The undermining of mutationism began almost at once, in 1902, as the statistician  Udny Yule  analysed Mendel's theory and showed that given full dominance of one allele over another, a 3:1 ratio of alleles would be sustained indefinitely. This meant that the recessive allele could remain in the population with no need to invoke mutation. He also showed that given multiple factors, Mendel's theory enabled continuous variation, as indeed Mendel had suggested, removing the central plank of the mutationist theory, and criticised Bateson's confrontational approach. [ 32 ]  However, the "excellent" [ 33 ]  paper did not prevent the  Mendelians and the biometricians  from falling out. [ 33 ]


The Swedish geneticist  H. Nilsson-Ehle  demonstrated in 1908, in a paper published in German in a Swedish journal,  Einige Ergebnisse von Kreuzungen bei Hafer und Weizen  (Observations on Crosses in Oats and Wheat), [ 34 ]  that continuous variation could readily be produced by multiple Mendelian genes. He found numerous Mendelian 3:1 ratios, implying a dominant and a recessive allele, in  oats  and  wheat ; a 15:1 ratio for a cross of oat varieties with black and white  glumes  respectively, implying two pairs of alleles (two Mendelian factors); and that crossing a red-grained Swedish velvet wheat with a white one gave in the third (F3) generation the complex signature of ratios expected of three factors at once, with 37 grains giving only red offspring, 8 giving 63:1 in their offspring, 12 giving 15:1, and 6 giving 3:1. There weren't any grains giving all white, but as he had only expected 1 of those in his sample, 0 was not an unlikely outcome. Genes could clearly combine in almost infinite combinations: ten of his factors allowed for almost 60,000 different forms, with no need to suppose that any new mutations were involved. The results implied that natural selection would work on Mendelian genes, helping to bring about the unification of Darwinian evolution and genetics. [ 35 ]


Similar work in America by  Edward East  on  maize  in 1910 [ 36 ]  showed the same thing for biologists without access to Nilsson-Ehle's work. [ 37 ]  On the same theme, the mathematician  Ronald Fisher  published " The Correlation Between Relatives on the Supposition of Mendelian Inheritance " in 1918, [ 38 ]  again showing that continuous variation could readily be produced by multiple Mendelian genes. It showed, too, that Mendelian inheritance had no essential link with mutationism: Fisher stressed that small variations (per gene) would be sufficient for natural selection to drive evolution. [ 39 ]


Starting in 1906,  William Castle   carried out a long study of the effect of selection on coat colour in  rats . The  piebald  or hooded pattern was recessive to the grey  wild type . He crossed hooded rats with the black-backed Irish type, and then back-crossed the offspring with pure hooded rats. The dark stripe on the back was bigger. He then tried selecting different groups for bigger or smaller stripes for 5 generations, and found that it was possible to change the characteristics way beyond the initial range of variation. This effectively refuted de Vries's claim that continuous variation could not be inherited permanently, requiring new mutations. By 1911 Castle noted that the results could be explained by Darwinian selection on heritable variation of Mendelian genes. [ 40 ]


By 1912, after years of work on the genetics of  Drosophila  fruit flies,  Thomas Hunt Morgan  showed that these animals had many small Mendelian factors on which Darwinian evolution could work as if variation was fully continuous. The way was open for geneticists to conclude that Mendelism supported Darwinism. [ 41 ] [ 42 ]


De Vries's mutationism was dealt a serious if not fatal blow in 1918 by the American geneticist  Hermann Joseph Muller . He compared the behaviour of balanced lethals in  Drosophila  with De Vries's supposed mutations in  Oenothera , showing that they could work the same way. [ 43 ]  No actual mutations were involved, but infrequent  chromosome  crossovers accounted for the sudden appearance of traits which had been present in the genes all along. [ 44 ]


In 1927, Fisher explicitly attacked Punnett's 1915 theory of discontinuous evolution of mimicry. Fisher argued that selection acting on genes making small modifications to the butterfly's  phenotype  (its appearance) would allow the multiple forms of a polymorphism to be established. [ 39 ]


The understanding that Mendelian genetics could both preserve discrete variations indefinitely, and support continuous variation for natural selection to work on gradually, meant that most biologists from around 1918 onwards accepted natural selection as the driving force of evolution. [ 45 ]  Mutationism and other alternatives to evolution by natural selection did not however vanish entirely. [ 46 ] [ 47 ] [ 48 ]


Lev Berg  proposed a combination of mutationism and directed (orthogenetic) evolution in his 1922 book  Nomogenesis ; or, Evolution Determined by Law . He used evidence from  paleontology ,  zoology , and  botany  to argue that natural selection had limitations which set a direction for evolution. He claimed that speciation was caused by "mass transformation of a great number of individuals" by directed mass mutations. [ 49 ] [ 46 ]


In 1923, the botanist  John Christopher Willis  proposed that species were formed by large mutations, not gradual evolution by natural selection, [ 50 ] [ 51 ]  and that evolution was driven by  orthogenesis , which he called "differentiation", rather than by natural selection. [ 47 ]


In his 1940 book  The Material Basis of Evolution , the German geneticist  Richard Goldschmidt  argued for single-step speciation by macromutation, describing the organisms thus produced as "hopeful monsters". Goldschmidt's thesis was universally rejected and widely ridiculed by biologists, who favoured the  neo-Darwinian  explanations of Fisher,  J. B. S. Haldane  and  Sewall Wright . [ 48 ] [ 53 ]  However, interest in Goldschmidt's ideas has reawakened in the field of  evolutionary developmental biology . [ 54 ] [ 55 ] [ 56 ] [ 57 ] [ 58 ]


Contemporary biologists accept that mutation and selection both play roles in evolution; the mainstream view is that while mutation supplies material for selection in the form of variation, all non-random outcomes are caused by natural selection. [ 59 ]   Masatoshi Nei  argues instead that the production of more efficient genotypes by mutation is fundamental for evolution, and that evolution is often mutation-limited. [ 52 ] [ 60 ] [ 61 ] [ 62 ]  Nei's book received thoughtful reviews; while Wright [ 63 ]  rejected Nei's thinking as mistaken, Brookfield, [ 64 ]  Galtier, [ 65 ]  Weiss, [ 66 ]  Stoltzfus, [ 52 ]  and Wagner, [ 59 ]  although not necessarily agreeing with Nei's position, treated it as a relevant alternative view.


Reviewing the history of macroevolutionary theories, the American evolutionary biologist  Douglas J. Futuyma  notes that since 1970, two very different alternatives to Darwinian gradualism have been proposed, both by  Stephen Jay Gould : mutationism, and  punctuated equilibria . [ 67 ] [ 68 ]  Gould's macromutation theory gave a nod to his predecessor with an envisaged "Goldschmidt break" between evolution within a species and speciation. His advocacy of Goldschmidt was attacked with "highly unflattering comments" [ 67 ]  by  Brian Charlesworth [ 69 ]  and  Alan Templeton . [ 70 ]  Futuyma concludes, following other biologists reviewing the field such as K.Sterelny [ 71 ]  and A. Minelli, [ 72 ]  that essentially all the claims of evolution driven by large mutations could be explained within the Darwinian evolutionary synthesis. [ 67 ]   James A. Shapiro 's claim that molecular genetics undermines Darwinism has been described as mutationism and an extreme view by the zoologist Andy Gardner. [ 73 ]


Cases of  mutation bias  are cited by mutationism advocates of the  extended evolutionary synthesis  who have argued that mutation bias is an entirely novel evolutionary principle. This viewpoint has been criticized by Erik Svensson. [ 74 ]  A 2019 review by Svensson and David Berger concluded that "we find little support for mutation bias as an independent force in adaptive evolution, although it can interact with selection under conditions of small population size and when standing genetic variation is limited, entirely consistent with standard evolutionary theory." [ 75 ]  In contrast to Svensson and Berger a 2023 review by Arlin Stoltzfus and colleagues concluded that there is strong empirical evidence and theoretical arguments that mutation bias has predictable effects on genetic changes fixed in adaptation. [ 76 ]


Biologists at the start of the 20th century broadly agreed that evolution occurred, but felt that the mechanisms suggested by Darwin, including natural selection, would be ineffective. Large mutations looked likely to drive evolution quickly, and avoided the difficulty which had rightly worried Darwin, namely that blending inheritance would average out any small favourable changes. [ d ] [ 78 ]  Further, large saltatory mutation, able to create species in a single step, offered a ready explanation of why the fossil record should contain large discontinuities and times of rapid change. [ 79 ]


These discoveries were often framed by supporters of the mid-20th century  modern synthesis , such as  Julian Huxley  and  Ernst Mayr , as a controversy between the early geneticists—the "Mendelians"—including Bateson, Johannsen, de Vries, Morgan, and Punnett, who advocated Mendelism and mutation, and were understood as opponents of Darwin's original gradualist view, and the biometricians such as Pearson and Weldon, who opposed Mendelism and were more faithful to Darwin. In this version, little progress was made during the  eclipse of Darwinism , and the debate between mutationist geneticists such as de Vries and biometricians such as Pearson ended with the victory of the modern synthesis between about 1918 and 1950. [ 80 ] [ 81 ]  According to this account, the new  population genetics  of the 1940s demonstrated the explanatory power of natural selection, while mutationism, alongside  other non-Darwinian approaches  such as  orthogenesis  and  structuralism , was essentially abandoned. [ 82 ]  This view became dominant in the second half of the 20th century, and was accepted by both biologists and historians. [ 83 ]


