild-type phage. A mutant defective in gene 39 also shows increased sensitivity to inactivation by ultraviolet irradiation during the stage of phage infection after initiation of DNA replication when multiple copies of the phage chromosome are present.
== See also ==
GyrA RNA motif
== References ==
== External links ==
PDBe-KB P0AES4: an overview of all the structure information available in the PDB for Escherichia coli DNA gyrase subunit A
PDBe-KB P0A2I3: an overview of all the structure information available in the PDB for Salmonella enterica DNA gyrase subunit B | Wikipedia/DNA_gyrase |
Tus (terminus utilization substance), also known as a ter-binding protein, is a protein that binds to terminator sequences and acts as a counter-helicase when it comes in contact with an advancing helicase. The bound Tus protein effectively halts DNA polymerase movement. Tus helps end DNA replication in prokaryotes. They function by binding to DNA replication terminator sequences, thus preventing the passage of replication forks. The termination efficiency is affected by the affinity of a particular protein for the terminator sequence.
In E. coli (P16525), Tus binds to 10 closely related sites encoded in the chromosome, although only 6 are likely to be involved in replication termination. Each site is 23 base pairs. The sites are called Ter sites, and are designated TerA, TerB, ..., TerG. These binding sites are asymmetric, such that when a Tus-Ter complex (Tus protein bound to a Ter site) is encountered by a replication fork from one direction, the complex is dissociated and replication continues (permissive). But when encountered from the other direction, the Tus-Ter complex provides a much larger kinetic barrier and halts replication (non-permissive). The multiple Ter sites in the chromosome are oriented such that the two oppositely moving replication forks are both stalled in the desired termination region.
Bacillus subtilis utilize replication terminator protein (RTP) instead of Tus. This is a different protein family using a different structure to bind to DNA: InterPro: IPR003432.
== Structure ==
The Ter protein contains two domains. The N-terminal domain is composed of an alpha helices, beta sheet, and three loops. The C-terminal domain is made of two alpha helices and one beta sheet. Alternatively, CATH divides the structure into a big domain spanning the entire sequence and a small insertion that pops out and folds as a separate domain.
== Function ==
A DNA replication terminus (Ter) has a role in preventing progress of the DNA replication fork. Therefore, a DNA replication terminus site-binding protein binds to this site helping to block the DNA replication fork. There are two genes controlling Ter-binding activity, named tau and Tus.
== Further reading ==
"Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance."
"Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex."
"A molecular mousetrap determines polarity of termination of DNA replication in E. coli."
"Isolation and characterization of mutants of Tus, the replication arrest protein of Escherichia coli."
"Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli."
"Structure of a replication-terminator protein complexed with DNA."
Structure at protein data bank
== References == | Wikipedia/Ter_protein |
Tus (terminus utilization substance), also known as a ter-binding protein, is a protein that binds to terminator sequences and acts as a counter-helicase when it comes in contact with an advancing helicase. The bound Tus protein effectively halts DNA polymerase movement. Tus helps end DNA replication in prokaryotes. They function by binding to DNA replication terminator sequences, thus preventing the passage of replication forks. The termination efficiency is affected by the affinity of a particular protein for the terminator sequence.
In E. coli (P16525), Tus binds to 10 closely related sites encoded in the chromosome, although only 6 are likely to be involved in replication termination. Each site is 23 base pairs. The sites are called Ter sites, and are designated TerA, TerB, ..., TerG. These binding sites are asymmetric, such that when a Tus-Ter complex (Tus protein bound to a Ter site) is encountered by a replication fork from one direction, the complex is dissociated and replication continues (permissive). But when encountered from the other direction, the Tus-Ter complex provides a much larger kinetic barrier and halts replication (non-permissive). The multiple Ter sites in the chromosome are oriented such that the two oppositely moving replication forks are both stalled in the desired termination region.
Bacillus subtilis utilize replication terminator protein (RTP) instead of Tus. This is a different protein family using a different structure to bind to DNA: InterPro: IPR003432.
== Structure ==
The Ter protein contains two domains. The N-terminal domain is composed of an alpha helices, beta sheet, and three loops. The C-terminal domain is made of two alpha helices and one beta sheet. Alternatively, CATH divides the structure into a big domain spanning the entire sequence and a small insertion that pops out and folds as a separate domain.
== Function ==
A DNA replication terminus (Ter) has a role in preventing progress of the DNA replication fork. Therefore, a DNA replication terminus site-binding protein binds to this site helping to block the DNA replication fork. There are two genes controlling Ter-binding activity, named tau and Tus.
== Further reading ==
"Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance."
"Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex."
"A molecular mousetrap determines polarity of termination of DNA replication in E. coli."
"Isolation and characterization of mutants of Tus, the replication arrest protein of Escherichia coli."
"Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli."
"Structure of a replication-terminator protein complexed with DNA."
Structure at protein data bank
== References == | Wikipedia/Tus_protein |
A model lipid bilayer is any bilayer assembled in vitro, as opposed to the bilayer of natural cell membranes or covering various sub-cellular structures like the nucleus. They are used to study the fundamental properties of biological membranes in a simplified and well-controlled environment, and increasingly in bottom-up synthetic biology for the construction of artificial cells. A model bilayer can be made with either synthetic or natural lipids. The simplest model systems contain only a single pure synthetic lipid. More physiologically relevant model bilayers can be made with mixtures of several synthetic or natural lipids.
There are many different types of model bilayers, each having experimental advantages and disadvantages. The first system developed was the black lipid membrane or “painted” bilayer, which allows simple electrical characterization of bilayers but is short-lived and can be difficult to work with. Supported bilayers are anchored to a solid substrate, increasing stability and allowing the use of characterization tools not possible in bulk solution. These advantages come at the cost of unwanted substrate interactions which can denature membrane proteins.
== Black lipid membranes (BLM) ==
The earliest model bilayer system developed was the “painted” bilayer, also known as a “black lipid membrane.” The term “painted” refers to the process by which these bilayers are made. First, a small aperture is created in a thin layer of a hydrophobic material such as Teflon. Typically the diameter of this hole is a few tens of micrometers up to hundreds of micrometers. To form a BLM, the area around the aperture is first "pre-painted" with a solution of lipids dissolved in a hydrophobic solvent by applying this solution across the aperture with a brush, syringe, or glass applicator. The solvent used must have a very high partition coefficient and must be relatively viscous to prevent immediate rupture. The most common solvent used is a mixture of decane and squalene.
After allowing the aperture to dry, salt solution (aqueous phase) is added to both sides of the chamber. The aperture is then "painted" with a lipid solution (generally the same solution that was used for pre-painting). A lipid monolayer spontaneously forms at the interface between the organic and aqueous phases on either side of the lipid/solvent droplet. Because the walls of the aperture are hydrophobic the lipid/solvent solution wets this interface, thinning the droplet in the center. Once the two sides of the droplet come close enough together, the lipid monolayers fuse, rapidly excluding the small remaining volume of solution. At this point a bilayer is formed in the center of the aperture, but a significant annulus of solvent remains at the perimeter. This annulus is required to maintain stability by acting as a bridge between the ~5 nm bilayer and the tens of micrometers thick sheet in which the aperture is made.
The term “black” bilayer refers to the fact that they are dark in reflected light because the thickness of the membrane is only a few nanometers, so light reflecting off the back face destructively interferes with light reflecting off the front face. Indeed, this was one of the first clues that this technique produced a membrane of molecular-scale thickness. Black lipid membranes are also well suited to electrical characterization because the two chambers separated by the bilayer are both accessible, allowing simple placement of large electrodes. For this reason, electrical characterization is one of the most important methods used in conjunction with painted lipid bilayers. Simple measurements indicate when a bilayer forms and when it breaks, as an intact bilayer has a large resistance (>GΩ) and a large capacitance (~2 μF/cm2). More advanced electrical characterization has been particularly important in the study of voltage gated ion channels. Membrane proteins such as ion channels typically cannot be incorporated directly into the painted bilayer during formation because immersion in an organic solvent would denature the protein. Instead, the protein is solubilized with a detergent and added to the aqueous solution after the bilayer is formed. The detergent coating allows these proteins to spontaneously insert into the bilayer over a period of minutes. Additionally, initial experiments have been performed which combine electrophysiological and structural investigations of black lipid membranes. In another variation of the BLM technique, termed the bilayer punch, a glass pipet (inner diameter ~10-40 μm) is used as the electrode on one side of the bilayer in order to isolate a small patch of membrane. This modification of the patch clamp technique enables low noise recording, even at high potentials (up to 600 mV), at the expense of additional preparation time.
The main problems associated with painted bilayers are residual solvent and limited lifetime. Some researchers believe that pockets of solvent trapped between the two bilayer leaflets can disrupt normal protein function. To overcome this limitation, Montal and Mueller developed a modified deposition technique that eliminates the use of a heavy non-volatile solvent. In this method, the aperture starts out above the water surface, completely separating the two fluid chambers. On the surface of each chamber, a monolayer is formed by applying lipids in a volatile solvent such as chloroform and waiting for the solvent to evaporate. The aperture is then lowered through the air-water interface and the two monolayers from the separate chambers are folded down against each other, forming a bilayer across the aperture. The stability issue has proven more difficult to solve. Typically, a black lipid membrane will survive for less than an hour, precluding long-term experiments. This lifetime can be extended by precisely structuring the supporting aperture, chemically crosslinking the lipids or gelling the surrounding solution to mechanically support the bilayer. Work is ongoing in this area and lifetimes of several hours will become feasible.
== Supported lipid bilayers (SLB) ==
Unlike a vesicle or a cell membrane in which the lipid bilayer is rolled into an enclosed shell, a supported bilayer is a planar structure sitting on a solid support. Because of this, only the upper face of the bilayer is exposed to free solution. This layout has advantages and drawbacks related to the study of lipid bilayers. One of the greatest advantages of the supported bilayer is its stability. SLBs will remain largely intact even when subject to high flow rates or vibration and, unlike black lipid membranes, the presence of holes will not destroy the entire bilayer. Because of this stability, experiments lasting weeks and even months are possible with supported bilayers while BLM experiments are usually limited to hours. Another advantage of the supported bilayer is that, because it is on a flat hard surface, it is amenable to a number of characterization tools which would be impossible or would offer lower resolution if performed on a freely floating sample.
One of the clearest examples of this advantage is the use of mechanical probing techniques which require a direct physical interaction with the sample. Atomic force microscopy (AFM) has been used to image lipid phase separation, formation of transmembrane nanopores followed by single protein molecule adsorption, and protein assembly with sub-nm accuracy without the need for a labeling dye. More recently, AFM has also been used to directly probe the mechanical properties of single bilayers and to perform force spectroscopy on individual membrane proteins. These studies would be difficult or impossible without the use of supported bilayers since the surface of a cell or vesicle is relatively soft and would drift and fluctuate over time. Another example of a physical probe is the use of the quartz crystal microbalance (QCM) to study binding kinetics at the bilayer surface. Dual polarisation interferometry is a high resolution optical tool for characterising the order and disruption in lipid bilayers during interactions or phase transitions providing complementary data to QCM measurements.
Many modern fluorescence microscopy techniques also require a rigidly-supported planar surface. Evanescent field methods such as total internal reflection fluorescence microscopy (TIRF) and surface plasmon resonance (SPR) can offer extremely sensitive measurement of analyte binding and bilayer optical properties but can only function when the sample is supported on specialized optically functional materials. Another class of methods applicable only to supported bilayers is those based on optical interference such as fluorescence interference contrast microscopy (FLIC) and reflection interference contrast microscopy (RICM) or interferometric scattering microscopy (iSCAT). When the bilayer is supported on top of a reflective surface, variations in intensity due to destructive interference from this interface can be used to calculate with angstrom accuracy the position of fluorophores within the bilayer. Both evanescent and interference techniques offer sub-wavelength resolution in only one dimension (z, or vertical). In many cases, this resolution is all that is needed. After all, bilayers are very small only in one dimension. Laterally, a bilayer can extend for many micrometres or even millimeters. But certain phenomena like dynamic phase rearrangement do occur in bilayers on a lateral sub-micrometre length scale. A promising approach to studying these structures is near field scanning optical microscopy (NSOM). Like AFM, NSOM relies on the scanning of a micromachined tip to give a highly localized signal. But unlike AFM, NSOM uses an optical rather than physical interaction with the sample, potentially perturbing delicate structures to a lesser extent.
Another important capability of supported bilayers is the ability to pattern the surface to produce multiple isolated regions on the same substrate. This phenomenon was first demonstrated using scratches or metallic “corrals” to prevent mixing between adjacent regions while still allowing free diffusion within any one region. Later work extended this concept by integrating microfluidics to demonstrate that stable composition gradients could be formed in bilayers, potentially allowing massively parallel studies of phase segregation, molecular binding and cellular response to artificial lipid membranes. Creative utilization of the corral concept has also allowed studies of the dynamic reorganization of membrane proteins at the synaptic interface.
One of the primary limitations of supported bilayers is the possibility of unwanted interactions with the substrate. Although supported bilayers generally do not directly touch the substrate surface, they are separated by only a very thin water gap. The size and nature of this gap depends on the substrate material and lipid species but is generally about 1 nm for zwitterionic lipids supported on silica, the most common experimental system. Because this layer is so thin there is extensive hydrodynamic coupling between the bilayer and the substrate, resulting in a lower diffusion coefficient in supported bilayers than for free bilayers of the same composition. A certain percentage of the supported bilayer will also be completely immobile, although the exact nature of and reason for these “pinned” sites is still uncertain. For high quality liquid phase supported bilayers the immobile fraction is typically around 1-5%. To quantify the diffusion coefficient and mobile fraction, researchers studying supported bilayers will often report FRAP data.
Unwanted substrate interactions are a much greater problem when incorporating integral membrane proteins, particularly those with large domains sticking out beyond the core of the bilayer. Because the gap between bilayer and substrate is so thin these proteins will often become denatured on the substrate surface and therefore lose all functionality. One approach to circumvent this problem is the use of polymer tethered bilayers. In these systems the bilayer is supported on a loose network of hydrated polymers or hydrogel which acts as a spacer and theoretically prevents denaturing substrate interactions. In practice, some percentage of the proteins will still lose mobility and functionality, probably due to interactions with the polymer/lipid anchors. Research in this area is ongoing.
== Tethered bilayer lipid membranes (t-BLM) ==
The use of a tethered bilayer lipid membrane (t-BLM) further increases the stability of supported membranes by chemically anchoring the lipids to the solid substrate.Gold can be used as a substrate because of its inert chemistry and thiolipids for covalent binding to the gold. Thiolipids are composed of lipid derivatives, extended at their polar head-groups by hydrophilic spacers which terminate in a thiol or disulphide group that forms a covalent bond with gold, forming self assembled monolayers (SAM).
The limitation of the intra-membrane mobility of supported lipid bilayers can be overcome by introducing half-membrane spanning tether lipids with benzyl disulphide (DPL) and synthetic archaea analogue full membrane spanning lipids with phytanoly chains to stabilize the structure and polyethyleneglycol units as a hydrophilic spacer. Bilayer formation is achieved by exposure of the lipid coated gold substrate to outer layer lipids either in an ethanol solution or in liposomes.
The advantage of this approach is that because of the hydrophilic space of around 4 nm, the interaction with the substrate is minimal and the extra space allows the introduction of protein ion channels into the bilayer. Additionally the spacer layer creates an ionic reservoir that readily enables ac electrical impedance measurement across the bilayer.