A more recent view, advocated by the historians Arlin Stoltzfus and Kele Cable, is that Bateson, de Vries, Morgan and Punnett had by 1918 formed a synthesis of Mendelism and mutationism. The understanding achieved by these geneticists spanned the action of natural selection on  alleles  (alternative forms of a gene), the  Hardy–Weinberg equilibrium , the evolution of continuously-varying traits (like height), and the probability that a new mutation will  become fixed . In this view, the early geneticists accepted natural selection alongside mutation, but rejected Darwin's non-Mendelian ideas about variation and heredity, and the synthesis began soon after 1900. [ 81 ] [ 84 ]  The traditional claim that Mendelians rejected the idea of continuous variation outright is simply false; as early as 1902, Bateson and  Edith Saunders  wrote that "If there were even so few as, say, four or five pairs of possible allelomorphs, the various  homo-  and  hetero-zygous  combinations might, on seriation, give so near an approach to a continuous curve, that the purity of the elements would be unsuspected". [ 85 ]


Historians have interpreted the history of mutationism in different ways. [ 80 ] [ 86 ] [ 26 ] [ 87 ] The classical view is that mutationism, opposed to Darwin's gradualism, was an obvious error; the decades-long delay in synthesizing genetics and Darwinism is an "inexplicable embarrassment"; [ 88 ]  genetics led logically to the modern synthesis and mutationism was one of several anti-Darwinian "blind alleys" separate from the main line leading from Darwin to the present. [ 89 ]
A revisionist view is that mutationists accepted both mutation and selection, with broadly the same roles they have today, and early on accepted and indeed offered a correct explanation for continuous variation based on multiple genes, paving the way for gradual evolution. At the time of the Darwin centennial in Cambridge in 1909, mutationism and  Lamarckism  were contrasted with natural selection as  competing ideas ; 50 years later, at the 1959 University of Chicago centennial of the publication of  On the Origin of Species , mutationism was no longer seriously considered. [ 90 ] [ 83 ]


Neutral mutations  are changes in  DNA  sequence that are neither beneficial nor detrimental to the ability of an organism to survive and reproduce. In  population genetics ,  mutations  in which  natural selection  does not affect the spread of the mutation in a species are termed neutral mutations. Neutral mutations that are inheritable and not  linked  to any genes under selection will be lost or will replace all other  alleles  of the gene. That loss or  fixation  of the gene proceeds based on random sampling known as  genetic drift . A neutral mutation that is in  linkage disequilibrium  with other alleles that are under selection may proceed to loss or fixation via  genetic hitchhiking  and/or  background selection .


While many mutations in a  genome  may decrease an organism’s ability to survive and reproduce, also known as  fitness , those mutations are selected against and are not passed on to  future generations . The most commonly-observed mutations that are detectable as variation in the genetic makeup of organisms and populations appear to have no visible effect on the fitness of individuals and are therefore neutral. The identification and study of neutral mutations has led to the development of the  neutral theory of molecular evolution , which is an important and often-controversial theory that proposes that most molecular variation within and among species is essentially neutral and not acted on by selection. Neutral mutations are also the basis for using  molecular clocks  to identify such evolutionary events as  speciation  and  adaptive  or  evolutionary radiations .


Charles Darwin  commented on the idea of neutral mutation in his work, hypothesizing that mutations that do not give an advantage or disadvantage may fluctuate or become fixed apart from  natural selection . "Variations neither useful nor injurious would not be affected by natural selection, and would be left either a fluctuating element, as perhaps we see in certain polymorphic species, or would ultimately become fixed, owing to the nature of the organism and the nature of the conditions."  While Darwin is widely credited with introducing the idea of natural selection which was the focus of his studies, he also saw the possibility for changes that did not benefit or hurt an organism. [ 1 ]


Darwin's view of change being mostly driven by traits that provide advantage was widely accepted until the 1960s. [ 2 ]   While researching mutations that produce nucleotide substitutions in 1968,  Motoo Kimura  found that the rate of substitution was so high that if each mutation improved fitness, the gap between the most fit and typical genotype would be  implausibly  large.  However, Kimura explained this rapid rate of mutation by suggesting that the majority of mutations were neutral, i.e. had little or no effect on the fitness of the organism.  Kimura developed mathematical models of the behavior of neutral mutations subject to random genetic drift in biological populations.  This theory has become known as the neutral theory of molecular evolution. [ 3 ]


As technology has allowed for better analysis of genomic data, research has continued in this area. While natural selection may encourage adaptation to a changing environment, neutral mutation may push divergence of species due to nearly random genetic drift. [ 2 ]


Neutral mutation has become a part of the neutral theory of molecular evolution, proposed in the 1960s.  This theory suggests that neutral mutations are responsible for a large portion of DNA sequence changes in a species. For example, bovine and human insulin, while differing in  amino acid  sequence are still able to perform the same function.  The amino acid substitutions between species were seen therefore to be neutral or not impactful to the function of the protein.  Neutral mutation and the neutral theory of molecular evolution are not separate from natural selection but add to Darwin's original thoughts.  Mutations can give an advantage, create a disadvantage, or make no measurable difference to an organism's survival. [ 4 ]


A number of observations associated with neutral mutation were predicted in neutral theory including:  amino acids with similar biochemical properties should be substituted more often than biochemically different amino acids; synonymous base substitutions should be observed more often than nonsynonymous substitutions;  introns  should evolve at the same rate as synonymous mutations in coding  exons ;  and  pseudogenes  should also evolve at a similar rate. These predictions have been confirmed with the introduction of additional genetic data since the theory’s introduction. [ 2 ]


When an incorrect  nucleotide  is inserted during  replication  or  transcription  of a coding region, it can affect the eventual  translation  of the sequence into amino acids.  Since multiple  codons  are used for the same amino acids, a change in a single base may still lead to translation of the same amino acid.  This phenomenon is referred to as  degeneracy  and allows for a variety of codon combinations leading to the same amino acid being produced.  For example, the codes TCT, TCC, TCA, TCG, AGT, and AGC all code for the amino acid  serine .  This can be explained by the wobble concept.   Francis Crick  proposed this theory to explain why specific tRNA molecules could recognize multiple codons.  The area of the tRNA that recognizes the codon called the anticodon is able to bind multiple interchangeable bases at its 5' end due to its spatial freedom.  A fifth base called  inosine  can also be substituted on a tRNA and is able to bind with A, U, or C.  This flexibility allows for changes in bases in codons leading to translation of the same amino acid. [ 5 ]   The changing of a base in a codon without the changing of the translated amino acid is called a synonymous mutation.  Since the amino acid translated remains the same a synonymous mutation has traditionally been considered a neutral mutation. [ 6 ]    Some research has suggested that there is bias in selection of base substitution in synonymous mutation. This could be due to selective pressure to improve translation efficiency associated with the most available  tRNAs  or simply mutational bias. [ 7 ]    If these mutations influence the rate of translation or an organism’s ability to manufacture protein they may actually influence the fitness of the affected organism. [ 6 ]


While substitution of a base in a noncoding area of a  genome  may make little difference and be considered neutral, base substitutions in or around  genes  may impact the organism.  Some base substitutions lead to synonymous mutation and no difference in the amino acid translated as noted above.  However, a base substitution can also change the  genetic code  so that a different amino acid is translated.  This sort of substitution usually has a negative effect on the protein being formed and will be eliminated from the population through  purifying selection .  However, if the change has a positive influence, the mutation may become more and more common in a population until it becomes a fixed genetic piece of that population.  Organisms changing via these two options comprise the classic view of natural selection.  A third possibility is that the amino acid substitution makes little or no positive or negative difference to the affected protein. [ 13 ]  Proteins demonstrate some tolerance to changes in amino acid structure.  This is somewhat dependent on where in the protein the substitution takes place.  If it occurs in an important structural area or in the  active site , one amino acid substitution may inactivate or substantially change the functionality of the protein.  Substitutions in other areas may be nearly neutral and drift randomly over time. [ 14 ]


Neutral mutations are measured in population and evolutionary genetics often by looking at variation in populations. These have been measured historically by  gel electrophoresis  to determine  allozyme  frequencies. [ 15 ]  Statistical analyses of this data is used to compare variation to predicted values based on population size, mutation rates and effective population size. Early observations that indicated higher than expected  heterozygosity  and overall variation within the protein isoforms studied, drove arguments as to the role of selection in maintaining this variation versus the existence of variation through the effects of neutral mutations arising and their random distribution due to genetic drift. [ 16 ] [ 17 ] [ 18 ]  The accumulation of data based on observed polymorphism led to the formation of the neutral theory of evolution. [ 16 ]  According to the neutral theory of evolution, the rate of fixation in a population of a neutral mutation will be directly related to the rate of formation of the neutral allele. [ 19 ]


In Kimura’s original calculations, mutations with |2 N s |<1 or | s |≤1/(2N) are defined as neutral. [ 16 ] [ 18 ]  In this equation, N is the  effective population size  and is a quantitative measurement of the ideal population size that assumes such constants as equal sex ratios and no emigration, migration, mutation nor selection. [ 20 ]  Conservatively, it is often assumed that effective population size is approximately one fifth of the total population size. [ 21 ]   s  is the  selection coefficient  and is a value between 0 and 1.  It is a measurement of the contribution of a genotype to the next generation where a value of 1 would be completely selected against and make no contribution and 0 is not selected against at all. [ 22 ]  This definition of neutral mutation has been criticized due to the fact that very large effective population sizes can make mutations with small selection coefficients appear non neutral.  Additionally, mutations with high selection coefficients can appear neutral in very small populations. [ 18 ]   The testable hypothesis of Kimura and others showed that polymorphism within species are approximately that which would be expected in a neutral evolutionary model. [ 18 ] [ 23 ] [ 24 ]


For many molecular biology approaches, as opposed to mathematical genetics, neutral mutations are generally assumed to be those mutations that cause no appreciable effect on gene function.  This simplification eliminates the effect of minor allelic differences in fitness and avoids problems when a selection has only a minor effect. [ 18 ]


Early convincing evidence of this definition of neutral mutation was shown through the lower mutational rates in functionally important parts of genes such as cytochrome c versus less important parts [ 25 ]  and the functionally interchangeable nature of mammalian cytochrome c in in vitro studies. [ 26 ]  Nonfunctional pseudogenes provide more evidence for the role of neutral mutations in evolution.  The rates of mutation in mammalian globin pseudogenes has been shown to be much higher than rates in functional genes. [ 27 ] [ 28 ]  According to neo-Darwinian evolution, such mutations should rarely exist as these sequences are functionless and positive selection would not be able to operate. [ 18 ]


The  McDonald-Kreitman test [ 29 ]   has been used to study selection over long periods of evolutionary time. This is a statistical test that compares polymorphism in neutral and functional sites and estimates what fraction of substitutions have been acted on by positive selection. [ 30 ]   The test often uses synonymous substitutions in protein coding genes as the neutral component; however, synonymous mutations have been shown to be under purifying selection in many instances. [ 31 ] [ 32 ]


Molecular clocks  can be used to estimate the amount of time since divergence of two species and for placing evolutionary events in time. [ 33 ]   Pauling  and  Zuckerkandl , proposed the idea of the molecular clock in 1962 based on the observation that the random mutation process occurs at an approximate constant rate. Individual proteins were shown to have linear rates of amino acid changes over evolutionary time. [ 34 ]  Despite controversy from some biologists arguing that morphological evolution would not proceed at a constant rate, many amino acid changes were shown to accumulate in a constant fashion.  Kimura and  Ohta  explained these rates as part of the framework of the neutral theory. These mutations were reasoned to be neutral as positive selection should be rare and deleterious mutations should be eliminated quickly from a population. [ 35 ]   By this reasoning, the accumulation of these neutral mutations should only be influenced by the mutation rate.  Therefore, the neutral mutation rate in individual organisms should match the molecular evolution rate in species over evolutionary time.  The neutral mutation rate is affected by the amount of neutral sites in a protein or DNA sequence versus the amount of mutation in sites that are functionally constrained.  By quantifying these neutral mutations in protein and/or DNA and comparing them between species or other groups of interest, rates of divergence can be determined. [ 33 ] [ 36 ]


Molecular clocks have caused controversy due to the dates they derive for events such as explosive radiations seen after extinction events like the  Cambrian explosion  and the radiations of mammals and birds.  Two-fold differences exist in dates derived from molecular clocks and the fossil record.  While some paleontologists argue that molecular clocks are systemically inaccurate, others attribute the discrepancies to lack of robust fossil data and bias in sampling. [ 37 ]  While not without constancy and discrepancies with the fossil record, the data from molecular clocks have shown how evolution is dominated by the mechanisms of a neutral model and is less influenced by the action of natural selection. [ 33 ]


In  genetics , a  nonsense mutation  is a  point mutation  in a  sequence  of  DNA  that results in a  nonsense codon , or a premature  stop codon  in the  transcribed   mRNA , and leads to a truncated, incomplete, and possibly nonfunctional  protein  product. [ 1 ]  Nonsense mutations are not always harmful; [ 2 ]  the functional effect of a nonsense mutation depends on many aspects, such as the location of the  stop codon  within the coding  DNA . [ 2 ]  For example, the effect of a nonsense mutation depends on the proximity of the nonsense mutation to the original stop codon, and the degree to which functional subdomains of the protein are affected. [ 3 ]  As nonsense mutations leads to premature termination of  polypeptide chains ; they are also called chain termination mutations. [ 4 ]


Missense mutations  differ from nonsense mutations since they are  point mutations  that exhibit a single  nucleotide  change to cause substitution of a different  amino acid . A nonsense mutation also differs from a  nonstop mutation , which is a point mutation that removes a stop codon. About 10% of patients facing genetic diseases have involvement with nonsense mutations. [ 5 ]  Some of the diseases that these mutations can cause are  Duchenne muscular dystrophy  (DMD),  cystic fibrosis  (CF), [ 6 ]   spinal muscular atrophy  (SMA),  cancers ,  metabolic diseases , and  neurologic disorders. [ 5 ] [ 7 ]  The rate of nonsense mutations is variable from gene-to-gene and tissue-to-tissue, but gene silencing occurs in every patient with a nonsense mutation. [ 5 ]


The example above begins with a 5'  DNA sequence  with 24  nucleotides  (8 triplet codons) seen and its complementary strand shown below. The next row highlights the 5'  mRNA  strand, which is generated through  transcription . Lastly, the final row showcases which the  amino acids  that are  translated  from each respective  codon , with the eighth and final codon representing the  stop codon . The codons corresponding to the fourth amino acid,  Arginine  (Arg), are highlighted because they will undergo a nonsense mutation in the following figure of this example.


Now, suppose that a nonsense mutation was introduced at the fourth codon in the 5′ DNA sequence (CGA) causing the  cytosine  to be replaced with  thymine , yielding TGA in the 5′ DNA sequence and ACT in the complementary strand. Because ACT is transcribed as UGA, it is translated as a stop codon. This leads the remaining codons of the mRNA to not be translated into protein because the stop codon is prematurely reached during translation. This can yield a truncated ( i.e. , abbreviated) protein product, which quite often lacks the functionality of the normal, non-mutant protein. [ 1 ]


A AG (Lys)  →  U AG (stop)
 C AG (Gln)  →  U AG (stop)
 G AG (Glu)  →  U AG (stop)
 U C G (Ser)  →  U A G (stop)
 U G G (Trp)  →  U A G (stop)
 U U G (Leu)  →  U A G (stop)
 UA C  (Tyr)  →  UA G  (stop)
 UA U  (Tyr)  →  UA G  (stop)


A AA (Lys)  →  U AA (stop)
 C AA (Gln)  →  U AA (stop)
 G AA (Glu)  →  U AA (stop)
 U C A (Ser)  →  U A A (stop)
 U U A (Leu)  →  U A A (stop)
 UA C  (Tyr)  →  UA A  (stop)
 UA U  (Tyr)  →  UA A  (stop)


A GA (Arg)  →  U GA (stop)
 C GA (Arg)  →  U GA (stop)
 G GA (Gly)  →  U GA (stop)
 U C A (Ser)  →  U G A (stop)
 U U A (Leu)  →  U G A (stop)
 UG C  (Cys)  →  UG A  (stop)
 UG G  (Trp)  →  UG A  (stop)
 UG U  (Cys)  →  UG A  (stop)


Deleterious outcomes represent the majority of nonsense mutations and are the most common outcome that is observed naturally. Deleterious nonsense mutations decreases the overall  fitness  and  reproductive success  of the  organism . [ 8 ]  For example, a nonsense mutation occurring in a  gene  encoding a protein can cause structural or functional defects in the protein that disrupt  cellular biology.  Depending on the significance of the functions of this protein, this disruption now could be detrimental to the fitness and survival of that organism. [ 8 ]


When a nonsense mutation is neutral, it does not provide benefits or harm. These occur when the effects of the mutation are unnoticed. In other words, this means that the mutation does not positively or negatively affect the organism. As this effect is unnoticed, there is a lack of papers describing such mutations. An example of this type of nonsense mutation is one that occurs directly before the original stop codon for that given protein. [ 8 ]  Because this mutation occurred in such close proximity to the end of the protein chain, the impact of this change might not be as significant. This would suggest that this amino acid that was mutated did not have a large impact on the overall structure or function of the protein or the organism as a whole. This scenario is rare, but possible. [ 8 ]