== Vesicles ==
A vesicle is a lipid bilayer rolled up into a spherical shell, enclosing a small amount of water and separating it from the water outside the vesicle. Because of this fundamental similarity to the cell membrane, vesicles have been used extensively to study the properties of lipid bilayers. Another reason vesicles have been used so frequently is that they are relatively easy to make. If a sample of dehydrated lipid is exposed to water it will spontaneously form vesicles. These initial vesicles are typically multilamellar (many-walled) and are of a wide range of sizes from tens of nanometers to several micrometres. Methods such as sonication or extrusion through a membrane are needed to break these initial vesicles into smaller, single-walled vesicles of uniform diameter known as small unilamellar vesicles (SUVs). SUVs typically have diameters between 50 and 200 nm. Alternatively, rather than synthesizing vesicles it is possible to simply isolate them from cell cultures or tissue samples. Vesicles are used to transport lipids, proteins and many other molecules within the cell as well as into or out of the cell. These naturally isolated vesicles are composed of a complex mixture of different lipids and proteins so, although they offer greater realism for studying specific biological phenomena, simple artificial vesicles are preferred for studies of fundamental lipid properties.
Since artificial SUVs can be made in large quantities they are suitable for bulk material studies such as x-ray diffraction to determine lattice spacing and differential scanning calorimetry to determine phase transitions. Dual polarisation interferometry can measure unilamelar and multilamelar structures and insertion into and disruption of the vesicles in a label free assay format. Vesicles can also be labeled with fluorescent dyes to allow sensitive FRET-based fusion assays.
In spite of the fluorescent labeling, it is often difficult to perform detailed imaging on SUVs simply because they are so small. To combat this problem, researchers use giant unilamellar vesicles (GUVs). GUVs are large enough (1 - 200 μm) to be studied using traditional fluorescence microscopy and are within the same size range as most biological cells. Thus, they are used as mimicries of cell membranes for in vitro studies in molecular and cell biology. Many of the studies of lipid rafts in artificial lipid systems have been performed with GUVs for this reason. Compared to supported bilayers, GUVs present a more “natural” environment since there is no rigid surface that might induce defects, affect the properties of the membrane or denature proteins. Therefore, GUVs are frequently used to study membrane-remodeling and other protein-membrane interactions in vitro. A variety of methods exist to encapsulate proteins or other biological reactants within such vesicles, making GUVs an ideal system for the in vitro recreation (and investigation) of cell functions in cell-like model membrane environments. These methods include microfluidic methods, which allow for a high-yield production of vesicles with consistent sizes.
== Droplet Interface Bilayers ==
Droplet Interface Bilayers (DIBs) are phospholipid-encased droplets that form bilayers when they are put into contact. The droplets are surrounded by oil and phospholipids are dispersed in either the water or oil. As a result, the phospholipids spontaneously form a monolayer at each of the oil-water interfaces. DIBs can be formed to create tissue-like material with the ability to form asymmetric bilayers, reconstitute proteins and protein channels or made for use in studying electrophysiology. Extended DIB networks can be formed either by employing droplet microfluidic devices or using droplet printers.
== Micelles, bicelles and nanodiscs ==
Detergent micelles are another class of model membranes that are commonly used to purify and study membrane proteins, although they lack a lipid bilayer. In aqueous solutions, micelles are assemblies of amphipathic molecules with their hydrophilic heads exposed to solvent and their hydrophobic tails in the center. Micelles can solubilize membrane proteins by partially encapsulating them and shielding their hydrophobic surfaces from solvent.
Bicelles are a related class of model membrane, typically made of two lipids, one of which forms a lipid bilayer while the other forms an amphipathic, micelle-like assembly shielding the bilayer center from surrounding solvent molecules. Bicelles can be thought of as a segment of bilayer encapsulated and solubilized by a micelle. Bicelles are much smaller than liposomes, and so can be used in experiments such as NMR spectroscopy where the larger vesicles are not an option.
Nanodiscs consist of a segment of bilayer encapsulated by an amphipathic protein coat, rather than a lipid or detergent layer. Nanodiscs are more stable than bicelles and micelles at low concentrations, and are very well-defined in size (depending on the type of protein coat, between 10 and 20 nm). Membrane proteins incorporated into and solubilized by Nanodiscs can be studied by a wide variety of biophysical techniques.
== References == | Wikipedia/Model_lipid_bilayer |
DNA transposons are DNA sequences, sometimes referred to "jumping genes", that can move and integrate to different locations within the genome. They are class II transposable elements (TEs) that move through a DNA intermediate, as opposed to class I TEs, retrotransposons, that move through an RNA intermediate. DNA transposons can move in the DNA of an organism via a single-or double-stranded DNA intermediate. DNA transposons have been found in both prokaryotic and eukaryotic organisms. They can make up a significant portion of an organism's genome, particularly in eukaryotes. In prokaryotes, TE's can facilitate the horizontal transfer of antibiotic resistance or other genes associated with virulence. After replicating and propagating in a host, all transposon copies become inactivated and are lost unless the transposon passes to a genome by starting a new life cycle with horizontal transfer. DNA transposons do not randomly insert themselves into the genome, but rather show preference for specific sites.
With regard to movement, DNA transposons can be categorized as autonomous and nonautonomous. Autonomous ones can move on their own, while nonautonomous ones require the presence of another transposable element's gene, transposase, to move. There are three main classifications for movement for DNA transposons: "cut and paste," "rolling circle" (Helitrons), and "self-synthesizing" (Polintons). These distinct mechanisms of movement allow them to move around the genome of an organism. Since DNA transposons cannot synthesize DNA, they replicate using the host replication machinery. These three main classes are then further broken down into 23 different superfamilies characterized by their structure, sequence, and mechanism of action.
DNA transposons are a cause of gene expression alterations. As newly inserted DNA into active coding sequences, they can disrupt normal protein functions and cause mutations. Class II TEs make up about 3% of the human genome. Today, there are no active DNA transposons in the human genome. Therefore, the elements found in the human genome are called fossils.
== Mechanisms of action ==
=== Cut and paste ===
Traditionally, DNA transposons move around in the genome by a cut and paste method. The system requires a transposase enzyme that catalyzes the movement of the DNA from its current location in the genome and inserts it in a new location. Transposition requires three DNA sites on the transposon: two at each end of the transposon called terminal inverted repeats and one at the target site. The transposase will bind to the terminal inverted repeats of the transposon and mediate synapsis of the transposon ends. The transposase enzyme then disconnects the element from the flanking DNA of the original donor site and mediates the joining reaction that links the transposon to the new insertion site. The addition of the new DNA into the target site causes short gaps on either side of the inserted segment. Host systems repair these gaps resulting in the target sequence duplication (TSD) that are characteristic of transposition. In many reactions, the transposon is completely excised from the donor site in what is called a "cut and paste" transposition and inserted into the target DNA to form a simple insertion. Occasionally, genetic material not originally in the transposable element gets copied and moved as well.
=== Helitrons ===
Helitrons are also a group of eukaryotic class II TEs. Helitrons do not follow the classical "cut and paste" mechanism. Instead, they are hypothesized to move around the genome via a rolling circle like mechanism. This process involves making a nick to a circular strand by an enzyme, which separates the DNA into two single strands. The initiation protein then remains attached to the 5' Phosphate on the nicked strand, exposing the 3' hydroxyl of the complementary strand. This allows a polymerase enzyme to begin replication on the un-nicked strand. Eventually the entire strand is replicated at which point the newly synthesized DNA disassociates and is replicated in parallel with the original template strand. Helitrons encode an unknown protein which is thought to have HUH endonuclease function as well as 5' to 3' helicase activity. This enzyme would make a single stranded cut in the DNA which explains the lack of Target Site Duplications found in Helitrons. Helitrons were also the first class of transposable elements to be discovered computationally and marked a paradigm shift in the way that whole genomes were studied.
=== Polintons ===
Polintons are also a group of eukaryotic class II TEs. As one of the most complex known DNA transposons in eukaryotes, they make up the genomes of protists, fungi, and animals, such as the entamoeba, soybean rust, and chicken, respectively. They contain genes with homology to viral proteins and which are often found in eukaryotic genomes, like polymerase and retroviral integrase. However, there is no known protein functionally similarly to the viral capsid or envelope proteins. They share their many structural characteristics with linear plasmids, bacteriophages and adenoviruses, which replicate using protein-primed DNA polymerases. Polintons have been proposed to go through a similar self-synthesis by their polymerase. Polintons, 15–20 kb long, encode up to 10 individual proteins. For replication, they utilize a protein-primed DNA polymerase B, retroviral integrase, cysteine protease, and ATPase. First, during host genome replication, a single-stranded extra-chromosomal Polinton element is excised from the host DNA using the integrase, forming a racket-like structure. Second, the Polinton undergoes replication using the DNA polymerase B, with initiation started by a terminal protein, which may encoded in some linear plasmids. Once the double stranded Polinton is generated, the integrase serves to insert it into the host genome. Polintons exhibit high variability between difference species and may tightly regulated, resulting in a low frequency rate in many genomes.
== Classification ==
As of the most recent update in 2023, 31 superfamilies of DNA transposons were recognized and annotated in
Repbase, a database of repetitive DNA elements maintained by the Genetic Information Research Institute:
== Effects of transposons ==
DNA transposons, like all transposons, are quite impactful with respect to gene expression. A sequence of DNA may insert itself into a previously functional gene and create a mutation. This can happen in three distinct ways: 1. alteration of function, 2. chromosomal rearrangement, and 3. a source of novel genetic material. Since DNA transposons may happen to take parts of genomic sequences with them, exon shuffling may occur. Exon shuffling is the creation of novel gene products due to the new placement of two previously unrelated exons through transposition. Because of their ability to alter DNA expression, transposons have become an important target of research in genetic engineering.
== Examples ==
=== Maize ===
Barbara McClintock first discovered and described DNA transposons in Zea mays, during the 1940s; this is an achievement that would earn her the Nobel Prize in 1983. She described the Ac/Ds system where the Ac unit (activator) was autonomous but the Ds genomic unit required the presence of the activator in order to move. This TE is one of the most visually obvious as it was able to cause the maize to change color from yellow to brown/spotted on individual kernels.
=== Fruit flies ===
The Mariner/Tc1 transposon, found in many animals but studied in Drosophila was first described by Jacobson and Hartl. Mariner is well known for being able to excise and insert horizontally in to a new organism. Thousands of copies of the TE have been found interspersed in the human genome as well as other animals.
The Hobo transposons in Drosophila have been extensively studied due to their ability to cause gonadal dysgenesis. The insertion and subsequent expression of hobo-like sequences results in the loss of germ cells in the gonads of developing flies.
=== Bacteria ===
Bacterial transposons are especially good at facilitating horizontal gene transfer between microbes. Transposition facilitates the transfer and accumulation of antibiotic resistance genes. In bacteria, transposable elements can easily jump between the chromosomal genome and plasmids. In a 1982 study by Devaud et al., a multi-drug resistant strain of Acinetobacter was isolated and examined. Evidence pointed to the transfer of a plasmid in to the bacterium, where the resistance genes were transposed in to the chromosomal genome.
=== Genetic diversity ===
Transposons may have an effect on the promotion of genetic diversity of many organisms. DNA transposons can drive the evolution of genomes by promoting the relocation of sections of DNA sequences. As a result, this can alter gene regulatory regions and phenotypes. The discovery of transposons was made by Barbara McClintock who noticed that these elements could actually change the color of the maize plants she was studying, providing quick evidence of one outcome from transposon movement. Another example is the Tol2 DNA transposon in medaka fish that is said to be the result of their variety in pigmentation patterns. These examples show that transposons can greatly influence the process of evolution by rapidly inducing changes in the genome.
== Inactivation ==
All DNA transposons are inactive in the human genome. Inactivated, or silenced, transposons do not result in a phenotypic outcome and do not move around in the genome. Some are inactive because they have mutations that affect their ability to move between chromosomes, while others are capable of moving but remain inactive due to epigenetic defenses, like DNA methylation and chromatin remodeling. For example, chemical modifications of DNA can constrict certain areas of the genome such that transcription enzymes are unable to reach them. RNAi, specifically siRNA and miRNA silencing, is a naturally occurring mechanisms that, in addition to regulating eukaryotic gene expression, prevents transcription of DNA transposons. Another mode of inactivation is overproduction inhibition. When transposase exceeds a threshold concentration, transposon activity is decreased. Since transposase can form inactive or less active monomers that will decrease transposition activity overall, a decrease in the production of transposase will also occur when large copies of those less active elements increase in the host genome.
=== Horizontal transfer ===
Horizontal transfer refers to the movement of DNA information between cells of different organisms. Horizontal transfer can involve the movement of TEs from one organism into the genome of another. The insertion itself allows the TE to become an activated gene in the new host. Horizontal transfer is used by DNA transposons to prevent inactivation and complete loss of the transposon. This inactivation is termed vertical inactivation, meaning that the DNA transposon is inactive and remains as a fossil. This type of transfer is not the most common, but has been seen in the case of the wheat virulence protein ToxA, which was transferred between the different fungal pathogens Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana. Other examples include transfer between marine crustaceans, insects of different orders, and organisms of different phyla, such as humans and nematodes.
== Evolution ==
Eukaryotic genomes differ in TE content. Recently, a study of the different superfamilies of TEs reveals that there are striking similarities between the groups. It has been hypothesized that many of them are represented in two or more Eukaryotic supergroups. This means that divergence of the transposon superfamilies could even predate the divergence of Eukaryotic supergroups.
=== V(D)J recombination ===
V(D)J recombination, although not a DNA TE, is remarkably similar to transposons. V(D)J recombination is the process by which the large variation in antibody binding sites is created. In this mechanism, DNA is recombined in order to create genetic diversity. Because of this, it has been hypothesized that these proteins, particularly Rag1 and Rag2 are derived from transposable elements.
=== Extinction in the human genome ===
There is evidence suggesting that at least 40 human DNA transposon families were active during mammalian radiation and early primate lineage. Then, there was a pause in transpositional activity during the later portion of primate radiation, with a complete halt in transposon movement in an anthropoid primate ancestor. There is no evidence of any transposable element younger than about 37 million years.
== References ==
== External links ==
Dfam, a database of repeating DNA sequences
Repbase, a database and classification system for repeating DNA sequences
DNA transposon derived genes, in HGNC database | Wikipedia/DNA_transposon |
DnaB helicase is an enzyme in bacteria which opens the replication fork during DNA replication. Although the mechanism by which DnaB both couples ATP hydrolysis to translocation along DNA and denatures the duplex is unknown, a change in the quaternary structure of the protein involving dimerisation of the N-terminal domain has been observed and may occur during the enzymatic cycle. Initially when DnaB binds to dnaA, it is associated with dnaC, a negative regulator. After DnaC dissociates, DnaB binds dnaG.
The N-terminal has a multi-helical structure that forms an orthogonal bundle. The C-terminal domain contains an ATP-binding site and is therefore probably the site of ATP hydrolysis.
In eukaryotes, helicase function is provided by the MCM (Minichromosome maintenance) complex.
The DnaB helicase is the product of the dnaB gene. DnaB is expressed as a monomer and oligomerises into hexamer through N-terminal interactions. Replicative helicases have a central ring and that feature is conserved across bacterial to eukaryotes.
The energy for DnaB activity is provided by NTP hydrolysis. Mechanical energy moves the DnaB into the replication fork, physically splitting it in half.