Beneficial nonsense mutations are considered as the rarest of possible nonsense mutation outcomes. Beneficial nonsense mutations increase the overall fitness and reproductive success of an organism, opposite of the effects of a deleterious mutation. [ 2 ] [ 8 ]  Because a nonsense mutation introduces a premature stop codon within a sequence of DNA, it is extremely unlikely that this scenario can actually benefit the organism. [ 1 ]  An example of this would occur with a nonsense mutation that impacts a dysfunctional protein that releases  toxins . The stop codon that this mutation brings would stop this dysfunctional protein from properly carrying out its function. Stopping this protein from performing at full strength causes less toxin to be released and the fitness of the organism to be improved. These types of situations with nonsense mutations occur a lot less frequently than the deleterious outcomes. [ 8 ]


Nonsense-mediated mRNA decay


Despite an expected tendency for premature termination codons to yield shortened polypeptide products, in fact the formation of truncated proteins does not occur often  in vivo . Many organisms—including humans and lower species, such as  yeast —employ a  nonsense-mediated mRNA decay  pathway, which degrades mRNAs containing nonsense mutations before they are able to be translated into nonfunctional polypeptides.


tRNA Suppression


Because nonsense mutations result in altered mRNA with a premature stop codon, one way of suppressing the damage done to the final protein's function is to alter the tRNA that reads the mRNA. These  tRNA ’s are termed  suppressor tRNA's . If the stop codon is UAG, any other amino acid tRNA could be altered from its original  anticodon  to AUC so it will recognize the UAG codon instead. This will result in the protein not being truncated, but it may still have an altered amino acid. These suppressor tRNA mutations are only possible if the cell has more than one tRNA that reads a particular codon, otherwise the mutation would kill the cell. The only stop codons are UAG, UAA, and UGA. UAG and UAA suppressors read their respective stop codons instead of their original codon, but UAA suppressors also read UAG due to  wobble base  pairing. UGA suppressors are very rare. Another hurdle to pass in this technique is the fact that stop codons are also recognized by  release factors , so the tRNA still needs to compete with the release factors to keep the translation going. Because of this, suppression is usually only 10-40% successful. These suppressor tRNA mutations also target stop codons that are not mutations, causing some proteins to be much longer than they should be. Only bacteria and lower  eukaryotes  can survive with these mutations, mammal and insect cells die as a result of a suppressor mutation. [ 4 ]


For historical reasons the three stop codons were given names (see  Stop codons ): UAG is called the amber codon, UAA is called the ochre codon, and UGA is called the opal codon. [ 10 ]


Nonsense mutations comprise around 20% of single nucleotide substitutions within protein coding sequences that result in human disease. [ 12 ]  Nonsense mutation-mediated  pathology  is often attributed to reduced amounts of full-length protein, because only 5-25% of transcripts possessing nonsense mutations do not undergo  nonsense-mediated decay  (NMD). [ 13 ] [ 12 ]  Translation of the remaining nonsense-bearing mRNA may generate abbreviated protein variants with toxic effects. [ 14 ]


Twenty-three different single-point nucleotide substitutions are capable of converting a non-stop codon into a stop-codon, with the mutations  CGA

 ⟶

 {\displaystyle \longrightarrow }

 TGA and CAG

 ⟶

 {\displaystyle \longrightarrow }

 TAG being the most common disease-related substitutions characterized in the Human Gene Mutation Database (HGMD). [ 12 ]  As a result of different substitution frequencies for each nucleotide, the proportions of the three stop codons generated by disease-inducing nonsense mutations differs from stop codon distributions in non-diseased gene variants. [ 12 ]  Notably, the codon TAG is overrepresented, while the TGA and TAA codons are underrepresented in disease-related nonsense mutations. [ 12 ]


Translation termination efficiency is influenced by the specific stop codon sequence on the mRNA, with the UAA sequence yielding the highest termination. [ 15 ]  Sequences surrounding the stop codon also impact termination efficiency. [ 15 ]  Consequently, the underlying pathology of diseases caused by nonsense mutations is ultimately dependent on the identity of the mutated gene, and specific location of the mutation.


Examples of diseases induced by nonsense mutations include:


Nonsense mutations in other genes may also drive dysfunction of several tissue or organ systems:


SMAD8  is the eighth homolog of the ENDOGLIN gene family and is involved in the signaling between  TGF-b/BMP . It has been identified that novel nonsense mutations in SMAD8 are associated with  pulmonary arterial hypertension. [ 16 ]  The pulmonary system relies on SMAD1, SMAD5, and SMAD 8 to regulate pulmonary vascular function.  Downregulation  and loss of signals that are normally operated by SMAD8 contributed to  pathogenesis  in pulmonary arterial hypertension. [ 16 ]  The  ALK1  gene, a part of the TGF-B signaling family, was found to have been mutated while also down-regulating the SMAD8 gene in patients with pulmonary arterial hypertension. [ 16 ]  SMAD8 mutants were not  phosphorylated  by ALK1, disrupting interactions with SMAD4 that would normally allow for signaling in  wild-type  organisms. [ 16 ]


LGR4  binds  R-spondins  to activate the  Wnt signaling pathway . [ 17 ]  Wnt signaling regulates bone mass and  osteoblast   differentiation  and is important for the development of bone, heart, and muscle. [ 17 ]  An LGR4 nonsense mutation in a healthy population has been linked to low bone mass density and symptoms of  osteoporosis . LGR4  mutant  mice showed the observed low bone mass is not due to age-related bone loss. [ 17 ]  Mutations in LGR4 have been associated with family lineages with medical histories of rare bone disorders. [ 17 ]  Wild-type mice lacking LGR4 also displayed delayed  osteoblast  differentiation during development, showcasing the important role of LGR4 in bone mass regulation and development. [ 17 ]


Therapeutics for diseases caused by nonsense mutations attempt to recapitulate wild-type function by decreasing the efficacy of NMD, facilitating readthrough of the premature stop codon during translation, or editing the genomic nonsense mutation. [ 18 ]


Antisense oligonucleotides  to suppress the expression of NMD and translation termination proteins are being explored in animal models of nonsense mutation-induced disease. [ 18 ] [ 19 ]  Other RNA therapeutics under investigation include synthetic suppressor tRNAs that enable  ribosomes  to insert an amino acid, instead of initiating chain termination, upon encountering premature stop codons. [ 18 ]


CRISPR-Cas9  based single nucleotide substitutions have been used to generate amino acid codons from stop codons, achieving an editing success rate of 10% in cell cultures. [ 20 ]


Read-through has been achieved using small molecule drugs such as  aminoglycosides  and negamycin. [ 18 ]  An  oxadiazole ,  ataluren  (previously PTC124), facilitates the selective read-through of aberrant stop codons, rendering it a potential therapeutic against nonsense mutation-induced disease. [ 21 ]  Ataluren, sold under the tradename Translarna, is currently an approved treatment for Duchenne muscular dystrophy in the  European Economic area  and  Brazil . [ 22 ] [ 23 ]  However, phase III trials of Ataluren as a cystic fibrosis therapeutic have failed to meet their primary endpoints. [ 24 ] [ 25 ]


A  nonsynonymous substitution  is a  nucleotide   mutation  that alters the  amino acid  sequence of a  protein . Nonsynonymous substitutions differ from  synonymous substitutions , which do not alter amino acid sequences and are (sometimes)  silent mutations . As nonsynonymous substitutions result in a biological change in the organism, they are subject to  natural selection .


Nonsynonymous substitutions at a certain locus can be compared to the synonymous substitutions at the same locus to obtain the  K a /K s  ratio . This ratio is used to measure the evolutionary rate of gene sequences. [ 1 ]  If a gene has lower levels of nonsynonymous than synonymous nucleotide substitution, then it can be inferred to be functional because a  K a /K s  ratio  < 1 is a hallmark of sequences that are being constrained to code for proteins.


[ 2 ]  Nonsynonymous substitutions are also referred to as  replacement mutations .


There are several common types of nonsynonymous substitutions. [ 3 ]


Missense mutations  are nonsynonymous substitutions that arise from  point mutations , mutations in a single nucleotide that result in the substitution of a different  amino acid , resulting in a change to the protein encoded.