== E. coli dnaB ==
In E. coli, dnaB is a hexameric protein of six 471-residue subunits, which form a ring-shaped structure with threefold symmetry. During DNA replication, the lagging strand of DNA binds in the central channel of dnaB, and the second DNA strand is excluded. The binding of NTPs causes a conformational change and subsequent hydrolysis allows the dnaB to translocate along the DNA, thus mechanically forcing the separation of the DNA strands.
== Mechanism of initiation of replication ==
At least 10 different enzymes or proteins participate in the initiation phase of replication. They open the DNA helix at the origin and establish a prepriming complex for subsequent reactions. The crucial component in the initiation process is the DnaA protein, a member of the AAA+ ATPase protein family (ATPases associated with diverse cellular activities). Many AAA+ ATPases, including DnaA, form oligomers and hydrolyze ATP relatively slowly. This ATP hydrolysis acts as a switch mediating interconversion of the protein between two states. In the case of DnaA, the ATP-bound form is active and the ADP-bound form is inactive.
Eight DnaA protein molecules, all in the ATP-bound state, assemble to form a helical complex encompassing the R and I sites in oriC. DnaA has a higher affinity for the R sites than I sites, and binds R sites equally well in its ATP or ADP-bound form. The I sites, which bind only the ATP-bound DnaA, allow discrimination between the active and inactive forms of DnaA. The tight right-handed wrapping of the DNA around this complex introduces an effective positive supercoil. The associated strain in the nearby DNA leads to denaturation in the A:T-rich 'DUE' (DNA Unwinding Element) region. The complex formed at the replication origin also includes several DNA-binding proteins- Hu, IHF and FIS that facilitate DNA bending.
The DnaC protein, another AAA+ ATPase, then loads the DnaB protein onto the separated DNA strands in the denatured region. A hexamer of DnaC, each subunit bound to ATP, forms a tight complex with the hexameric, ring-shaped DnaB helicase. This DnaC-DnaB interaction opens the DnaB ring, the process being aided by a further interaction between DnaB and DnaA. Two of the ring-shaped DnaB hexamers are loaded in the DUE, one onto each DNA strand. The ATP bound to DnaC is hydrolyzed, releasing the DnaC and leaving the DnaB bound to the DNA.
Loading of the DnaB helicase is the key step in replication initiation. As a replicative helicase, DnaB migrates along the single-stranded DNA in the 5'→3' direction, unwinding the DNA as it travels. The DnaB helicases loaded onto the two DNA strands thus travel in opposite directions, creating two potential replication forks. All other proteins at the replication fork are linked directly or indirectly to DnaB.
== References ==
== External links ==
DnaB+Helicases at the U.S. National Library of Medicine Medical Subject Headings (MeSH) | Wikipedia/DnaB_helicase |
Helicases are a class of enzymes that are vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic double helix, separating the two hybridized nucleic acid strands (hence helic- + -ase), via the energy gained from ATP hydrolysis. There are many helicases, representing the great variety of processes in which strand separation must be catalyzed. Approximately 1% of eukaryotic genes code for helicases.
The human genome codes for 95 non-redundant helicases: 64 RNA helicases and 31 DNA helicases. Many cellular processes, such as DNA replication, transcription, translation, recombination, DNA repair and ribosome biogenesis involve the separation of nucleic acid strands that necessitates the use of helicases. Some specialized helicases are also involved in sensing viral nucleic acids during infection and fulfill an immunological function. Genetic mutations that affect helicases can have wide-reaching impacts for an organism, due to their significance in many biological processes.
== Function ==
Helicases are often used to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases. They also function to remove nucleic acid-associated proteins and catalyze homologous DNA recombination. Metabolic processes of RNA such as translation, transcription, ribosome biogenesis, RNA splicing, RNA transport, RNA editing, and RNA degradation are all facilitated by helicases. Helicases move incrementally along one nucleic acid strand of the duplex with a directionality and processivity specific to each particular enzyme.
Helicases adopt different structures and oligomerization states. Whereas DnaB-like helicases unwind DNA as ring-shaped hexamers, other enzymes have been shown to be active as monomers or dimers. Studies have shown that helicases may act passively, waiting for uncatalyzed unwinding to take place and then translocating between displaced strands, or can play an active role in catalyzing strand separation using the energy generated in ATP hydrolysis. In the latter case, the helicase acts comparably to an active motor, unwinding and translocating along its substrate as a direct result of its ATPase activity. Helicases may process much faster in vivo than in vitro due to the presence of accessory proteins that aid in the destabilization of the fork junction.
=== Activation barrier in helicase activity ===
Enzymatic helicase action, such as unwinding nucleic acids, is achieved through the lowering of the activation barrier (
B
{\displaystyle B}
) of each specific action. The activation barrier is a result of various factors, and can be defined by
B
=
N
(
Δ
G
bp
−
G
int
−
G
f
)
{\displaystyle B=N(\Delta G_{\text{bp}}-G_{\text{int}}-G_{\text{f}})}
where
N
{\displaystyle N}
= number of unwound base pairs (bps),
Δ
G
bp
{\displaystyle \Delta G_{\text{bp}}}
= free energy of base pair formation,
G
int
{\displaystyle G_{\text{int}}}
= reduction of free energy due to helicase, and
G
f
{\displaystyle G_{\text{f}}}
= reduction of free energy due to unzipping forces.
Factors that contribute to the height of the activation barrier include: specific nucleic acid sequence of the molecule involved, the number of base pairs involved, tension present on the replication fork, and destabilization forces.
=== Active and passive helicases ===
The size of the activation barrier to overcome by the helicase contributes to its classification as an active or passive helicase. In passive helicases, a significant activation barrier exists (defined as
B
>
k
B
T
{\displaystyle B>k_{\text{B}}T}
, where
k
B
{\displaystyle k_{\text{B}}}
is the Boltzmann constant and
T
{\displaystyle T}
is temperature of the system). Due to this significant activation barrier, its unwinding progression is affected largely by the sequence of nucleic acids within the molecule to unwind, and the presence of destabilization forces acting on the replication fork. Certain nucleic acid combinations will decrease unwinding rates (i.e. guanine and cytosine), while various destabilizing forces can increase the unwinding rate. In passive systems, the rate of unwinding (
V
u
n
{\displaystyle V_{un}}
) is less than the rate of translocation (
V
t
r
a
n
s
{\displaystyle V_{trans}}
) (translocation along the single-strand nucleic acid, ssNA), due to its reliance on the transient unraveling of the base pairs at the replication fork to determine its rate of unwinding.
In active helicases,
B
<
k
B
T
{\displaystyle B<k_{\text{B}}T}
, where the system lacks a significant barrier, as the helicase can destabilize the nucleic acids, unwinding the double-helix at a constant rate, regardless of the nucleic acid sequence. In active helicases,
V
un
{\displaystyle V_{\text{un}}}
is closer to
V
trans
{\displaystyle V_{\text{trans}}}
, due to the active helicase ability to directly destabilize the replication fork to promote unwinding.
Active helicases show similar behaviour when acting on both double-strand nucleic acids, dsNA, or ssNA, in regards to the rates of unwinding and rates of translocation, where in both systems
V
un
{\displaystyle V_{\text{un}}}
and
V
trans
{\displaystyle V_{\text{trans}}}
are approximately equal.
These two categories of helicases may also be modeled as mechanisms. In such models, the passive helicases are conceptualized as Brownian ratchets, driven by thermal fluctuations and subsequent anisotropic gradients across the DNA lattice. The active helicases, in contrast, are conceptualized as stepping motors – also known as powerstroke motors – utilizing either a conformational "inch worm" or a hand-over-hand "walking" mechanism to progress. Depending upon the organism, such helix-traversing progress can occur at rotational speeds in the range of 5,000 to 10,000 R.P.M.
== History of DNA helicases ==
DNA helicases were discovered in E. coli in 1976. This helicase was described as a "DNA unwinding enzyme" that is "found to denature DNA duplexes in an ATP-dependent reaction, without detectably degrading". The first eukaryotic DNA helicase discovered was in 1978 in the lily plant. Since then, DNA helicases were discovered and isolated in other bacteria, viruses, yeast, flies, and higher eukaryotes. To date, at least 14 different helicases have been isolated from single celled organisms, 6 helicases from bacteriophages, 12 from viruses, 15 from yeast, 8 from plants, 11 from calf thymus, and approximately 25 helicases from human cells. Below is a history of helicase discovery:
1976 – Discovery and isolation of E. coli-based DNA helicase
1978 – Discovery of the first eukaryotic DNA helicases, isolated from the lily plant
1982 – "T4 gene 41 protein" is the first reported bacteriophage DNA helicase
1985 – First mammalian DNA helicases isolated from calf thymus
1986 – SV40 large tumor antigen reported as a viral helicase (1st reported viral protein that was determined to serve as a DNA helicase)
1986 – ATPaseIII, a yeast protein, determined to be a DNA helicase
1988 – Discovery of seven conserved amino acid domains determined to be helicase motifs
1989 – Designation of DNA helicase Superfamily I and Superfamily II
1989 – Identification of the DEAD box helicase family
1990 – Isolation of a human DNA helicase
1992 – Isolation of the first reported mitochondrial DNA helicase (from bovine brain)
1996 – Report of the discovery of the first purified chloroplast DNA helicase from the pea
2002 – Isolation and characterization of the first biochemically active malarial parasite DNA helicase – Plasmodium cynomolgi.
== Structural features ==
The common function of helicases accounts for the fact that they display a certain degree of amino acid sequence homology; they all possess sequence motifs located in the interior of their primary structure, involved in ATP binding, ATP hydrolysis and translocation along the nucleic acid substrate. The variable portion of the amino acid sequence is related to the specific features of each helicase.
The presence of these helicase motifs allows putative helicase activity to be attributed to a given protein, but does not necessarily confirm it as an active helicase. Conserved motifs do, however, support an evolutionary homology among enzymes. Based on these helicase motifs, a number of helicase superfamilies have been distinguished.
== Superfamilies ==
Helicases are classified in 6 groups (superfamilies) based on their shared sequence motifs. Helicases not forming a ring structure are in superfamilies 1 and 2, and ring-forming helicases form part of superfamilies 3 to 6. Helicases are also classified as α or β depending on if they work with single or double-strand DNA; α helicases work with single-strand DNA and β helicases work with double-strand DNA. They are also classified by translocation polarity. If translocation occurs 3’-5’ the helicase is type A; if translocation occurs 5’-3’ it is type B.
Superfamily 1 (SF1): This superfamily can be further subdivided into SF1A and SF1B helicases. In this group helicases can have either 3’-5’ (SF1A subfamily) or 5’-3’(SF1B subfamily) translocation polarity. The most known SF1A helicases are Rep and UvrD in gram-negative bacteria and PcrA helicase from gram-positive bacteria. The most known Helicases in the SF1B group are RecD and Dda helicases. They have a RecA-like-fold core.
Superfamily 2 (SF2): This is the largest group of helicases that are involved in varied cellular processes. They are characterized by the presence of nine conserved motifs: Q, I, Ia, Ib, and II through VI. This group is mainly composed of DEAD-box RNA helicases. Some other helicases included in SF2 are the RecQ-like family and the Snf2-like enzymes. Most of the SF2 helicases are type A with a few exceptions such as the XPD family. They have a RecA-like-fold core.
Superfamily 3 (SF3): Superfamily 3 consists of AAA+ helicases encoded mainly by small DNA viruses and some large nucleocytoplasmic DNA viruses. They have a 3’-5’ translocation directionality, meaning that they are all type A helicases. The most known SF3 helicase is the papilloma virus E1 helicase.
Superfamily 4 (SF4): All SF4 family helicases have a type B polarity (5’-3’). They have a RecA fold. The most studied SF4 helicase is gp4 from bacteriophage T7.
Superfamily 5 (SF5): Rho proteins conform the SF5 group. They have a RecA fold.
Superfamily 6 (SF6): They contain the core AAA+ that is not included in the SF3 classification. Some proteins in the SF6 group are: mini chromosome maintenance MCM, RuvB, RuvA, and RuvC.
All helicases are members of a P-loop, or Walker motif-containing family.
== Helicase disorders and diseases ==
=== ATRX helicase mutations ===
The ATRX gene encodes the ATP-dependent helicase, ATRX (also known as XH2 and XNP) of the SNF2 subgroup family, that is thought to be responsible for functions such as chromatin remodeling, gene regulation, and DNA methylation. These functions assist in prevention of apoptosis, resulting in cortical size regulation, as well as a contribution to the survival of hippocampal and cortical structures, affecting memory and learning. This helicase is located on the X chromosome (Xq13.1-q21.1), in the pericentromeric heterochromatin and binds to heterochromatin protein 1. Studies have shown that ATRX plays a role in rDNA methylation and is essential for embryonic development. Mutations have been found throughout the ATRX protein, with over 90% of them being located in the zinc finger and helicase domains. Mutations of ATRX can result in X-linked-alpha-thalassaemia-mental retardation (ATR-X syndrome).
Various types of mutations found in ATRX have been found to be associated with ATR-X, including most commonly single-base missense mutations, as well as nonsense, frameshift, and deletion mutations. Characteristics of ATR-X include: microcephaly, skeletal and facial abnormalities, mental retardation, genital abnormalities, seizures, limited language use and ability, and alpha-thalassemia. The phenotype seen in ATR-X suggests that the mutation of ATRX gene causes the downregulation of gene expression, such as the alpha-globin genes. It is still unknown what causes the expression of the various characteristics of ATR-X in different patients.
=== XPD helicase point mutations ===
XPD (Xeroderma pigmentosum factor D, also known as protein ERCC2) is a 5'-3', Superfamily II, ATP-dependent helicase containing iron-sulphur cluster domains. Inherited point mutations in XPD helicase have been shown to be associated with accelerated aging disorders such as Cockayne syndrome (CS) and trichothiodystrophy (TTD). Cockayne syndrome and trichothiodystrophy are both developmental disorders involving sensitivity to UV light and premature aging, and Cockayne syndrome exhibits severe mental retardation from the time of birth. The XPD helicase mutation has also been implicated in xeroderma pigmentosum (XP), a disorder characterized by sensitivity to UV light and resulting in a several 1000-fold increase in the development of skin cancer.
XPD is an essential component of the TFIIH complex, a transcription and repair factor in the cell. As part of this complex, it facilitates nucleotide excision repair by unwinding DNA. TFIIH assists in repairing damaged DNA such as sun damage. A mutation in the XPD helicase that helps form this complex and contributes to its function causes the sensitivity to sunlight seen in all three diseases, as well as the increased risk of cancer seen in XP and premature aging seen in trichothiodystrophy and Cockayne syndrome.
XPD helicase mutations leading to trichothiodystrophy are found throughout the protein in various locations involved in protein-protein interactions. This mutation results in an unstable protein due to its inability to form stabilizing interactions with other proteins at the points of mutations. This, in turn, destabilizes the entire TFIIH complex, which leads to defects with transcription and repair mechanisms of the cell.
It has been suggested that XPD helicase mutations leading to Cockayne syndrome could be the result of mutations within XPD, causing rigidity of the protein and subsequent inability to switch from repair functions to transcription functions due to a "locking" in repair mode. This could cause the helicase to cut DNA segments meant for transcription. Although current evidence points to a defect in the XPD helicase resulting in a loss of flexibility in the protein in cases of Cockayne syndrome, it is still unclear how this protein structure leads to the symptoms described in Cockayne syndrome.