Nonsense mutations  are nonsynonymous substitutions that arise when a mutation in the DNA sequence causes a protein to terminate prematurely by changing the original amino acid to a  stop codon . Another type of mutation that deals with stop codons is known as a  nonstop mutation  or readthrough mutation, which occurs when a stop codon is exchanged for an amino acid codon, causing the protein to be longer than specified. [ 3 ]


Studies have shown that diversity among nonsynonymous substitutions is significantly lower than among synonymous substitutions. [ 4 ]  This is due to the fact that nonsynonymous substitutions are subject to much higher selective pressures than synonymous mutations. [ 5 ]  Motoo Kimura (1968) determined that calculated mutation rates were impossibly high, unless most of the mutations that occurred were either neutral or "nearly neutral". [ 3 ]  He determined that if this were true,  genetic drift  would be a more powerful factor in  molecular evolution  than natural selection. [ 6 ]  The "nearly neutral" theory proposes that molecular evolution acting on nonsynonymous substitutions is driven by mutation, genetic drift, and very weak natural selection, and that it is extremely sensitive to population size. [ 7 ]  In order to determine whether natural selection is taking place at a certain loci, the  McDonald–Kreitman test  can be performed. [ 8 ]  The test consists of comparing ratios of synonymous and nonsynonymous genes between closely related species to the ratio of synonymous to nonsynonymous polymorphisms within species. If the ratios are the same, then  Neutral theory of molecular evolution  is true for that loci, and evolution is proceeding primarily through genetic drift. If there are more nonsynonymous substitutions between species than within a species, positive natural selection is occurring on beneficial alleles and natural selection is taking place. [ 3 ]  Nonsynonymous substitutions have been found to be more common in loci involving pathogen resistance, reproductive loci involving sperm competition or egg-sperm interactions, and genes that have replicated and gained new functions, indicating that positive selection is taking place. [ 3 ]


Research on accurately modeling rates of mutation has been conducted for many years. A recent paper by Ziheng Yang and Rasmus Nielsen compared various methods and developed a new modeling method. They found that the new method was preferable for its smaller biases, which make it useful for large scale screening, but that the maximum-likelihood model was preferable in most scenarios because of its simplicity, and its flexibility in comparing multiple sequences while taking into account phylogeny. [ 9 ]


Further research by Yang and Nielsen found that nonsynonymous to synonymous substitution ratios varied across loci in differing evolutionary lineages. During their study of nuclear loci of primates, even-toed ungulates, and rodents, they found that the ratio varied significantly at 22 of the 48 loci studied. This result provides strong evidence against a strictly neutral theory of  molecular evolution , which states that mutations are mostly neutral or deleterious, and provides support for theories that include advantageous mutations. [ 10 ]


NSP1  (NS53), the product of  rotavirus  gene 5, is a  nonstructural  RNA-binding protein that contains a cysteine-rich region and is a component of early replication intermediates. RNA- folding  predictions suggest that this region of the NSP1  mRNA  can interact with itself, producing a stem-loop structure similar to that found near the 5'-terminus of the NSP1  mRNA . [ 1 ]


The carboxyl-half of the  rotavirus  nonstructural protein NSP1 is not required for virus replication. [ 2 ]


NSP1 could play a role in host range restriction. [ 3 ]


The cysteine-rich region of NSP1 is not considered essential for genome segment reassortment with heterologous virus. [ 4 ]


NSP1 interacts with  IRF3  in the infected cell. NSP1 is an antagonist of the IFN-signaling pathway. [ 5 ]


Interferon regulatory factor 3 ( IRF3 ) is a key transcription factor involved in the induction of interferon (IFN) in response to viral infection. NSP1 binds to and targets IRF3 for  proteasome  degradation early post-infection. IRF3 degradation is dependent on the presence of NSP1 and the integrity of the N-terminal zinc-binding domain, coupled with the regulated stability of IRF3 and NSP1 by the proteasome, collectively support the hypothesis that NSP1 is an E3 ubiquitin ligase. [ 6 ]


NSP1 could mediates the degradation of  IRF3 ,  IRF5 , and  IRF7  by recognizing a common element of IRF proteins, thereby allowing NSP1 to act as a broad-spectrum antagonist of IRF function. [ 7 ]


NSP1 also inhibits activation of  NFkappaB [ 8 ]


NSP1 inhibits cellular apoptosis by directly interacting p85 subunit of PI3K and thus activating PI3K/Akt survival pathway during early stages of rotavirus infection. [ 9 ] [ 10 ]


An  obligate carrier  is an individual who may be clinically unaffected but who must carry a  gene mutation  based on analysis of the family history; usually applies to disorders inherited in an  autosomal  recessive and X-linked recessive manner. [ 1 ] [ 2 ]


In  X-linked recessive  disorders, only females can be the  carriers  of the recessive mutation, making them obligate carriers of this type of disease. Females acquire one  X-chromosome  from their father and one from their mother, and this means they can either be  heterozygous  for the mutated  allele  or homozygous. If heterozygous, she is a carrier of the mutated allele because the disease is recessive. If homozygous, she has the disease. An affected father with an X-linked recessive trait will always pass the trait on to the daughter. Therefore, all daughters of an affected male are obligate carriers. On the other hand, a carrier mother has a 50% chance of passing her mutated X-chromosome to the daughter. [ 3 ]  This makes all daughters of carrier mothers possible carriers but not necessarily obligate carriers. Males cannot be obligate or possible carriers of X-linked recessive traits because they only have one X-chromosome, and so are always phenotypically affected when receiving the mutated X-chromosome from their mother. [ 4 ]


Females that are heterozygous for X-linked recessive disorders are obligate carriers, but can never be  phenotypically  affected, and this is because of  X-inactivation . Heterozygous females have an X-chromosome from each parent; one with a mutated gene and one with a functional copy of the same gene. When the mutated chromosome is randomly inactivated in order to maintain the  copy number , presence of the functional copy results in a normal  phenotype . [ 5 ]  Males only have one copy of any gene on the X-chromosome, and because they do not undergo X-inactivation, they only have the mutated gene. As a result, these types of diseases most commonly phenotypically affect males and rarely females.


Hemophilia, or  haemophilia , is an X-linked recessive disorder that impairs the body's control over  blood clotting .  Haemophilia A  and  Haemophilia B  arise from mutations in the genes for  factor VIII  and  factor IX , respectively. [ 6 ]  Females with this disease are almost exclusively unaffected, obligate carriers. The mutations can be passed on to offspring by mothers and fathers, but the phenotype is only expressed in males that inherit the mutation. [ 7 ]  All daughters of a hemophiliac father are obligate carriers of the disease.


In an  autosomal recessive  disease, if an individual is heterozygous for the mutant allele, they are a carrier because the disease is recessive. If homozygous, they have the disease. All offspring of an affected individual are either heterozygous or homozygous for the mutated allele. Consequently, all unaffected (heterozygous) offspring of an affected individual are obligate carriers of the disease because they will necessarily carry the mutated allele. [ 8 ]


Due to the predictable patterns of heritable disorders, techniques can be used to detect past, present, and future disease prevalence in individuals among a family. Specifically,  pedigrees  and laboratory methods are used to search for and predict obligate carriers for a specific disease such as hemophilia. After analysis of family history, one way to be completely sure that an individual is an obligate carrier is through  genetic tests , such as mutational analysis. This allows professionals to see if the specific  mutation  exists in the chromosome of the individual. [ 9 ] [ 10 ]  In potential hemophiliacs, factor  assays  are used to measure the amount of blood clotting in an individual. However, some carriers might have completely normal clotting levels and so this method is not always useful. [ 9 ]   Genetic counselling  informs patients that may have a family history of a certain disease about their risk of disease and potential risk in their children.


Phenotypic heterogeneity  describes different mutations in the same  gene  that can sometimes give rise to strikingly different  phenotypes .


E.g., certain loss-of-function mutations in the  RET gene , which encodes a receptor  tyrosine   kinase , can cause dominantly inherited failure of development of  colonic   ganglia , leading to defective colonic  motility  and severe chronic constipation ( Hirschsprung disease ). [ 1 ] [ 2 ] [ 3 ]


A  point accepted mutation  — also known as a PAM — is the replacement of a single  amino acid  in the  primary structure  of a  protein  with another single amino acid, which is accepted by the processes of  natural selection . This definition does not include all  point mutations  in the  DNA  of an organism. In particular,  silent mutations  are not point accepted mutations, nor are mutations that are lethal or that are rejected by natural selection in other ways.


A  PAM matrix  is a  matrix  where each column and row represents one of the twenty standard amino acids. In  bioinformatics , PAM matrices are sometimes used as  substitution matrices  to score  sequence alignments  for proteins. Each entry in a PAM matrix indicates the likelihood of the amino acid of that row being replaced with the amino acid of that column through a series of one or more point accepted mutations during a specified evolutionary interval, rather than these two amino acids being aligned due to chance. Different PAM matrices correspond to different lengths of time in the evolution of the protein sequence.


The genetic instructions of every replicating  cell  in a living organism are contained within its DNA. [ 1 ]  Throughout the cell's lifetime, this information is transcribed and replicated by cellular mechanisms to produce proteins or to provide instructions for daughter cells during  cell division , and the possibility exists that the DNA may be altered during these processes. [ 1 ] [ 2 ]  This is known as a  mutation . At the molecular level, there are regulatory systems that correct most — but not all — of these changes to the DNA before it is replicated. [ 2 ] [ 3 ]


One of the possible mutations that occurs is the replacement of a single  nucleotide , known as a point mutation. If a point mutation occurs within an expressed region of a  gene , an  exon , then this will change the  codon  specifying a particular amino acid in the protein produced by that gene. [ 2 ]  Despite the redundancy in the  genetic code , there is a possibility that this mutation will then change the amino acid that is produced during  translation , and as a consequence the structure of the protein will be changed.