In xeroderma pigmentosa, the XPD helicase mutation exists at the site of ATP or DNA binding. This results in a structurally functional helicase able to facilitate transcription, however it inhibits its function in unwinding DNA and DNA repair. The lack of a cell's ability to repair mutations, such as those caused by sun damage, is the cause of the high cancer rate in xeroderma pigmentosa patients.
=== RecQ family mutations ===
RecQ helicases (3'-5') belong to the Superfamily II group of helicases, which help to maintain stability of the genome and suppress inappropriate recombination. Deficiencies and/or mutations in RecQ family helicases display aberrant genetic recombination and/or DNA replication, which leads to chromosomal instability and an overall decreased ability to proliferate. Mutations in RecQ family helicases BLM, RECQL4, and WRN, which play a role in regulating homologous recombination, have been shown to result in the autosomal recessive diseases Bloom syndrome (BS), Rothmund–Thomson syndrome (RTS), and Werner syndrome (WS), respectively.
Bloom syndrome is characterized by a predisposition to cancer with early onset, with a mean age-of-onset of 24 years. Cells of Bloom syndrome patients show a high frequency of reciprocal exchange between sister chromatids (SCEs) and excessive chromosomal damage. There is evidence to suggest that BLM plays a role in rescuing disrupted DNA replication at replication forks.
Werner syndrome is a disorder of premature aging, with symptoms including early onset of atherosclerosis and osteoporosis and other age related diseases, a high occurrence of sarcoma, and death often occurring from myocardial infarction or cancer in the 4th to 6th decade of life. Cells of Werner syndrome patients exhibit a reduced reproductive lifespan with chromosomal breaks and translocations, as well as large deletions of chromosomal components, causing genomic instability.
Rothmund-Thomson syndrome, also known as poikiloderma congenitale, is characterized by premature aging, skin and skeletal abnormalities, rash, poikiloderma, juvenile cataracts, and a predisposition to cancers such as osteosarcomas. Chromosomal rearrangements causing genomic instability are found in the cells of Rothmund-Thomson syndrome patients. RecQ is a family of DNA helicase enzymes that are found in various organisms including bacteria, archaea, and eukaryotes (like humans). These enzymes play important roles in DNA metabolism during DNA replication, recombination, and repair. There are five known RecQ helicase proteins in humans: RecQ1, BLM, WRN, RecQ4, and RecQ5. Mutations in some of these genes are associated with genetic disorders. For instance, mutations in the BLM gene cause Bloom syndrome, which is characterized by increased cancer risk and other health issues. Mutations in the WRN gene lead to Werner syndrome, a condition characterized by premature aging and an increased risk of age-related diseases. RecQ helicases are crucial for maintaining genomic stability and integrity. They help prevent the accumulation of genetic abnormalities that can lead to diseases like cancer. Genome integrity depends on the RecQ DNA helicase family, which includes DNA repair, recombination, replication, and transcription processes. Genome instability and early aging are conditions that arise from mutations in human RecQ helicases. RecQ helicase Sgs1 is missing in yeast cells, making them useful models for comprehending human cell abnormalities and the RecQ helicase function. The RecQ helicase family member, RECQ1, is connected to a small number of uncommon genetic cancer disorders in individuals. It participates in transcription, the cell cycle, and DNA repair. According to recent research, missense mutations in the RECQ1 gene may play a role in the development of familial breast cancer. DNA helicases are frequently attracted to regions of DNA damage and are essential for cellular DNA replication, recombination, repair, and transcription. Chemical manipulation of their molecular processes can change the rate at which cancer cells divide, as well as, the efficiency of transactions and cellular homeostasis. Small-molecule-induced entrapment of DNA helicases, a type of DNA metabolic protein, may have deleterious consequences on rapidly proliferating cancer cells, which could be effective in cancer treatment.
During meiosis DNA double-strand breaks and other DNA damages in a chromatid are repaired by homologous recombination using either the sister chromatid or a homologous non-sister chromatid as template. This repair can result in a crossover (CO) or, more frequently, a non-crossover (NCO) recombinant. In the yeast Schizosaccharomyces pombe the FANCM-family DNA helicase FmI1 directs NCO recombination formation during meiosis. The RecQ-type helicase Rqh1 also directs NCO meiotic recombination. These helicases, through their ability to unwind D-loop intermediates, promote NCO recombination by the process of synthesis-dependent strand annealing.
In the plant Arabidopsis thaliana, FANCM helicase promotes NCO and antagonizes the formation of CO recombinants. Another helicase, RECQ4A/B, also independently reduces COs. It was suggested that COs are restricted because of the long term costs of CO recombination, that is, the breaking up of favourable genetic combinations of alleles built up by past natural selection.
== RNA helicases ==
RNA helicases are essential for most processes of RNA metabolism such as ribosome biogenesis, pre-mRNA splicing, and translation initiation. They also play an important role in sensing viral RNAs. RNA helicases are involved in the mediation of antiviral immune response because they can identify foreign RNAs in vertebrates. About 80% of all viruses are RNA viruses and they contain their own RNA helicases. Defective RNA helicases have been linked to cancers, infectious diseases and neuro-degenerative disorders. Some neurological disorders associated with defective RNA helicases are: amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebellar ataxia type-2, Alzheimer disease, and lethal congenital contracture syndrome.
RNA helicases and DNA helicases can be found together in all the helicase superfamilies except for SF6. All the eukaryotic RNA helicases that have been identified up to date are non-ring forming and are part of SF1 and SF2. On the other hand, ring-forming RNA helicases have been found in bacteria and viruses. However, not all RNA helicases exhibit helicase activity as defined by enzymatic function, i.e., proteins of the Swi/Snf family. Although these proteins carry the typical helicase motifs, hydrolize ATP in a nucleic acid-dependent manner, and are built around a helicase core, in general, no unwinding activity is observed.
RNA helicases that do exhibit unwinding activity have been characterized by at least two different mechanisms: canonical duplex unwinding and local strand separation. Canonical duplex unwinding is the stepwise directional separation of a duplex strand, as described above, for DNA unwinding. However, local strand separation occurs by a process wherein the helicase enzyme is loaded at any place along the duplex. This is usually aided by a single-strand region of the RNA, and the loading of the enzyme is accompanied with ATP binding. Once the helicase and ATP are bound, local strand separation occurs, which requires binding of ATP but not the actual process of ATP hydrolysis. Presented with fewer base pairs the duplex then dissociates without further assistance from the enzyme. This mode of unwinding is used by the DEAD/DEAH box helicases.
An RNA helicase database is currently available online that contains a comprehensive list of RNA helicases with information such as sequence, structure, and biochemical and cellular functions.
== Diagnostic tools for helicase measurement ==
=== Measuring and monitoring helicase activity ===
Various methods are used to measure helicase activity in vitro. These methods range from assays that are qualitative (assays that usually entail results that do not involve values or measurements) to quantitative (assays with numerical results that can be utilized in statistical and numerical analysis). In 1982–1983, the first direct biochemical assay was developed for measuring helicase activity. This method was called a "strand displacement assay".
Strand displacement assay involves the radiolabeling of DNA duplexes. Following helicase treatment, the single-strand DNA is visually detected as separate from the double-strand DNA by non-denaturing PAGE electrophoresis. Following detection of the single-strand DNA, the amount of radioactive tag that is on the single-strand DNA is quantified to give a numerical value for the amount of double-strand DNA unwinding.The strand displacement assay is acceptable for qualitative analysis, its inability to display results for more than a single time point, its time consumption, and its dependence on radioactive compounds for labeling warranted the need for development of diagnostics that can monitor helicase activity in real time.
Other methods were later developed that incorporated some, if not all of the following: high-throughput mechanics, the use of non-radioactive nucleotide labeling, faster reaction time/less time consumption, real-time monitoring of helicase activity (using kinetic measurement instead of endpoint/single point analysis). These methodologies include: "a rapid quench flow method, fluorescence-based assays, filtration assays, a scintillation proximity assay, a time resolved fluorescence resonance energy transfer assay, an assay based on flashplate technology, homogenous time-resolved fluorescence quenching assays, and electrochemiluminescence-based helicase assays". With the use of specialized mathematical equations, some of these assays can be utilized to determine how many base paired nucleotides a helicase can break per hydrolysis of 1 ATP molecule.
Commercially available diagnostic kits are also available. One such kit is the "Trupoint" diagnostic assay from PerkinElmer, Inc. This assay is a time-resolved fluorescence quenching assay that utilizes the PerkinElmer "SignalClimb" technology that is based on two labels that bind in close proximity to one another but on opposite DNA strands. One label is a fluorescent lanthanide chelate, which serves as the label that is monitored through an adequate 96/384 well plate reader. The other label is an organic quencher molecule. The basis of this assay is the "quenching" or repressing of the lanthanide chelate signal by the organic quencher molecule when the two are in close proximity – as they would be when the DNA duplex is in its native state. Upon helicase activity on the duplex, the quencher and lanthanide labels get separated as the DNA is unwound. This loss in proximity negates the quenchers ability to repress the lanthanide signal, causing a detectable increase in fluorescence that is representative of the amount of unwound DNA and can be used as a quantifiable measurement of helicase activity.
The execution and use of single-molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single-molecule resolution.
=== Determining helicase polarity ===
Helicase polarity, which is also deemed "directionality", is defined as the direction (characterized as 5'→3' or 3'→5') of helicase movement on the DNA/RNA single-strand along which it is moving. This determination of polarity is vital in f.ex. determining whether the tested helicase attaches to the DNA leading strand, or the DNA lagging strand. To characterize this helicase feature, a partially duplex DNA is used as the substrate that has a central single-strand DNA region with different lengths of duplex regions of DNA (one short region that runs 5'→3' and one longer region that runs 3'→5') on both sides of this region. Once the helicase is added to that central single-strand region, the polarity is determined by characterization on the newly formed single-strand DNA.
== See also ==
Chromodomain helicase DNA binding protein: CHD1, CHD1L, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CHD8, CHD9
DEAD box/DEAD/DEAH box helicase: DDX3X, DDX5, DDX6, DDX10, DDX11, DDX12, DDX58, DHX8, DHX9, DHX37, DHX40, DHX58
ASCC3, BLM, BRIP1, DNA2, FBXO18, FBXO30, HELB, HELLS, HELQ, HELZ, HFM1, HLTF, IFIH1, NAV2, PIF1, RECQL, RTEL1, SHPRH, SMARCA4, SMARCAL1, WRN, WRNIP1
RNA helicase database
== References ==
== External links ==
DNA+Helicases at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
RNA+Helicases at the U.S. National Library of Medicine Medical Subject Headings (MeSH) | Wikipedia/DNA_helicase |
In cell biology, eukaryotes possess a regulatory system that ensures that DNA replication occurs only once per cell cycle.
A key feature of the DNA replication mechanism in eukaryotes is that it is designed to replicate relatively large genomes rapidly and with high fidelity. Replication is initiated at multiple origins of replication on multiple chromosomes simultaneously so that the duration of S phase is not limited by the total amount of DNA. This flexibility in genome size comes at a cost: there has to be a high-fidelity control system that coordinates multiple replication origins so that they are activated only once during each S phase. If this were not the case, daughter cells might inherit an excessive amount of any DNA sequence, which could lead to many harmful effects.
== The replication origin ==
Replication in eukaryotes begins at replication origins, where complexes of initiator proteins bind and unwind the helix. In eukaryotes, it is still unclear what exact combinations of DNA sequence, chromatin structure, and other factors define these sites. The relative contribution of these factors varies between organisms. Yeast origins are defined primarily by DNA sequence motifs, while origin locations in other organisms seem to be defined by local chromatin structure.
=== Yeast ===
Origins in budding yeast are defined by the autonomously replicating sequence (ARS), a short stretch of DNA (100-200 bp) that can initiate replication when transferred to any sequence of DNA. The ARS contains several specific sequence elements. One of these is the A element (ACS), an 11 bp consensus sequence rich in adenines and thymines that is essential for initiation. Single base-pair mutations in the ACS can abolish initiation activity. The ORC, a component of the initiation complex, binds the ACS in vivo throughout the cell cycle, and in vitro in an ATP dependent manner. When a few of these sequences are deleted, DNA is still copied from other intact origins, but when many are deleted, chromosome replication slows down dramatically. Still, presence of an ACS sequence is not sufficient to identify an origin of replication. Only about 30% of ACS sequences present in the genome are the sites of initiation activity. Origins in fission yeast contain long stretches of DNA rich in thymines and adenines that are important for origin function, but do not exhibit strong sequence similarity.
=== Animals ===
In animals, no highly conserved sequence elements have been found to direct origin activity, and it has proved difficult to identify common features of replication origins. At some loci, initiation occurs within small, relatively definable stretches of DNA, while at others, larger initiation zones of 10–50 kb seem to direct origin activity. At the sequence level, AT rich elements and CpG islands have been found at origins, but their importance or role is not yet clear. At the level of DNA structure, bent DNA and loop formation have been identified as origin features. Features identified at the chromatin level include nucleosome free regions, histone acetylation and DNAse sensitive sites.
== The pre-replication complex ==
Before DNA replication can start, the pre-replicative complex assembles at origins to load helicase onto DNA. The complex assembles in late mitosis and early G1. Assembly of these pre-replicative complexes (pre-RCs) is regulated in a manner that coordinates DNA replication with the cell cycle.
=== Components of the pre-RC ===
==== The ORC ====
The ORC is a six subunit complex that binds DNA and provides a site on the chromosome where additional replication factors can assemble. It was identified in S. cerevisiae by its ability to bind the conserved A and B1 elements of yeast origins. It is a conserved feature of the replication system in Eukaryotes. Studies in Drosophila showed that recessive lethal mutations in multiple drosophila ORC subunits reduces the amount of BrdU (a marker of active replication), incorporated. Studies in Xenopus extracts show that immuno-depletion of ORC subunits inhibits DNA replication of Xenopus sperm nuclei. In some organisms, the ORC appears to associate with chromatin throughout the cell cycle, but in others it dissociates at specific stages of the cell cycle.
==== Cdc6 and Cdt1 ====
Cdc6 and Cdt1 assemble on the ORC and recruit the Mcm proteins. Homologs for these two S. cerevisiae proteins have been found in all eukaryotes. Studies have shown that these proteins are necessary for DNA replication. Mutations in S. pombe cdt1 blocked DNA replication.
==== The Mcm Complex ====
Mcm 2-7 form a six-subunit complex and is thought to have helicase activity. Deletion of any single subunit of the complex has a lethal phenotype in yeast. Studies in Xenopus revealed the Mcm2-7 complex is a critical component of DNA replication machinery. Inactivation of temperature sensitive mutants of any of the Mcm proteins in "S. cerevisiae" caused DNA replication to halt if inactivation occurred during S phase, and prevented initiation of replication if inactivation occurred earlier. Although biochemical data support the hypothesis that the Mcm complex is a helicase, helicase activity was not detected in all species, and some studies suggest that some of the mcm subunits act together as the helicase, while other subunits act as inhibitors of this activity. If this is true, activation of the Mcm complex probably involves rearrangement of the subunits.
== Regulation of pre-RC complex assembly ==
A two-step mechanism ensures that DNA is replicated only once per cycle. Assembly of the pre-RC complex (licensing) is limited to late mitosis and early G1 because it can occur only when CDK activity is low, and APC activity is high. Origin firing occurs only in S phase, when the APC is inactivated, and CDKs are activated.
=== Yeast ===
In budding yeast, CDK is the key regulator of pre-RC assembly. Evidence for this is that inactivation of CDKs in cells arrested in G2/M or in S phase drives reassembly of pre-RCs. CDK acts by inhibiting the individual components of the pre-RC. CDK phosphorylates Cdc6 to mark it for degradation by the SCF in late G1 and early S phase. CDK also induces export of Mcm complexes and Cdt1 from the nucleus. Evidence that CDKs regulate the localization of Mcm2-7 is that inactivation of CDKs in nocodazole arrested cells induced accumulation of Mcm2-7 in the nucleus. Cdt1 is also exported because it binds to the Mcm complex. In Mcm depleted cells, Cdt1 did not accumulate in the nucleus. Conversely, when a nuclear localization signal was attached to Mcm7, Mcm2-7 and Cdt1 were always found in the nucleus. Export of Mcm from the nucleus prevents loading of new Mcm complexes but does not affect the complexes that have already been loaded onto the DNA.
CDK also phosphorylates ORC proteins. It has been suggested that phosphorylation affects the ability of the ORC to bind other components of the pre-RC.
To get substantial re-replication of DNA, regulation of all three components, Cdc6, Mcm2-7 and the ORC has to be prevented. Having multiple mechanisms to prevent re-replication is beneficial because it the regulatory network continues to function even if one of the components fails.
=== Animals ===
Geminin is an important inhibitor of pre-Rc assembly is metazoan cells.
Geminin was identified in a screen for APC/C substrates in Xenopus. Studies have shown that Geminin prevents pre_RC assembly by binding to cdt1 and preventing its association with the pre-RC. Since geminin is degraded by the APC/C, pre-Rc assembly can proceed only when APC/C activity is high, which occurs in G1.
The importance of CDKs in preventing re-licensing in metazoan cells is still unclear. Some studies have shown that under some conditions, CDKs can also promote licensing. In G0 mammalian cells, APC mediated degradation of Cdc6 prevents licensing. However, when the cells transition into a proliferative state, CDK phosphorylates Cdc6 to stabilizes it and allow it to accumulate and bind to origins before licensing inhibitors such as geminin accumulate.
== Activation of replication origins ==
While pre-RC complexes mark potential sites for origin activation, further proteins and complexes must assemble at these sites to activate replication (origin firing). The following events must occur in order to activate the origin: the DNA helix has to open, the helicase must be activated, and DNA polymerases and the rest of the replicative machinery have to load onto the DNA. These events depend on the assembly of several proteins to form the pre-initiation complex at the replication origins loaded with pre-replicative complexes. Assembly of the pre-initiation complex depends on the activities of S-Cdks and the protein kinase Cdc7. The pre-initiation complex activates the Mcm helicase and recruits DNA polymerase.
When the cell commits to a new cell cycle, after passing through the Start checkpoint, G1 and G1/S cyclin CDK complexes are activated. These activate the expression of the replicative machinery and of S-Cdk cylin complexes. S-Cdks and G1/S Cdks act to activate replication origins. At the same time, S-Cdks suppress formation of new pre-RCs during S phase, G2 and early M, when S cyclin levels remains high. Cdc7 is activated in late G1 and is required throughout S phase for origin firing. Mutations in this protein in budding yeast, and in its homolog in fission yeast block initiation of replication. Cdc7 is highly conserved – related proteins have been identified in frogs and humans. DNA replication is inhibited when Cdc7 homologs are inhibited with antibodies in frog or human cells. It is not known whether CDKs and Cdc7 just regulate protein assembly at origins, or whether they directly activate components of the pre-initiation complex.
=== Role of CdK ===
In S. cerevisiae, the S cyclins Clb5 and Clb6 play and important role in initiating replication. In frog embryos, cyclin E-Cdk2 is primarily responsible for activating origins. Removal of cyclin E with antibodies blocks replication. Cyclin E-CDk2 is also important in Drosophila. Levels of cyclin E rise during S phase and activate Cdk2.
=== Role of Cdc7 ===
Cdc7 levels remain relatively constant throughout the cell cycle, but its activity varies. Its activity is low in G1, increases in late G1, and remains high till late mitosis. Dbf4 is the key regulator of Cdc7 activity – association Cdc7 with Dbf4 activates its kinase activity. In a similar manner to cyclin levels, dbf4 levels fluctuate throughout the cell cycle. In vitro biochemical studies have shown that Cdc7-Dbf4 phosphorylates individual components of the Mcm complex. It also seems to be involved in the recruitment of Cdc45 to chromatin at the time of initiation. In Xenopus eggs, Cdc45 has been shown to interact with DNA polymerase α, and in yeast, mutations in Cdc45 prevent assembly of DNA pol α at origins, suggesting that Cdc45 recruits DNA pol α to chromatin in a Cdc7/Dbf4 dependent manner.
== References == | Wikipedia/Control_of_chromosome_duplication |
dnaQ is the gene encoding the ε subunit of DNA polymerase III in Escherichia coli. The ε subunit is one of three core proteins in the DNA polymerase complex. It functions as a 3’→5’ DNA directed proofreading exonuclease that removes incorrectly incorporated bases during replication. dnaQ may also be referred to as mutD.
== Biological role ==
Missense mutations in the dnaQ gene lead to the induction of the SOS DNA repair mechanism. Mutating the essential amino acid in the catalytic center of the ε subunit leads to complete loss of function.
Overexpression of the ε subunit decreases the incidence of mutations with exposure to UV, proving that the epsilon subunit has an essential function in DNA editing and preventing the initiation of SOS DNA repair.
The ε subunit has also been proven to have some impact on the growth rate of E. coli. Silencing of the dnaQ gene is correlated to significantly reduced growth.
== Interactions ==
The ε subunit is stabilized by the θ subunit within the complete polymerase complex.
The gene encodes two functional domains: the N-terminus of the gene product binds the θ subunit and carries out the exonuclease function and the C-terminus binds the α subunit responsible for polymerase activity.
A Q-linker peptide of 22 residues has been identified that links the α subunit to the ε subunit, conferring flexibility that sets the α:ε complex apart from other more restricted multi-domain proofreading polymerases.
There is interaction between the missense suppressor glycine tRNA encoded by the mutA gene that is correlated to significantly increased mutation rate in cells that express the gene. The uncharged MutA tRNA possesses complementarity to a region in the 5' end of the dnaQ mRNA. This allows it to act as an antisense mRNA that directs the degradation of the dnaQ transcript and thus, a lower abundance of the subunit and increased frequency of mutation. More recently, it was suggested that the tRNA directs replacement of essential glutamate residues with glycine, leading to aberrant ε subunits and resulting in an increase in mutations. Studies with T4 bacteriophage and E. coli with defective dnaQ genes give evidence that the mutA tRNA may not have any effect on the transcription of the dnaQ gene but may affect the translation of the gene product.
== Related sequences ==
Sequences have been found in other organisms that encode gene products with a similar function to dnaQ:
In Mycobaterium tuberculosis, the gene dnaE1 encodes a polymerase and histidinol-phosphatase (PHP) domain that carries out the 3’→5’ exonuclease and proofreading function.
TREX1, the major 3'→5' exonuclease in humans, was initially called DNase III because it showed sequence homology with dnaQ in E. coli and with eukaryotic DNA polymerase epsilon and to possess biochemical characteristics that associate with the capability of DNA proofreading. It is responsible for metabolizing both single stranded DNA (ssDNA) and double stranded DNA (dsDNA) with mismatched 3' ends and is directed by endogenous retroelements.
== Evolution of dnaQ ==
During DNA replication in bacteria two key functions are expressed. The first is a DNA polymerizing function of DNA polymerase, and the second is a 3’ to 5’ exonuclease editing function. Both of these functions may be encoded within one gene, or alternatively the two functions may be encoded by separate genes. Two bacterial species that had diverged early in the course of evolution showing each of the alternative patterns were studied. In the Gram-negative bacteria Salmonella typhimurium the 3’ to 5’ editing function employed during DNA replication is encoded by gene dnaQ which specifies a 3’ to 5’ exonuclease subunit, one of the three separately encoded core proteins of the DNA polymerase III holoenzyme. The complete nucleotide sequence of dnaQ of S. typhimurium was determined. The DNA polymerase from the early diverging bacterial species, Buchnera aphidicola, was also sequenced. In this case, the DNA polymerase encoded by the DNA III (polC) gene contains both DNA polymerase and 3’ to 5’ exonuclease domains. This arrangement is in contrast to S. typhimurium in which these domains are encoded in separate genes. Based on the sequence homologies found between the DNA regions encoding the 3’ to 5’ editing functions in these bacteria, it was proposed that a last common ancestor of S. typhimurium and B. aphidicola had a single gene containing both 3’ to 5’ exonuclease and DNA polymerase domains. The evolutionary divergence of these bacteria (about 0.25 to 1.2 billion years ago), appears to have been associated with the separation of the DNA polymerase gene function from the 3’ to 5’ exonuclease editing gene function in the lineage containing S. typhimurium.
== References ==
== External links ==
dnaQ+protein,+E+coli at the U.S. National Library of Medicine Medical Subject Headings (MeSH) | Wikipedia/DnaQ |
DnaA is a protein that activates initiation of DNA replication in bacteria. Based on the Replicon Model, a positively active initiator molecule contacts with a particular spot on a circular chromosome called the replicator to start DNA replication. It is a replication initiation factor which promotes the unwinding of DNA at oriC. The DnaA proteins found in all bacteria engage with the DnaA boxes to start chromosomal replication. The onset of the initiation phase of DNA replication is determined by the concentration of DnaA. DnaA accumulates during growth and then triggers the initiation of replication. Replication begins with active DnaA binding to 9-mer (9-bp) repeats upstream of oriC. Binding of DnaA leads to strand separation at the 13-mer repeats. This binding causes the DNA to loop in preparation for melting open by the helicase DnaB.
== Function ==
DnaA consists mainly in two different forms, the active ATP-form and the inactive ADP. The level of active DnaA within a cell is low immediately after a cell has divided. Although the active form of DnaA requires ATP, the formation of the oriC/DnaA complex and subsequent DNA unwinding does not require ATP hydrolysis.
The oriC site in E. coli has three AT rich 13 base pair regions (DUEs) followed by four 9 bp regions with the sequence TTAT(C or A)CA(C or A)A. DnaA molecules bind to the 9 bp regions, which wrap around the proteins causing the DNA at the AT-rich region to unwind. There are currently 11 DnaA binding sites identified within oriC, to which DnaA binds with differential affinity. When DNA replication is about to commence, DnaA occupies all of the high and low affinity binding sites. The denatured AT-rich region allows for the recruitment of DnaB (helicase), which complexes with DnaC (helicase loader). DnaC helps the helicase to bind to and to properly accommodate the ssDNA at the 13 bp region; this is accomplished by ATP hydrolysis, after which DnaC is released. Single-strand binding proteins (SSBs) stabilize the single DNA strands in order to maintain the replication bubble. DnaB is a 5'→3' helicase, so it travels on the lagging strand. It associates with DnaG (a primase) to form the only primer for the leading strand and to add RNA primers on the lagging strand. The interaction between DnaG and DnaB is necessary to control the longitude of Okazaki fragments on the lagging strand. DNA polymerase III is then able to start DNA replication.
DnaA is made up of four domains: the first is the N-terminal that associates with regulatory proteins, the second is a helical linker region, the third domain is a AAA+ region that binds to ATP, and the fourth domain is the C-terminal DNA binding region. DnaA contains two conserved regions: the first is located in the central part of the protein and corresponds to the ATP-binding domain, the second is located in the C-terminal half and is involved in DNA-binding.
== DnaA mutants ==
The first strains to have the dnaA gene mutated were the temperature-sensitive K-12 strains CRT46 and CRT83, with the corresponding strain numbers beingdnaA46 and dnaA83. In contrary to dnaA mutants, the PC2 strain has a mutation in the dnaC gene, which codes for the loading factor for the DNA helicase dnaB.
== Synthesis ==
DnaA has the ability to bind its own promoter. When DnaA binds to its own promoter it blocks RNA polymerase from binding the promoter and inhibits initiation of transcription. In this way, DnaA is able to regulate its own expression. This process is called autoregulation.
== Regulation ==
Each cell division cycle triggers a new round of chromosome replication with the accumulation of DnaA, the initiator protein, on the OriC region of DNA. It is crucial to regulate DnaA-ATP monomer interactions with oriC during helicase loading and unwinding of origin DNA for precise timing. DnaA recognition sites in Escherichia coli are arranged in OriC to facilitate staged pre-replication complex assembling, with DnaA interacting with low affinity sites as it oligomerizes to fill the gaps between high affinity sites as it oligomerizes. There may be numerous gap-filling strategies to link OriC functions to bacterial lifestyles in nature, which may account for the wide variability of OriC DnaA recognition site patterns. The two forms of DnaA, the active ATP- and ADP-form are regulated. The ATP-form is converted to the ADP-form through either Regulatory inactivation of DnaA (RIDA), which in turn consists of the Hda protein and the β sliding clamp (DnaN) and datA-dependent DnaA-ATP hydrolysis. The ADP-form is converted to the ATP-form by DnaA-reactivating sequences 1 and 2 (DARS1 and DARS2).
Regulation of DnaA binding to DNA at OriC
Since DNA replication must occur irreversibly and only once per cycle, the binding behavior of DnaA complexes to OriC is a highly regulated, and therefore dependent on many other cellular mechanisms. While all OriC sites are bound at replication initiation, there are three high-affinity binding sites–R1, R2, and R4–that are typically occupied by DnaA for the majority of the cell cycle, thus making their binding somewhat less dependent of other events happening within the cell at a given point in time. By contrast, the lower affinity sites are typically only bound to DnaA complexes right before replication begins. There are currently eight identified sites with lower DnaA/OriC binding affinity: R5 (or R5M), I1, I2, I3 and R3, tau2, C1, C2 and C3. Between the R1 and R2 high affinity sites exist the R5M, tau2, I1, and I2 low affinity sites, and C3, C2, 13, and C1 exist between the R2 and R4 sites. The I sites, tau2, C2, and C3 sites preferentially bind with and are more efficient at binding to DnaA in its ATP-bound active form (DnaA-ATP) prior to DNA strand separation, whereas the R1-R5 sites and C1 site have not demonstrated a preference for binding with DnaA-ATP over DnaA-ADP. OriC binding with active DnaA-ATP complexes at the lower affinity I sites, as well as the tau2, C2, and C3, sites is required for the strand separation process to initiate in a time regulated manner, meaning DnaA-ATP cannot be substituted with inactive DnaA-ADP complexes to initiate replication properly and with sufficient regulation. Recent studies suggest that while OriC sites bound entirely to DnaA-ADP complexes are capable of preparing the cell for DNA replication, they struggle to maintain the healthy and consistent replication frequency regulation cells continuing OriC sites bound to DnaA-ATP complexes achieve, perhaps explaining why some sites bind preferentially to the active DnaA conformation over the inactive conformation. Two other proteins, an integration host factor (IHF) protein and a DnaA initiator associating (DiaA) protein, help facilitate the binding of DnaA-ATP complexes to the OriC sites and set the stage for replication initiation to occur.