The functionality of a protein is highly dependent on its structure. [ 4 ]  Changing a single amino acid in a protein may reduce its ability to carry out this function, or the mutation may even change the function that the protein carries out. [ 2 ]  Changes like these may severely impact a crucial function in a cell, potentially causing the cell — and in extreme cases, the organism — to die. [ 5 ]  Conversely, the change may allow the cell to continue functioning albeit differently, and the mutation can be passed on to the organism's offspring. If this change does not result in any significant physical disadvantage to the offspring, the possibility exists that this mutation will persist within the population. The possibility also exists that the change in function becomes advantageous. In either case, while being subjected to the processes of natural selection, the point mutation has been accepted into the genetic pool.


The 20 amino acids translated by the genetic code vary greatly by the physical and chemical properties of their side chains. [ 4 ]  However, these amino acids can be categorised into groups with similar physicochemical properties. [ 4 ]  Substituting an amino acid with another from the same category is more likely to have a smaller impact on the structure and function of a protein than replacement with an amino acid from a different category. Consequently, acceptance of point mutations depends heavily on the amino acid being replaced in the mutation, and the replacement amino acid. The PAM matrices are a mathematical tool that account for these varying rates of acceptance when evaluating the similarity of proteins during alignment.


The term  accepted point mutation  was initially used to describe the mutation phenomenon. However, the acronym PAM was preferred over APM due to readability, and so the term  point accepted mutation  is used more regularly. [ 6 ]  Because the value

 n

 {\displaystyle n}

  in the PAM n  matrix represents the number of mutations per 100 amino acids, which can be likened to a percentage of mutations, the term  percentage accepted mutation  is sometimes used.


It is important to distinguish between point accepted mutations (PAMs), point accepted mutation matrices (PAM matrices) and the PAM n  matrix. The term 'point accepted mutation' refers to the mutation event itself. However, 'PAM matrix' refers to one of a family of matrices which contain scores representing the likelihood of two amino acids being aligned due to a series of mutation events, rather than due to random chance. The 'PAM n  matrix' is the PAM matrix corresponding to a time frame long enough for

 n

 {\displaystyle n}

  mutation events to occur per 100 amino acids.


PAM matrices were introduced by  Margaret Dayhoff  in 1978. [ 7 ]  The calculation of these matrices was based on 1572 observed mutations in the  phylogenetic trees  of 71 families of closely related proteins. The proteins to be studied were selected on the basis of having high similarity with their predecessors. The protein alignments included were required to display at least 85% identity. [ 6 ] [ 8 ]  As a result, it is reasonable to assume that any aligned mismatches were the result of a single mutation event, rather than several at the same location.


Each PAM matrix has twenty rows and twenty columns — one representing each of the twenty amino acids translated by the genetic code. The value in each cell of a PAM matrix is related to the probability of a row amino acid before the mutation being aligned with a column amino acid afterwards. [ 6 ] [ 7 ] [ 8 ]  From this definition, PAM matrices are an example of a  substitution matrix .


For each branch in the phylogenetic trees of the protein families, the number of mismatches that were observed were recorded and a record kept of the two amino acids involved. [ 7 ]  These counts were used as entries below the main diagonal of the matrix

 A

 {\displaystyle A}

 . Since the vast majority of protein samples come from organisms that are alive today (extant species), the 'direction' of a mutation cannot be determined. That is, the amino acid present before the mutation cannot be distinguished from the amino acid that replaced it after the mutation. Because of this, the matrix

 A

 {\displaystyle A}

  is assumed to be  symmetric , and the entries of

 A

 {\displaystyle A}

  above the main diagonal are computed on this basis. The entries along the diagonal of

 A

 {\displaystyle A}

  do not correspond to mutations and can be left unfilled.


In addition to these counts, data on the mutability and the frequency of the amino acids was obtained. [ 6 ] [ 7 ]  The mutability of an amino acid is the ratio of the number of mutations it is involved in and the number of times it occurs in an alignment. [ 7 ]  Mutability measures how likely an amino acid is to mutate acceptably.  Asparagine , an amino acid with a small  polar  side chain, was found to be the most mutable of the amino acids. [ 7 ]   Cysteine  and  tryptophan  were found to be the least mutable amino acids. [ 7 ]  The side chains for cysteine and tryptophan have less common structures: cysteine's side chain contains sulfur which participates in  disulfide bonds  with other cysteine molecules, and tryptophan's side chain is large and  aromatic . [ 4 ]  Since there are several small polar amino acids, these extremes suggest that amino acids are more likely to acceptably mutate if their physical and chemical properties are more common among alternative amino acids. [ 6 ] [ 8 ]


For the

 j

 {\displaystyle j}

 th amino acid, the values

 m
 (
 j
 )

 {\displaystyle m(j)}

  and

 f
 (
 j
 )

 {\displaystyle f(j)}

  are its mutability and frequency. The frequencies of the amino acids are normalised so that they sum to 1. If total number of occurrences of the

 j

 {\displaystyle j}

 th amino acid is

 n
 (
 j
 )

 {\displaystyle n(j)}

 , and

 N

 {\displaystyle N}

  is the total number of all amino acids, then


Based on the definition of mutability as the ratio of mutations to occurrences of an amino acid


or


The mutation matrix

 M

 {\displaystyle M}

  is constructed so that the entry

 M
 (
 i
 ,
 j
 )

 {\displaystyle M(i,j)}

  represents the probability of the

 j

 {\displaystyle j}

 th amino acid mutating into the

 i

 {\displaystyle i}

 th amino acid. The non-diagonal entries are computed by the equation [ 7 ]


where

 λ

 {\displaystyle \lambda }

  is a constant of proportionality. However, this equation does not compute the diagonal entries. Each column in the matrix

 M

 {\displaystyle M}

  lists each of the twenty possible outcomes for an amino acid — it can mutate into one of the 19 other amino acids, or remain unchanged. Since the non-diagonal entries listing the probabilities of each of the 19 mutations are known, and the sum of the probabilities of these twenty outcomes must be 1, this last probability can be calculated by


which simplifies to [ 7 ]


Substituting in the expression for the non-diagonal entries mutation matrix:


Since the values of

 λ

 {\displaystyle \lambda }

  and

 m
 (
 j
 )

 {\displaystyle m(j)}

  are constants that don't change with the value of

 i

 {\displaystyle i}


And thus cancellation reveals that


A result of particular significance is that for the non-diagonal entries


Which means that for all entries in the mutation matrix


The probabilities contained in

 M

 {\displaystyle M}

  vary as some unknown function of the amount of time that a protein sequence is allowed to mutate for. Instead of attempting to determine this relationship, the values of

 M

 {\displaystyle M}

  are calculated for a short time frame, and the matrices for longer periods of time are calculated by assuming mutations follow a  Markov chain  model. [ 9 ] [ 10 ]  The base unit of time for the PAM matrices is the time required for 1 mutation to occur per 100 amino acids, sometimes called 'a PAM unit' or 'a PAM' of time. [ 6 ]  This is precisely the duration of mutation assumed by the PAM 1  matrix.


The constant

 λ

 {\displaystyle \lambda }

  is used to control the proportion of amino acids that are unchanged. By using only alignments of proteins that had at least 85% similarity, it could be reasonably assumed that the mutations observed were direct, without any intermediate states. This means that scaling down these counts by a common factor would provide an accurate estimate of the mutation counts had the similarity been closer to 100%. It also means that the number of mutations per 100 amino acids, the

 n

 {\displaystyle n}

  in PAM n  is equal to the number of mutated amino acids per 100 amino acids.


To find the mutation matrix for the PAM 1  matrix, the requirement that 99% of the amino acids in a sequence are conserved is imposed. The quantity

 n
 (
 j
 )
 M
 (
 j
 ,
 j
 )

 {\displaystyle n(j)M(j,j)}

  is equal to the number of conserved amino acid

 j

 {\displaystyle j}

  units, and so the total number of conserved amino acids is


The value of

 λ

 {\displaystyle \lambda }

  needed to be pick to produce 99% identity after mutation is then given by the equation


This

 λ

 {\displaystyle \lambda }

  value can then be used in the mutation matrix for the PAM 1  matrix.


The Markov chain model of protein mutation relates the mutation matrix for PAM n ,

 M

 n

 {\displaystyle M_{n}}

 , to the mutation matrix for the PAM 1  matrix,

 M

 1

 {\displaystyle M_{1}}

  by the simple relationship


The PAM n  matrix is constructed from the ratio of the probability of point accepted mutations replacing the

 j

 {\displaystyle j}

 th amino acid  with the

 i

 {\displaystyle i}

 th amino acid, to the probability of these amino acids being aligned by chance.
The entries of the PAM n  matrix are given by the equation [ 11 ] [ 12 ]


Note that in Gusfield's book, the entries

 M
 (
 i
 ,
 j
 )

 {\displaystyle M(i,j)}

  and

 PAM

 n

 (
 i
 ,
 j
 )

 {\displaystyle {\text{PAM}}_{n}(i,j)}

  are related to the probability of the

 i

 {\displaystyle i}

 th amino acid mutating into the

 j

 {\displaystyle j}

 th amino acid. [ 11 ]  This is the origin of the different equation for the entries of the PAM matrices.