IHF plays a key functional role positively regulating the binding of DnaA complexes to the lower affinity OriC sites as the cell prepares for replication, essentially evening the playing field between the high and low affinity OriC sites in terms of their ability to bind with DnaA complexes. Cooperative binding is thought to be a mechanism in which the high-affinity sites supply the lower-affinity sites within their vicinity with DnaA-ATP complexes in the moments leading up to replication initiation. While DnaA can saturate all OriC binding sites in systems lacking IHF, a much higher concentration of DnaA is needed in the cellular environment for this to be achieved. However, in these situations, cells also experience a loss of synchronization in their replication initiation timing, indicating how important IHF is for maintaining consistent regulation of this process in cells and preventing a lag in the initiation of replication. When IHF is present in a cellular system, IHF enhances DnaA binding to low affinity OriC sites without any need for increasing the baseline concentration of DnaA present, further highlighting its importance in maintaining replication initiation timing.
Conformationally, IHF assists in promoting the process of DnaA-ATP complexes binding to the low affinity OriC binding sites at the right time by binding to a different site on OriC ahead of replication initiation, causing DNA it to bend in such a way that facilitates efficient binding with DnaA-ATP complexes. Prior to IHF binding to OriC, a different protein, factor for inversion stimulation (FIS) protein, is bound to DNA for the majority of the cell cycle (with the exception of the events leading up to replication initiation), inhibiting the binding of IHF to DNA. Consequently, the binding of DnaA complexes to the lower affinity OriC sites is also inhibited, thus, preventing the chromosomal replication process from starting prematurely and thereby demonstrating how FIS positively regulates the maintenance of a consistent cell cycle progression via inhibition. As FIS binding to OriC weakens, IHF begins to bind to OriC, therefore increasing the low affinity sites’ ability to bind to DnaA-ATP complexes, concurrent with IHF binding. The switch from FIS binding to IHF binding to DNA is hypothesized to be brought about by the generation of more DnaA-ATP complexes, promoted by the existence of the high affinity sites bound to DnaA while FIS is concurrently bound to DNA, which are then recruited to the high affinity region and build up, exerting a conformational stress on bound FIS (especially by accumulation at the R2 site, as it is closest to the FIS binding site), thereby deteriorating its binding ability with DNA. As a result, IHF can take advantage of the weakened state of FIS binding so that it can then bind to its own respective OriC site, causing DNA to bend and essentially align the accumulated DnaA-ATP complexes better with the low affinity binding sites, thus aiding in the facilitation of their binding with DnaA-ATP. In the absence of the switch-like behavior that occurs with the transition from FIS to IHF binding to DNA, cells are unable to maintain a control over the sequence of events that ensure replication initiation happens both irreversibly and only once per cell cycle.
DiaA positively regulates the replication initiation timeline by facilitating the binding of DnaA-ATP complexes on OriC sites. DiaA binds to DnaA in its tetrameric form (consisting of four DiaA protomers (individual proteins) bound to one another), specifically to the first domain of DnaA–in the same region where another protein, replicative DNA helicase (DnaB), is presumed to bind with DnaA. Due to its tetrameric structure, DiaA has the ability to bind to multiple DnaA-ATP complexes at a time, as each protamer within the homotetramer consists of an DnaA-ATP binding site. This beneficial characteristic of DiaA tetramers can aid in promoting the cooperative binding behavior of transferring DnaA-ATP molecules to different sites on the OriC region of DNA as the cell prepares to undergo chromosomal replication. DiaA also negatively regulates the chromosomal replication process by inhibiting the binding of the DnaB protein, whose presence and function is required for chromosomal replication, from binding to DnaA-ATP complexes assembled on OriC, therefore helping to preserve the inflexible regulation sequence of events needed for a controlled replication process and prevents asynchronous initiation within the overall cycle cycle. Thus, taken together, IHF and DiaA, along with the proteins they interact with in their respective binding mechanisms, are very both important for helping DnaA-ATP complexes bind to all the identified binding sites on OriC, including the low affinity sites, within a timely manner that ensures replication initiation occurs irreversibly and only a single time during the cell cycle.
Once replication initiation has occurred and DNA has undergone strand separation successfully, a different process commences to make sure DnaA-ATP cannot bind directly to DNA again with a protein that negatively regulates replication initiation–the locus of datA–SeqA. When DNA unwinds post-initiation, new replication forks are generated, a process that subsequently leads to the unbinding of DnaA complexes from the OriC sites. DNA’s GATC sites within OriC and at the region where the dnaA promoter exists become hemimethylated, and therefore experience a reduced ability to function and express the same way as they would while methylated. SeqA is able to physically prevent replication from starting up again prematurely by binding to the hemimethylated GATA sites on OriC–many of which somewhat overlap with a couple of the low affinity binding DnaA binding sites, as well as IHF’s binding site on OriC–essentially shielding IHF and DnaA from binding to OriC. However, the high affinity OriC DnaA complex binding sites are not blocked by SeqA binding to DNA, thus explaining how DnaA stays bound to the three high affinity sites throughout the majority of the cycle cycle duration. When GATC sites are bound to SeqA while hemimethylated, they are limited in their ability to synthesize new DnaA proteins as well, thus causing DnaA concentration within the cell to decline post initiation. Thus, with these sites blocked by SeqA, DnaA-ATP binding to some of the lower affinity sites is not possible for a combination of reasons. In studies performed with strains lacking the ability to produce SeqA, cells were unable to synchronously initiate replication once per cycle, mirroring the effects of what happens when cells lack IHF. Since the binding of the low affinity sites on OriC are basically the key event that kick start replication initiation and DNA’s unraveling, SeqA’s blocking of DnaA-ATP complex binding during the majority of the cell cycle is vital for keep cells healthy by maintaining a consistent cycle.
== DnaA protein structure ==
There are four disciplines within the DnaA protein. An initial comparison of Escherichia coli and Bacillus subtilis proteins led to the discovery of a sphere structure, which revealed a relatively conserved N-terminus and a largely conserved large C-terminus separated by a region that was mostly variable. As an example, the Enterobacterial proteins have nearly identical N- and C-terminal sequences, however they are characterized by numerous amino acid adjustments, elisions, and insertions in the variable regions. There is an AAA+ family ATPase motif and an independent DNA binding sphere in the C-terminal region. It was determined by NMR that Escherichia coli sphere IV had a crystal-clear structure when complexed with a DnaA- box. As a result, it was confirmed that the DNA list is intermediated by a combination of a helix-turn-helix motif and an introductory circle. When bound to ATP, but not to ADP, DnaA forms a super-helical structure with four monomers per turn. The structure of sphere I has been determined from three additional bacterial species and Escherichia coli by NMR.
== Autoregulation of DnaA protein synthesis ==
The research on dnaA(Ts) mutants provided the first proof that the dnaA gene is autoregulated. DnaA protein is still produced at non-permissive temperatures where it is inactive, but in some mutants it can be made active again by returning to a temperature that is conducive to development. This reversible initiation capacity—which was larger than anticipated given the mass gain of the culture—could be seen in the absence of protein synthesis at the permissive temperature and suggested that the DnaA protein synthesis was derepressed at the high growth temperature. These results prompted a thorough investigation of the dnaA46 mutant under permissive, intermediate, and non-permissive development conditions. The study's findings revealed that as growth temperature increased, the DnaA46 protein's activity decreased, leading to progressively decreasing DNA and origin concentrations at intermediate temperatures. An increase in initiation capacity was seen concurrently with a decrease in DnaA protein activity. Hansen and Rasmussen (1977) argued that the DnaA protein had a positive effect in replication initiation aing transcripts entering the dnaA gene were found as a result of sequencing the dnaA promoter region and the dnaA gene. The DnaA promoter region has nine GATC sites within 225 base pairs, and a sequence that is similar to nd a negative role in its own synthesis based on these observations. Two promoters providrepetitions (DnaA-boxes) in the oriC region was found between the two promoters. According to several studies, the DnaA protein negatively regulates both promoters. In these research, it was discovered that the dnaA transcription was upregulated by 4- to 5-fold at non-permissive temperatures in dnaATs mutants and repressed by the same amount when DnaA protein was overproduced. The autoregulation of the dnaA gene requires the DnaA-box. The sequence of the dnaA2p promoter region has some intriguing characteristics that can be seen more clearly. This promoter contains two GATC sites, one in the 10 sequence and the other in the 35 sequence, and both in vivo and in vitro, methylation increases transcription from this promoter by a factor of two. In addition, DnaA protein binds to regions upstream of the dnaA2p promoter with a high affinity.
== See also ==
Origin recognition complex
== References ==
== Further reading ==
== External links ==
DnaA+protein,+Bacteria at the U.S. National Library of Medicine Medical Subject Headings (MeSH) | Wikipedia/DnaA |
Cell division cycle 7-related protein kinase is an enzyme that in humans is encoded by the CDC7 gene. The Cdc7 kinase is involved in regulation of the cell cycle at the point of chromosomal DNA replication. The gene CDC7 appears to be conserved throughout eukaryotic evolution; this means that most eukaryotic cells have the Cdc7 kinase protein.
== Function ==
The product encoded by this gene is predominantly localized in the nucleus and is a cell division cycle protein with kinase activity.
The protein is a serine-threonine kinase that is activated by another protein called either Dbf4 in the yeast Saccharomyces cerevisiae or ASK in mammals. The Cdc7/Dbf4 complex adds a phosphate group to the minichromosome maintenance (MCM) protein complex allowing for the initiation of DNA replication in mitosis (as explained in the Cdc7 and Replication section below).
Although expression levels of the protein appear to be constant throughout the cell cycle, the protein kinase activity appears to increase during S phase. It has been suggested that the protein is essential for initiation of DNA replication and that it plays a role in regulating cell cycle progression. Overexpression of this gene product may be associated with neoplastic transformation for some tumors. Additional transcript sizes have been detected, suggesting the presence of alternative splicing.
== Cell cycle regulation ==
The gene, CDC7, is involved in the regulation of cell cycle because of the gene product Cdc7 kinase. The protein is expressed at constant levels throughout the cell cycle. The gene coding for the Dbf4 or ASK protein is regulated during the different phases of cell cycle. The concentration of Dbf4 at the G1/S transition of the cell cycle is higher than the concentration at the M/G1 transition. This tells us that Dbf4 is expressed around the time for replication; right after replication is over, the protein levels drop. Because the two proteins, Cdc7 and Dbf4, must form a complex before activating the MCM complex, the regulation of one protein is sufficient for both.
It has been shown that CDC7 is important for replication. There are several ways its expression can be altered that leads to problems. In mouse embryonic stem cells (ESCs), Cdc7 is needed for proliferation. Without the CDC7 gene DNA synthesis is stopped, and the ESCs do not grow. With the loss of function of Cdc7 in ESCs the S phase is stopped at the G2/M checkpoint. Recombinational repair (RR) is done at this point to try to fix the CDC7 gene so replication can occur. By copying and replacing the altered area with a very similar area on the sister homolog chromosome, the gene can be replicated as if nothing was ever wrong on the chromosome. However, when the cell enters this arrested state, levels of p53 may increase. These increased levels of p53 may initiate cell death.
== Replication ==
After chromatin undergoes changes in telophase of mitosis, the hexameric protein complex of MCM proteins 2-7 forms part of the pre-replication complex (pre-RC) by binding to the chromatin and other aiding proteins (Cdc6 and Cdt1). Mitosis occurs during M phase of the cell cycle and has a number of stages; telophase is the end stage of mitosis when the replication of chromosomes is complete, but separation has not occurred.
The Cdc7/Dbf4 kinase complex, along with another serine-threonine kinase, cyclin-dependent kinase (Cdk), phosphorylates the pre-RC which activates it at the G1/S transition. The Dbf4 tethers itself to part of the pre-RC, the origin recognition complex (ORC). Since Cdc7 is attached to the Dbf4 protein the entire complex is held in place during replication. This activation of MCM 2 leads to helicase activity of the MCM complex at the origin of replication. This is most likely due to the change in conformation allowing the remainder of replication machinery proteins to be loaded. DNA replication can begin after all the necessary proteins are in place.
== Interactions ==
CDC7 has been shown to interact with:
DBF4,
MCM5,
MCM4,
MCM7,
ORC1L, and
ORC6L.
== Ligands ==
Inhibitors
XL-413
== References ==
== Further reading == | Wikipedia/Cell_division_cycle_7-related_protein_kinase |
Double-stranded RNA viruses (dsRNA viruses) are a polyphyletic group of viruses that have double-stranded genomes made of ribonucleic acid. The double-stranded genome is used as a template by the viral RNA dependent RNA polymerase (RdRp) to transcribe a positive-strand RNA functioning as messenger RNA (mRNA) for the host cell's ribosomes, which translate it into viral proteins. The positive-strand RNA can also be replicated by the RdRp to create a new double-stranded viral genome.
A distinguishing feature of the dsRNA viruses is their ability to carry out transcription of the dsRNA segments within the capsid, and the required enzymes are part of the virion structure.
Double-stranded RNA viruses are classified into two phyla, Duplornaviricota and Pisuviricota (specifically class Duplopiviricetes), in the kingdom Orthornavirae and realm Riboviria. The two phyla do not share a common dsRNA virus ancestor, but evolved their double strands two separate times from positive-strand RNA viruses. In the Baltimore classification system, dsRNA viruses belong to Group III.
Virus group members vary widely in host range (animals, plants, fungi, and bacteria), genome segment number (one to twelve), and virion organization (T-number, capsid layers, or turrets). Double-stranded RNA viruses include the rotaviruses, known globally as a common cause of gastroenteritis in young children, and bluetongue virus, an economically significant pathogen of cattle and sheep. The family Reoviridae is the largest and most diverse dsRNA virus family in terms of host range.
== Classification ==
Two clades of dsRNA viruses exist: the phylum Duplornaviricota and the class Duplopiviricetes, which is in the phylum Pisuviricota. Both are included in the kingdom Orthornavirae in the realm Riboviria. Based on phylogenetic analysis of RdRp, the two clades do not share a common dsRNA ancestor but are instead separately descended from different positive-sense, single-stranded RNA viruses. In the Baltimore classification system, which groups viruses together based on their manner of mRNA synthesis, dsRNA viruses are group III.
=== Duplornaviricota ===
Duplornaviricota contains most dsRNA viruses, including reoviruses, which infect a diverse range of eukaryotes, and cystoviruses, which are the only dsRNA viruses known to infect prokaryotes. Apart from RdRp, viruses in Duplornaviricota also share icosahedral capsids that contain 60 homo- or heterodimers of the capsid protein organized on a pseudo T=2 lattice. The phylum is divided into three classes: Chrymotiviricetes, which primarily contains fungal and protozoan viruses, Resentoviricetes, which contains reoviruses, and Vidaverviricetes, which contains cystoviruses.
=== Duplopiviricetes ===
The class Duplopiviricetes is the second clade of dsRNA viruses and is in the phylum Pisuviricota, which also contains positive-sense single-stranded RNA viruses. Duplopiviricetes mostly contains plant and fungal viruses and includes the following four families: Amalgaviridae, Hypoviridae, Partitiviridae, and Picobirnaviridae.
== Notes on selected species ==
=== Reoviridae ===
Reoviridae are currently classified into nine genera. The genomes of these viruses consist of 10 to 12 segments of dsRNA, each generally encoding one protein. The mature virions are non-enveloped. Their capsids, formed by multiple proteins, have icosahedral symmetry and are arranged generally in concentric layers.
==== Orthoreoviruses ====
The orthoreoviruses (reoviruses) are the prototypic members of the virus Reoviridae family and representative of the turreted members, which comprise about half the genera. Like other members of the family, the reoviruses are non-enveloped and characterized by concentric capsid shells that encapsidate a segmented dsRNA genome. In particular, reovirus has eight structural proteins and ten segments of dsRNA. A series of uncoating steps and conformational changes accompany cell entry and replication. High-resolution structures are known for almost all of the proteins of mammalian reovirus (MRV), which is the best-studied genotype. Electron cryo-microscopy (cryoEM) and X-ray crystallography have provided a wealth of structural information about two specific MRV strains, type 1 Lang (T1L) and type 3 Dearing (T3D).
==== Cypovirus ====
The cytoplasmic polyhedrosis viruses (CPVs) form the genus Cypovirus of the family Reoviridae. CPVs are classified into 14 species based on the electrophoretic migration profiles of their genome segments. Cypovirus has only a single capsid shell, which is similar to the orthoreovirus inner core. CPV exhibits striking capsid stability and is fully capable of endogenous RNA transcription and processing. The overall folds of CPV proteins are similar to those of other reoviruses. However, CPV proteins have insertional domains and unique structures that contribute to their extensive intermolecular interactions. The CPV turret protein contains two methylase domains with a highly conserved helix-pair/β-sheet/helix-pair sandwich fold but lacks the β-barrel flap present in orthoreovirus λ2. The stacking of turret protein functional domains and the presence of constrictions and A spikes along the mRNA release pathway indicate a mechanism that uses pores and channels to regulate the highly coordinated steps of RNA transcription, processing, and release.
==== Rotavirus ====
Rotavirus is the most common cause of acute gastroenteritis in infants and young children worldwide. This virus contains a dsRNA genome and is a member of the Reoviridae family. The genome of rotavirus consists of eleven segments of dsRNA. Each genome segment codes for one protein with the exception of segment 11, which codes for two proteins. Among the twelve proteins, six are structural and six are non-structural proteins.
It is a double-stranded RNA non-enveloped virus. When at least two rotavirus genomes are present in a host cell, the genome segments may undergo reassortment to form progeny viruses with new gene combinations., or they may undergo intragenic homologous recombination. Some pathogenic rotovirus lineages that infect humans appear to have evolved through multiple interspecies reassortment events. Intragenic homologous recombination also appears to be a significant driver of rotovirus diversity and evolution. Intragenic recombination may occur when the VP1 RNA-dependent RNA polymerase replicates part of one template strand before switching to another.
==== Bluetongue virus ====
The members of genus Orbivirus within the Reoviridae family are arthropod borne viruses and are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at the molecular and structural levels. BTV, like other members of the family, is a complex non-enveloped virus with seven structural proteins and a RNA genome consisting of 10 variously sized dsRNA segments.
==== Phytoreoviruses ====
Phytoreoviruses are non-turreted reoviruses that are major agricultural pathogens, particularly in Asia. One member of this family, Rice Dwarf Virus (RDV), has been extensively studied by electron cryomicroscopy and x-ray crystallography. From these analyses, atomic models of the capsid proteins and a plausible model for capsid assembly have been derived. While the structural proteins of RDV share no sequence similarity to other proteins, their folds and the overall capsid structure are similar to those of other Reoviridae.
=== Saccharomyces cerevisiae virus L-A ===
The L-A dsRNA virus of the yeast Saccharomyces cerevisiae has a single 4.6 kb genomic segment that encodes its major coat protein, Gag (76 kDa) and a Gag-Pol fusion protein (180 kDa) formed by a -1 ribosomal frameshift. L-A can support the replication and encapsidation in separate viral particles of any of several satellite dsRNAs, called M dsRNAs, each of which encodes a secreted protein toxin (the killer toxin) and immunity to that toxin. L-A and M are transmitted from cell to cell by the cytoplasmic mixing that occurs in the process of mating. Neither is naturally released from the cell or enters cells by other mechanisms, but the high frequency of yeast mating in nature results in the wide distribution of these viruses in natural isolates. Moreover, the structural and functional similarities with dsRNA viruses of mammals has made it useful to consider these entities as viruses.
=== Infectious bursal disease virus ===
Infectious bursal disease virus (IBDV) is the best-characterized member of the family Birnaviridae. These viruses have bipartite dsRNA genomes enclosed in single layered icosahedral capsids with T = 13l geometry. IBDV shares functional strategies and structural features with many other icosahedral dsRNA viruses, except that it lacks the T = 1 (or pseudo T = 2) core common to the Reoviridae, Cystoviridae, and Totiviridae. The IBDV capsid protein exhibits structural domains that show homology to those of the capsid proteins of some positive-sense single-stranded RNA viruses, such as the nodaviruses and tetraviruses, as well as the T = 13 capsid shell protein of the Reoviridae. The T = 13 shell of the IBDV capsid is formed by trimers of VP2, a protein generated by removal of the C-terminal domain from its precursor, pVP2. The trimming of pVP2 is performed on immature particles as part of the maturation process. The other major structural protein, VP3, is a multifunctional component lying under the T = 13 shell that influences the inherent structural polymorphism of pVP2. The virus-encoded RNA-dependent RNA polymerase, VP1, is incorporated into the capsid through its association with VP3. VP3 also interacts extensively with the viral dsRNA genome.
=== Bacteriophage Φ6 ===
Bacteriophage Φ6, is a member of the Cystoviridae family. It infects Pseudomonas bacteria (typically plant-pathogenic P. syringae). It has a three-part, segmented, double-stranded RNA genome, totalling ~13.5 kb in length. Φ6 and its relatives have a lipid membrane around their nucleocapsid, a rare trait among bacteriophages. It is a lytic phage, though under certain circumstances has been observed to display a delay in lysis which may be described as a "carrier state".
== Anti-virals ==
Since cells do not produce double-stranded RNA during normal nucleic acid metabolism, natural selection has favored the evolution of enzymes that destroy dsRNA on contact. The best known class of this type of enzymes is Dicer. It is hoped that broad-spectrum anti-virals could be synthesized that take advantage of this vulnerability of double-stranded RNA viruses.
== See also ==
Animal virology
List of viruses
RNA virus
TLR3
Virology
Virus classification
== References ==
== Bibliography == | Wikipedia/DsDNA-RT_virus |
High-energy phosphate can mean one of two things:
The phosphate-phosphate (phosphoanhydride/phosphoric anhydride/macroergic/phosphagen) bonds formed when compounds such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP) are created.
The compounds that contain these bonds, which include the nucleoside diphosphates and nucleoside triphosphates, and the high-energy storage compounds of the muscle, the phosphagens. When people speak of a high-energy phosphate pool, they speak of the total concentration of these compounds with these high-energy bonds.
== Description ==
High-energy phosphate bonds are usually pyrophosphate bonds, acid anhydride linkages formed by taking phosphoric acid derivatives and dehydrating them. As a consequence, the hydrolysis of these bonds is exergonic under physiological conditions, releasing Gibbs free energy.
Except for PPi → 2 Pi, these reactions are, in general, not allowed to go uncontrolled in the human cell but are instead coupled to other processes needing energy to drive them to completion. Thus, high-energy phosphate reactions can:
provide energy to cellular processes, allowing them to run
couple processes to a particular nucleoside, allowing for regulatory control of the process
drive a reaction out of equilibrium (drive it to the right) by promoting one direction of the reaction faster than the equilibrium can relax.
The one exception is of value because it allows a single hydrolysis, ATP + H2O → AMP + PPi, to effectively supply the energy of hydrolysis of two high-energy bonds, with the hydrolysis of PPi being allowed to go to completion in a separate reaction. The AMP is regenerated to ATP in two steps, with the equilibrium reaction ATP + AMP ↔ 2ADP, followed by regeneration of ATP by the usual means, oxidative phosphorylation or other energy-producing pathways such as glycolysis.
Often, high-energy phosphate bonds are denoted by the character '~'. In this "squiggle" notation, ATP becomes A-P~P~P. The squiggle notation was invented by Fritz Albert Lipmann, who first proposed ATP as the main energy transfer molecule of the cell, in 1941. Lipmann's notation emphasizes the special nature of these bonds. Stryer states: ATP is often called a high energy compound and its phosphoanhydride bonds are referred to as high-energy bonds. There is nothing special about the bonds themselves. They are high-energy bonds in the sense that free energy is released when they are hydrolyzed, for the reasons given above. Lipmann’s term "high-energy bond" and his symbol ~P (squiggle P) for a compound having a high phosphate group transfer potential are vivid, concise, and useful notations. In fact Lipmann's squiggle did much to stimulate interest in bioenergetics.
The term 'high energy' with respect to these bonds can be misleading because the negative free energy change is not due directly to the breaking of the bonds themselves. The breaking of these bonds, like the breaking of most bonds, is endergonic and consumes energy rather than releasing it. The negative free energy change comes instead from the fact that the bonds formed after hydrolysis - or the phosphorylation of a residue by ATP - are lower in energy than the bonds present before hydrolysis. (This includes all of the bonds involved in the reaction, not just the phosphate bonds themselves). This effect is due to a number of factors including increased resonance stabilization and solvation of the products relative to the reactants, and destabilization of the reactants due to electrostatic repulsion between neighboring phosphorus atoms.
== References ==
== Further reading ==
McGilvery, R. W. and Goldstein, G., Biochemistry - A Functional Approach, W. B. Saunders and Co, 1979, 345–351.
Nicholls, David; Ferguson, Stuart (2002). "The myth of the 'high-energy phosphate bond'". Bioenergetics 3 (3rd ed.). San Diego, CA: Academic. ISBN 978-0-12-518121-1. | Wikipedia/High-energy_phosphate |
A DNA unwinding element (DUE or DNAUE) is the initiation site for the opening of the double helix structure of the DNA at the origin of replication for DNA synthesis. It is A-T rich and denatures easily due to its low helical stability, which allows the single-strand region to be recognized by origin recognition complex.
DUEs are found in both prokaryotic and eukaryotic organisms, but were first discovered in yeast and bacteria origins, by Huang Kowalski. The DNA unwinding allows for access of replication machinery to the newly single strands. In eukaryotes, DUEs are the binding site for DNA-unwinding element binding (DUE-B) proteins required for replication initiation. In prokaryotes, DUEs are found in the form of tandem consensus sequences flanking the 5' end of DnaA binding domain. The act of unwinding at these A-T rich elements occurs even in absence of any origin binding proteins due to negative supercoiling forces, making it an energetically favourable action. DUEs are typically found spanning 30-100 bp of replication origins.
== Function ==
The specific unwinding of the DUE allows for initiation complex assembly at the site of replication on single-stranded DNA, as discovered by Huang Kowalski. The DNA helicase and associated enzymes are now able to bind to the unwound region, creating a replication fork start. The unwinding of this duplex strand region is associated with a low free energy requirement, due to helical instability caused by specific base-stacking interactions, in combination with counteracting supercoiling. Negative supercoiling allows the DNA to be stable upon melting, driven by reduction of torsional stress. Found in the replication origins of both bacteria and yeast, as well as present in some mammalian ones. Found to be between 30 and 100 bp long.
== Prokaryotes ==
In prokaryotes, most of the time DNA replication is occurring from one single replication origin on one single strand of DNA sequence. Whether this genome is linear or circularized, bacteria have own machinery necessary for replication to occur.
=== Process ===
In bacteria, the protein DnaA is the replication initiator. It gets loaded onto oriC at a DnaA box sequence where it binds and assembles filaments to open duplex and recruit DnaB helicase with the help of DnaC. DnaA is highly conserved and has two DNA binding domains. Just upstream to this DnaA box, is three tandem 13-mer sequences. These tandem sequences, labelled L, M, R from 5' to 3' are the bacterial DUEs. Two out of three of these A-T rich regions (M and R) become unwound upon binding of DnaA to DnaA box, via close proximity to unwinding duplex. The final 13-mer sequence L, farthest from this DnaA box eventually gets unwound upon DnaB helicase encircling it. This forms a replication bubble for DNA replication to then proceed.
Archaea use a simpler homolog of the eukaryotic origin recognition complex to find the origin of replication, at sequences termed the origin recognition box (ORB).
=== Favourability ===
Unwinding of these three DUEs is a necessary step for DNA replication to initiate. The distant pull from duplex melting at the DnaA box sequence is what induces further melting at the M and R DUE sites. The more distant L site is then unwound by DnaB binding. Unwinding of these 13-mer sites is independent of oriC-binding proteins. It is the generation of negative supercoiling that causes the unwinding.
The rates of DNA unwinding in the three E. coli DUEs were experimentally compared through nuclear resonance spectroscopy. In physiological conditions, the opening efficiency of each of the A-T rich sequences differed from one another. Largely due to the different distantly surrounding sequences.
Additionally, melting of AT/TA base pairs were found to be much faster than that of GC/CG pairs (15-240s−1 vs. ~20s−1). This supports the idea that A-T sequences are evolutionarily favoured in DUE elements due to their ease of unwinding.
=== Consensus Sequence ===
The three 13-mer sequences identified as DUEs in E. coli, are well-conserved at the origin of replication of all documented enteric bacteria. A general consensus sequence was made via comparison of conserved bacteria to form an 11 base sequence, GATCTnTTnTTTT. E. coli contains 9 bases of the 11 base consensus sequence in its oriC, within the 13-mer sequences. These sequences are found exclusively at the single origin of replication; not anywhere else within the genome sequence.
== Eukaryotes ==
Eukaryotic replication mechanisms work in relatively similar ways to that of prokaryotes, but is under more finely-tuned regulation. There is a need to ensure that each DNA molecule is replicated only once and that this is occurring in the proper location at the proper time. Operates in response to extracellular signals that coordinate initiation of division, differently from tissue to tissue. External signals trigger replication in S phase via production of cyclins which activate cyclin-dependent kinases (CDK) to form complexes.
DNA replication in eukaryotes initiates upon origin recognition complex (ORC) binding to the origin. This occurs at G1 cell phase serving to drive the cell cycle forward into S phase. This binding allows for further factor binding to create a pre-replicative complex (pre-RC). Pre-RC triggered to initiate when cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) bind to it. Initiation complexes then allow for recruitment of MCM helicase activator Cdc45 and subsequent unwinding of duplex at origin.
Replication in eukaryotes is initiated at multiple sites on the sequence, forming multiple replication forks simultaneously. This efficiency is required with the large genomes that they need to replicate.
In eukaryotes, nucleosome structures can complicate replication initiation. They can block access of DUE-B's to the DUE, thus suppressing transcription initiation. Can impede on rate. The linear nature of eukaryotic DNA, vs prokaryotic circular DNA, though, is easier to unwind its duplex once has been properly unwound from nucleosome. Activity of DUE can be modulated by transcription factors like ABF1.
=== Yeast ===
A common yeast model system that well-represents eukaryotic replication is Saccharomyces cerevisiae. It possesses autonomously replicating sequences (ARSs) that are transformed and maintained well in a plasmid. Some of these ARSs are seen to act as replication origins. These ARSs are composed of three domains A, B, and C. The A domain is where the ARS consensu s sequence resides, coined an ACS. The B domain contains the DUE. Lastly, the C domain is necessary for facilitating protein-protein interactions. ARSs are found distributed across 16 chromosomes, repeated every 30–40 kb.
Between species, these ARS sequences are variable, but their A, B, and C domains are well conserved. Any alterations in the DUE (domain B) causes lower overall function of the ARS as a whole in replication initiation. This was found via studies using imino exchange and NMR spectroscopy.