When using the PAM n  matrix to score an alignment of two proteins, the following assumption is made:


When the alignment of the

 i

 {\displaystyle i}

 th and

 j

 {\displaystyle j}

 th amino acids is considered, the score indicates the relative likelihoods of the alignment due to the proteins being related or due to random chance.


While the mutation probability matrix

 M

 {\displaystyle M}

  is not symmetric, each of the PAM matrices are. [ 6 ] [ 7 ]  This somewhat surprising property is a result of the relationship that was noted for the mutation probability matrix:


In fact, this relationship holds for all positive integer powers of the matrix

 M

 {\displaystyle M}

 :


This generalisation can be proven using  mathematical induction . Suppose that for a matrix

 M

 {\displaystyle M}


And that for a positive integer

 k

 {\displaystyle k}


By expansion of the  matrix product

 M

 k
 +
 1

 =

 M

 k

 ⋅
 M

 {\displaystyle M^{k+1}=M^{k}\cdot M}

 ,


Using the property we have assumed of the matrix

 M

 {\displaystyle M}


And using the property for the matrix

 M

 k

 {\displaystyle M^{k}}


In this case, it is only known at first that the result holds for

 k
 =
 1

 {\displaystyle k=1}

 . However, the above argument shows that the property also holds for

 k
 =
 2

 {\displaystyle k=2}

 . This new knowledge then shows that the property also holds for

 k

 {\displaystyle k}

  and this repeats to show that the property holds for all positive integers

 k

 {\displaystyle k}

 .


As a result, the entries of the PAM n  matrix are symmetric, since


The value

 n

 {\displaystyle n}

  represents the number of mutations that occur per 100 amino acids, however this value is rarely accessible and often estimated. However, when comparing two proteins it is easy to calculate

 m

 {\displaystyle m}

  instead, which is the number of mutated amino acids per 100 amino acids. Despite the random nature of mutation, these values can be approximately related by [ 13 ]


Mutations in the primary structure of a protein can occur anywhere along the sequence. If it is assumed the distribution of the mutations among amino acid positions is uniform, the problem is analogous to a distribution of "balls into bins", a common problem in  combinatorics . In a case where

 K

 {\displaystyle K}

  balls (i.e. mutations) are distributed amongst

 N

 {\displaystyle N}

  bins (amino acid positions), the number of bins containing at least one ball,

 M

 {\displaystyle M}

  has a distribution with a mean given by [ 14 ]


If the rate of mutation is

 n

 {\displaystyle n}

  mutations per 100 amino acids, then


And if there are

 m

 {\displaystyle m}

  mutated amino acids per 100 amino acids, then it is approximately equal to


Now

 m

 {\displaystyle m}

  and

 n

 {\displaystyle n}

  can be related by


For large values of

 N

 {\displaystyle N}

 , an assumption that can be reasonably made for typical proteins, this expression is approximately equal to


The validity of these estimates can be verified by counting the number of amino acids that remain unchanged under the action of the matrix

 M

 {\displaystyle M}

 . The total number of unchanged amino acids for the time interval of the PAM n  matrix is


and so the proportion of unchanged amino acids is


A PAM250 is a commonly used scoring matrix for sequence comparison. Only the lower half of the matrix needs to be computed, since by their construction, PAM matrices are required to be symmetric. Each of the 20 amino acid are shown down the top and side of the matrix, with 3 additional  ambiguous amino acids . The amino acids are most commonly shown listed alphabetically, or listed in groups. These  groups  are the characteristics shared among the amino acids. [ 7 ]


The  molecular clock hypothesis  predicts that the rate of amino acid substitution in a particular protein will be approximately constant over time, though this rate may vary between protein families. [ 13 ]  This suggests that the number of mutations per amino acid in a protein increases approximately linearly with time.


Determining the time at which two proteins diverged is an important task in  phylogenetics .  Fossil records  are often used to establish the position of events on the timeline of the Earth's evolutionary history, but the application of this source is  limited . However, if the rate at which the molecular clock of protein family ticks — that is, the rate at which the number of mutations per amino acid increases — is known, then knowing this number of mutations would allow the date of divergence to be found.


Suppose the date of divergence for two related proteins, taken from organisms living today, is sought. The two proteins have both been accumulating accepted mutations since the date of divergence, and so the total number of mutations per amino acid separating them is approximately twice that which separates them from their  common ancestor . If a range of PAM matrices are used to align two proteins that are known to be related, then the value of

 n

 {\displaystyle n}

  in the PAM n  matrix which results in the best score is most likely to correspond to the mutations per amino acid separating the two proteins. Halving this value and dividing by the rate at which accepted mutations accumulate in the protein family provides an estimate of the time of divergence of these two proteins from their common ancestor. That is, the time of divergence in  myr  is [ 13 ]


Where

 K

 {\displaystyle K}

  is the number of mutations per amino acid, and

 r

 {\displaystyle r}

  is the rate of accepted mutation accumulation in mutations per amino acid site per million years.


PAM matrices are also used as a scoring matrix when comparing DNA sequences or protein sequences to judge the quality of the alignment. This form of scoring system is utilized by a wide range of alignment software including  BLAST . [ 15 ]


Although the PAM log-odds matrices were the first scoring matrices used with BLAST, the PAM matrices have largely been replaced by the  BLOSUM  matrices. Although both matrices produce similar scoring outcomes they were generated using differing methodologies. The BLOSUM matrices were generated directly from the amino acid differences in aligned blocks that have diverged to varying degrees the PAM matrices reflect the  extrapolation  of evolutionary information based on closely related sequences to longer timescales. [ 16 ]  Since scoring information for the PAM and BLOSUM matrices were generated in very different ways the numbers associated with the matrices have fundamentally different meanings; the numbers for PAM matrices increase for comparisons among more divergent proteins whereas the numbers for the BLOSUM matrices decrease. [ 17 ]  However, all amino acid substitution matrices can be compared in an information theoretic framework [ 18 ]  using their relative entropy.


A  point mutation  is a genetic  mutation  where a single nucleotide base is changed, inserted or deleted from a  DNA  or  RNA  sequence of an organism's genome. [ 1 ]  Point mutations have a variety of effects on the downstream protein product—consequences that are moderately predictable based upon the specifics of the mutation. These consequences can range from no effect (e.g.  synonymous mutations ) to deleterious effects (e.g.  frameshift mutations ), with regard to protein production, composition, and function.


Point mutations usually take place during  DNA replication . DNA replication occurs when one double-stranded DNA molecule creates two single strands of DNA, each of which is a template for the creation of the complementary strand. A single point mutation can change the whole DNA sequence. Changing one  purine  or  pyrimidine  may change the amino acid that the  nucleotides  code for.


Point mutations may arise from spontaneous  mutations  that occur during  DNA replication . The rate of mutation may be increased by  mutagens . Mutagens can be physical, such as radiation from  UV rays ,  X-rays  or extreme heat, or chemical (molecules that misplace base pairs or disrupt the helical shape of DNA). Mutagens associated with cancers are often studied to learn about cancer and its prevention.


There are multiple ways for point mutations to occur. First,  ultraviolet (UV) light  and higher-frequency light have ionizing capability, which in turn can affect DNA. Reactive oxygen molecules with free radicals, which are a byproduct of cellular metabolism, can also be very harmful to DNA. These reactants can lead to both single-stranded and double-stranded DNA breaks. Third, bonds in DNA eventually degrade, which creates another problem to keep the integrity of DNA to a high standard. There can also be replication errors that lead to substitution, insertion, or deletion mutations.


In 1959  Ernst Freese  coined the terms "transitions" or "transversions" to categorize different types of point mutations. [ 2 ] [ 3 ]  Transitions are replacement of a  purine  base with another  purine  or replacement of a  pyrimidine  with another pyrimidine. Transversions are replacement of a purine with a pyrimidine or vice versa. There is a systematic difference in mutation rates for  transitions  (Alpha) and  transversions  (Beta).  Transition mutations are about ten times more common than transversions.


Nonsense mutations  include stop-gain and start-loss. Stop-gain is a mutation that results in a premature  termination codon  ( a stop was gained ), which signals the end of translation.  This interruption causes the protein to be abnormally shortened. The number of amino acids lost mediates the impact on the protein's functionality and whether it will function whatsoever. [ 4 ]  Stop-loss is a mutation in the original termination codon ( a stop was lost ), resulting in abnormal extension of a protein's carboxyl terminus. Start-gain creates an AUG start codon upstream of the original start site. If the new AUG is near the original start site, in-frame within the processed transcript and downstream to a ribosomal binding site, it can be used to initiate translation. The likely effect is additional amino acids added to the amino terminus of the original protein. Frame-shift mutations are also possible in start-gain mutations, but typically do not affect translation of the original protein. Start-loss is a point mutation in a transcript's AUG start codon, resulting in the reduction or elimination of protein production.