=== Mammals ===
DUEs found in some mammalian replication origins to date. In general, very little mammalian origins of replication have been well-analyzed, so difficult to determine how prevalent the DUEs are, in their defined replication origins.
Human cells still have very little detailing of their origins. It is known that replication initiates in large initiation zone areas, associated with known proteins like the c-myc and β-globin gene. Ones with DUEs thought to act in nearly same way as yeast cells.
DUE in origin of plasmids in mammalian cells, SV40, found to be associated with a T-ag hexamer, that introduces opposite supercoiling to increase favourability of strand unwinding.
Mammals with DUEs have shown evidence of structure-forming abilities that provide single-stranded stability of unwound DNA. These include cruciforms, intramolecular triplexes, and more.
== DUE-binding proteins ==
DNA unwinding element proteins (DUE-Bs) are found in eukaryotes.
They act to initiate strand separation by binding to DUE. DUE-B sequence homologs found among a variety of animal species- fish, amphibians, and rodents. DUE-B's have disordered C-terminal domains that bind to the DUE by recognition of this C-terminus. No other sequence specificity involved in this interaction. Confirmed by inducing mutations along length of DUE-B sequence, but in all cases dimerization abilities remaining intact. Upon binding DNA, C-terminus becomes ordered, imparting a greater stability against protease degradation. DUE-B's are 209 residues in total, 58 of which are disordered until bound to DUE. DUE-B's hydrolyze ATP In order to function. Also possess similar sequence to aminoacyl-tRNA synthetase, and were previously classified a such. DUE-Bs form homodimers that create an extended beta-sheet secondary structure extending across it. Two of these homodimers come together to form the overall asymmetric DUE-B structure.
In formation of the pre-RC, Cdc45 is localized to the DUE for activity via interaction with a DUE-B. Allowing for duplex unwinding and replication initiation.
In humans, DUE-B's are 60 amino acids longer than its yeast ortholog counterparts. Both localized mainly in the nucleus.
DUE-B levels are in consistent quantity, regardless of cell cycle. In S phase though, DUE-Bs can be temporarily phosphorylated to prevent premature replication. DUE-B activity is covalently controlled. The assembly of these DUE-Bs at the DUE regions is dependent on local kinase and phosphatase activity. DUE-B's can also be down-regulated by siRNAs and have been implicated in extended G1 stages.
== Mutation Implications ==
Mutations that impair the unwinding at DUE sites directly impede DNA replication activity. This can be a result of deletions/changes in the DUE region, the addition of reactive reagents, or the addition of specific nuclease. DUE sites are relatively insensitive to point mutations though, maintaining their activity in when altering bases in protein binding sites. In many cases, DUE activity can be partially regained by increasing temperature. Can be regained by the re-addition of DUE site as well.
If there is a severe enough mutation to DUE causing it to no longer be bound to DUE-B, Cdc45 cannot associate and will not bind to c-myc transcription factor. This can be recovered in disease-related (ATTCT)(n) length expansions of the DUE sequence. If DUE activity regained in excess, could cause dysregulated origin formation and cell cycle progression.
In eukaryotes, when DUE-B's are knocked out, the cell will not go into S phase of its cycle, where DNA replication occurs. Increased apoptosis will result. But, activity can be rescued by re-addition of the DUE-B's, even from a different species. This is because DUE-B's are homologous between species. For example, if DUE-B in Xenopus egg are mutated, no DNA replication will occur, but can be saved by addition of HeLa DUE-B's to regain full functionality.
== References == | Wikipedia/DNA_unwinding_element |
DNA replication stress refers to the state of a cell whose genome is exposed to various stresses. The events that contribute to replication stress occur during DNA replication, and can result in a stalled replication fork.
There are many events that contribute to replication stress, including:
Misincorporation of ribonucleotides
Unusual DNA structures
Conflicts between replication and transcription
Insufficiency of essential replication factors
Common fragile sites
Overexpression or constitutive activation of oncogenes
Chromatin inaccessibility
ATM and ATR are proteins that help to alleviate replication stress. Specifically, they are kinases that are recruited and activated by DNA damage. The stalled replication fork can collapse if these regulatory proteins fail to stabilize it. When this occurs, reassembly of the fork is initiated in order to repair the damaged DNA end.
== Replication fork ==
The replication fork consists of a group of proteins that influence the activity of DNA replication. In order for the replication fork to stall, the cell must possess a certain number of stalled forks and arrest length. The replication fork is specifically paused due to the stalling of helicase and polymerase activity, which are linked together. In this situation, the fork protection complex (FPC) is recruited to help maintain this linkage.
In addition to stalling and maintaining the fork structure, protein phosphorylation can also create a signal cascade for replication restart. The protein Mrc1, which is part of the FPC, transmits the checkpoint signal by interacting with kinases throughout the cascade. When there is a loss of these kinases (from replication stress), an excess of ssDNA is produced, which is necessary for the restarting of replication.
== Replication block removal ==
DNA interstrand cross-links (ICLs) cause replication stress by blocking replication fork progression. This blockage leads to failure of DNA strand separation and a stalled replication fork. Repair of ICLs can be accomplished by sequential incisions, and homologous recombination. In vertebrate cells, replication of an ICL-containing chromatin template triggers recruitment of more than 90 DNA repair and genome maintenance factors. Analysis of the proteins recruited to stalled replication forks revealed a specific set of DNA repair factors involved in the replication stress response. Among these proteins, SLF1 and SLF2 were found to physically link the SMC5/6 DNA repair protein complex to RAD18. The SMC5/6 complex is employed in homologous recombination, and its linkage to RAD18 likely allows recruitment of SMC5/6 to ubiquitination products at sites of DNA damage.
=== Replication-coupled repair ===
Mechanisms that process damaged DNA in coordination with the replisome in order to maintain replication fork progression are considered to be examples of replication-coupled repair. In addition to the repair of DNA interstrand crosslinks, indicated above, multiple DNA repair processes operating in overlapping layers can be recruited to faulty sites depending on the nature and location of the damage. These repair processes include (1) removal of misincorporated bases; (2) removal of misincorporated ribonucleotides; (3) removal of damaged bases (e.g. oxidized or methylated bases) that block the replication polymerase; (4) removal of DNA-protein crosslinks; and (5) removal of double-strand breaks. Such repair pathways can function to protect stalled replication forks from degradation and allow restart of broken forks, but when deficient can cause replication stress.
=== Single-strand break repair ===
Singe-strand breaks are one of the most common forms of endogenous DNA damage. Replication fork collapse at leading strand nicks generates resected single-ended double-strand breaks that can be repaired by homologous recombination.
== Causation ==
Replication stress is induced from various endogenous and exogenous stresses, which are regularly introduced to the genome. These stresses include, but are not limited to, DNA damage, excessive compacting of chromatin (preventing replisome access), over-expression of oncogenes, or difficult-to-replicate genome structures. Replication stress can lead to genome instability, cancer, and ageing. Uncoordinated replication–transcription conflicts and unscheduled R-loop accumulation are significant contributors.
=== Specific events ===
The events that lead to genome instability occur in the cell cycle prior to mitosis, specifically in the S phase. Disturbance to this phase can generate negative effects, such as inaccurate chromosomal segregation, for the upcoming mitotic phase. The two processes that are responsible for damage to the S phase are oncogenic activation and tumor suppressor inactivation. They have both been shown to speed up the transition from the G1 phase to the S phase, leading to inadequate amounts of DNA replication components. These losses can contribute to the DNA damage response (DDR). Replication stress can be an indicative characteristic for carcinogenesis, which typically lacks DNA repair systems. A physiologically short duration of the G1 phase is also typical of fast replicating progenitors during early embryonic development.
== Applications in cancer ==
Normal replication stress occurs at low to mild levels and induces genomic instability, which can lead to tumorigenesis and cancer progression. However, high levels of replication stress have been shown to kill cancer cells.
In one study, researchers sought to determine the effects of inducing high levels of replication stress on cancer cells. The results showed that with further loss of checkpoints, replication stress is increased to a higher level. With this change, the DNA replication of cancer cells may be incomplete or incorrect when entering into the mitotic phase, which can eventually result in cell death through mitotic catastrophe.
Another study examined how replication stress affected APOBEC3B activity. APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3) has been seen to mutate the cancer genome in various cancer types. Results from this study show that weakening oncogenic signaling or intensifying DNA replication stress can alter carcinogenic potential, and can be manipulated therapeutically.
== References == | Wikipedia/DNA_replication_stress |
Prokaryotic DNA replication is the process by which a prokaryote duplicates its DNA into another copy that is passed on to daughter cells. Although it is often studied in the model organism E. coli, other bacteria show many similarities. Replication is bi-directional and originates at a single origin of replication (OriC). It consists of three steps: Initiation, elongation, and termination.
== Initiation ==
All cells must finish DNA replication before they can proceed for cell division. Media conditions that support fast growth in bacteria also couples with shorter inter-initiation time in them, i.e. the doubling time in fast growing cells is less as compared to the slow growth. In other words, it is possible that in fast growth conditions the grandmother cells starts replicating its DNA for grand daughter cell. For the same reason, the initiation of DNA replication is highly regulated. Bacterial origins regulate orisome assembly, a nuclei-protein complex assembled on the origin responsible for unwinding the origin and loading all the replication machinery. In E. coli, the direction for orisome assembly are built into a short stretch of nucleotide sequence called as origin of replication (oriC) which contains multiple binding sites for the initiator protein DnaA (a highly homologous protein amongst bacterial kingdom). DnaA has four domains with each domain responsible for a specific task. There are 11 DnaA binding sites/boxes on the E. coli origin of replication out of which three boxes R1, R2 and R4 (which have a highly conserved 9 bp consensus sequence 5' - TTATC/ACACA ) are high affinity DnaA boxes. They bind to DnaA-ADP and DnaA-ATP with equal affinities and are bound by DnaA throughout most of the cell cycle and forms a scaffold on which rest of the orisome assembles. The rest eight DnaA boxes are low affinity sites that preferentially bind to DnaA-ATP. During initiation, DnaA bound to high affinity DnaA box R4 donates additional DnaA to the adjacent low affinity site and progressively fill all the low affinity DnaA boxes. Filling of the sites changes origin conformation from its native state. It is hypothesized that DNA stretching by DnaA bound to the origin promotes strand separation which allows more DnaA to bind to the unwound region. The DnaC helicase loader then interacts with the DnaA bound to the single-stranded DNA to recruit the DnaB helicase, which will continue to unwind the DNA as the DnaG primase lays down an RNA primer and DNA Polymerase III holoenzyme begins elongation.
=== Regulation ===
Chromosome replication in bacteria is regulated at the initiation stage. DnaA-ATP is hydrolyzed into the inactive DnaA-ADP by RIDA (Regulatory Inactivation of DnaA), and converted back to the active DnaA-ATP form by DARS (DnaA Reactivating Sequence, which is itself regulated by Fis and IHF). However, the main source of DnaA-ATP is synthesis of new molecules. Meanwhile, several other proteins interact directly with the oriC sequence to regulate initiation, usually by inhibition. In E. coli these proteins include DiaA, SeqA, IciA, HU, and ArcA-P, but they vary across other bacterial species. A few other mechanisms in E. coli that variously regulate initiation are DDAH (datA-Dependent DnaA Hydrolysis, which is also regulated by IHF), inhibition of the dnaA gene (by the SeqA protein), and reactivation of DnaA by the lipid membrane.
== Elongation ==
Once priming is complete, DNA polymerase III holoenzyme is loaded into the DNA and replication begins. The catalytic mechanism of DNA polymerase III involves the use of two metal ions in the active site, and a region in the active site that can discriminate between deoxyribonucleotides and ribonucleotides. The metal ions are general divalent cations that help the 3' OH initiate a nucleophilic attack onto the alpha phosphate of the deoxyribonucleotide and orient and stabilize the negatively charged triphosphate on the deoxyribonucleotide. Nucleophilic attack by the 3' OH on the alpha phosphate releases pyrophosphate, which is then subsequently hydrolyzed (by inorganic phosphatase) into two phosphates. This hydrolysis drives DNA synthesis to completion.
Furthermore, DNA polymerase III must be able to distinguish between correctly paired bases and incorrectly paired bases. This is accomplished by distinguishing Watson-Crick base pairs through the use of an active site pocket that is complementary in shape to the structure of correctly paired nucleotides. This pocket has a tyrosine residue that is able to form van der Waals interactions with the correctly paired nucleotide. In addition, dsDNA (double-stranded DNA) in the active site has a wider major groove and shallower minor groove that permits the formation of hydrogen bonds with the third nitrogen of purine bases and the second oxygen of pyrimidine bases. Finally, the active site makes extensive hydrogen bonds with the DNA backbone. These interactions result in the DNA polymerase III closing around a correctly paired base. If a base is inserted and incorrectly paired, these interactions could not occur due to disruptions in hydrogen bonding and van der Waals interactions.
DNA is read in the 3' → 5' direction, therefore, nucleotides are synthesized (or attached to the template strand) in the 5' → 3' direction. However, one of the parent strands of DNA is 3' → 5' while the other is 5' → 3'. To solve this, replication occurs in opposite directions. Heading towards the replication fork, the leading strand is synthesized in a continuous fashion, only requiring one primer. On the other hand, the lagging strand, heading away from the replication fork, is synthesized in a series of short fragments known as Okazaki fragments, consequently requiring many primers. The RNA primers of Okazaki fragments are subsequently degraded by RNase H and DNA Polymerase I (exonuclease), and the gaps (or nicks) are filled with deoxyribonucleotides and sealed by the enzyme ligase.
=== Rate of replication ===
The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli. During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.
== Termination ==
Termination of DNA replication in E. coli is completed through the use of termination sequences and the Tus protein. These sequences allow the two replication forks to pass through in only one direction, but not the other.
DNA replication initially produces two catenated or linked circular DNA duplexes, each comprising one parental strand and one newly synthesised strand (by nature of semiconservative replication). This catenation can be visualised as two interlinked rings which cannot be separated. Topoisomerase 2 in E. coli unlinks or decatenates the two circular DNA duplexes by breaking the phosphodiester bonds present in two successive nucleotides of either parent DNA or newly formed DNA and thereafter the ligating activity ligates that broken DNA strand and so the two DNA get formed.
== Other prokaryotic replication models ==
=== Rolling circle replication ===
Rolling circle replication is seen in bacterial conjugation where the same circulartemplate DNA rotates and around it the new strand develops.
When conjugation is initiated by a signal, the relaxase enzyme creates a nick in one of the strands of the conjugative plasmid at the oriT. Relaxase may work alone or in a complex of over a dozen proteins known collectively as a relaxosome. In the F-plasmid system the relaxase enzyme is called TraI and the relaxosome consists of TraI, TraY, TraM and the integrated host factor IHF. The nicked strand, or T-strand, is then unwound from the unbroken strand and transferred to the recipient cell in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated either independent of conjugative action (vegetative replication beginning at the oriV) or in concert with conjugation (conjugative replication similar to the rolling circle replication of lambda phage). Conjugative replication may require a second nick before successful transfer can occur. A recent report claims to have inhibited conjugation with chemicals that mimic an intermediate step of this second nicking event.
=== D-loop replication ===
D-loop replication is mostly seen in organellar DNA, where a triple-stranded displacement loop is formed.
== References == | Wikipedia/Prokaryotic_DNA_replication |
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