Missense mutations  code for a different amino acid.  A missense mutation changes a codon so that a different protein is created, a non-synonymous change. [ 4 ]  Conservative mutations result in an amino acid change.  However, the properties of the amino acid remain the same (e.g., hydrophobic, hydrophilic, etc.).  At times, a change to one amino acid in the protein is not detrimental to the organism as a whole.  Most proteins can withstand one or two point mutations before their function changes. Non-conservative mutations result in an amino acid change that has different properties than the  wild type .  The protein may lose its function, which can result in a disease in the organism. For example,  sickle-cell disease  is caused by a single point mutation (a missense mutation) in the beta- hemoglobin   gene  that converts a GAG  codon  into GUG, which encodes the  amino acid   valine  rather than  glutamic acid . The protein may also exhibit a "gain of function" or become activated, such is the case with the mutation changing a valine to glutamic acid in the  BRAF  gene; this leads to an activation of the RAF protein which causes unlimited proliferative signalling in cancer cells. [ 5 ]  These are both examples of a non-conservative (missense) mutation.


Silent mutations  code for the same amino acid (a " synonymous substitution ").  A silent mutation does not affect the functioning of the  protein . A single nucleotide can change, but the new codon specifies the same amino acid, resulting in an unmutated protein.  This type of change is called synonymous change since the old and new codon code for the same amino acid.  This is possible because 64 codons specify only 20 amino acids.  Different codons can lead to differential protein expression levels, however. [ 4 ]


Sometimes the term  point mutation  is used to describe  insertions  or deletions of a single base pair (which has more of an adverse effect on the synthesized protein due to the nucleotides' still being read in triplets, but in different frames: a mutation called a  frameshift mutation ). [ 4 ]


Point mutations that occur in non-coding sequences are most often without consequences, although there are exceptions. If the mutated base pair is in the  promoter  sequence of a gene, then the expression of the gene may change. Also, if the mutation occurs in the splicing site of an  intron , then this may interfere with correct splicing of the transcribed  pre-mRNA .


By altering just one amino acid, the entire  peptide  may change, thereby changing the entire protein. The new protein is called a  protein variant . If the original protein functions in cellular reproduction then this single point mutation can change the entire process of cellular reproduction for this organism.


Point  germline mutations  can lead to beneficial as well as harmful traits or diseases. This leads to  adaptations  based on the environment where the organism lives. An advantageous mutation can create an advantage for that organism and lead to the trait's being passed down from generation to generation, improving and benefiting the entire population.  The scientific theory of  evolution  is greatly dependent on point mutations in  cells . The theory explains the diversity and history of living organisms on Earth. In relation to point mutations, it states that beneficial mutations allow the organism to thrive and reproduce, thereby passing its positively affected mutated genes on to the next generation. On the other hand, harmful mutations cause the organism to die or be less likely to reproduce in a phenomenon known as  natural selection .


There are different short-term and long-term effects that can arise from mutations. Smaller ones would be a halting of the cell cycle at numerous points.   This means that a codon coding for the amino acid  glycine  may be changed to a stop codon, causing the proteins that should have been produced to be deformed and unable to complete their intended tasks. Because the mutations can affect the DNA and thus the  chromatin , it can prohibit mitosis from occurring due to the lack of a complete chromosome.  Problems can also arise during the processes of transcription and replication of DNA.  These all prohibit the cell from reproduction and thus lead to the death of the cell.  Long-term effects can be a permanent changing of a chromosome, which can lead to a mutation.  These mutations can be either beneficial or detrimental.   Cancer  is an example of how they can be detrimental. [ 6 ]


Other effects of point mutations, or single nucleotide polymorphisms in DNA, depend on the location of the mutation within the gene.  For example, if the mutation occurs in the region of the gene responsible for coding, the amino acid sequence of the encoded protein may be altered, causing a change in the function, protein localization, stability of the protein or protein complex.  Many methods have been proposed to predict the effects of missense mutations on proteins. Machine learning algorithms train their models to distinguish known disease-associated from neutral mutations whereas other methods do not explicitly train their models but almost all methods exploit the evolutionary conservation assuming that changes at conserved positions tend to be more deleterious. While majority of methods provide a binary classification of effects of mutations into damaging and benign, a new level of annotation is needed to offer an explanation of why and how these mutations damage proteins. [ 7 ]


Moreover, if the mutation occurs in the region of the gene where transcriptional machinery binds to the protein, the mutation can affect the binding of the transcription factors because the short nucleotide sequences recognized by the transcription factors will be altered.  Mutations in this region can affect rate of efficiency of gene transcription, which in turn can alter levels of mRNA and, thus, protein levels in general.


Point mutations can have several effects on the behavior and reproduction of a protein depending on where the mutation occurs in the amino acid sequence of the protein. If the mutation occurs in the region of the gene that is responsible for coding for the protein, the amino acid may be altered.  This slight change in the sequence of amino acids can cause a change in the function, activation of the protein meaning how it binds with a given enzyme, where the protein will be located within the cell, or the amount of free energy stored within the protein.


If the mutation occurs in the region of the gene where transcriptional machinery binds to the protein, the mutation can affect the way in which transcription factors bind to the protein.  The mechanisms of transcription bind to a protein through recognition of short nucleotide sequences.  A mutation in this region may alter these sequences and, thus, change the way the transcription factors bind to the protein.  Mutations in this region can affect the efficiency of gene transcription, which controls both the levels of mRNA and overall protein levels. [ 8 ]


Point mutations in multiple tumor suppressor proteins cause  cancer . For instance, point mutations in Adenomatous Polyposis Coli promote tumorigenesis. [ 9 ]  A novel assay,  Fast parallel proteolysis (FASTpp) , might help swift screening of specific stability defects in individual cancer patients. [ 10 ]


Neurofibromatosis  is caused by point mutations in the  Neurofibromin 1 [ 11 ] [ 12 ]  or  Neurofibromin 2  gene. [ 13 ]


Sickle-cell anemia  is caused by a point mutation in the β-globin chain of hemoglobin, causing the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine at the sixth position.


The β-globin gene is found on the short arm of chromosome 11. The association of two wild-type α-globin subunits with two mutant β-globin subunits forms hemoglobin S (HbS). Under low-oxygen conditions (being at high altitude, for example), the absence of a polar amino acid at position six of the β-globin chain promotes the non-covalent polymerisation (aggregation) of hemoglobin, which distorts red blood cells into a sickle shape and decreases their elasticity. [ 14 ]


Hemoglobin  is a protein found in red blood cells, and is responsible for the transportation of oxygen through the body. [ 15 ]  There are two subunits that make up the hemoglobin protein:  beta-globins  and  alpha-globins . [ 16 ]
Beta-hemoglobin is created from the genetic information on the HBB, or "hemoglobin, beta" gene found on chromosome 11p15.5. [ 17 ]   A single point mutation in this polypeptide chain, which is 147 amino acids long, results in the disease known as Sickle Cell Anemia. [ 18 ]
Sickle-cell anemia is an autosomal recessive disorder that affects 1 in 500 African Americans, and is one of the most common blood disorders in the United States. [ 17 ]   The single replacement of the sixth amino acid in the beta-globin, glutamic acid, with valine results in deformed red blood cells.  These sickle-shaped cells cannot carry nearly as much oxygen as normal red blood cells and they get caught more easily in the capillaries, cutting off blood supply to vital organs.  The single nucleotide change in the beta-globin means that even the smallest of exertions on the part of the carrier results in severe pain and even heart attack.  Below is a chart depicting the first thirteen amino acids in the normal and abnormal  sickle cell  polypeptide chain. [ 18 ]


The cause of  Tay–Sachs disease  is a genetic defect that is passed from parent to child. This genetic defect is located in the HEXA gene, which is found on chromosome 15.


The HEXA gene makes part of an enzyme called beta-hexosaminidase A, which plays a critical role in the nervous system. This enzyme helps break down a fatty substance called GM2 ganglioside in nerve cells.
Mutations in the HEXA gene disrupt the activity of beta-hexosaminidase A, preventing the breakdown of the fatty substances. As a result, the fatty substances accumulate to deadly levels in the brain and spinal cord.  The buildup of GM2 ganglioside causes progressive damage to the nerve cells.  This is the cause of the signs and symptoms of Tay-Sachs disease. [ 19 ]


In  molecular biology ,  repeat-induced point mutation  or  RIP  is a process by which  DNA  accumulates  G : C  to  A : T   transition  mutations. Genomic evidence indicates that RIP occurs or has occurred in a variety of fungi [ 20 ]  while experimental evidence indicates that RIP is active in  Neurospora crassa , [ 21 ]   Podospora anserina , [ 22 ]   Magnaporthe grisea , [ 23 ]   Leptosphaeria maculans , [ 24 ]   Gibberella zeae [ 25 ]  and  Nectria haematococca . [ 26 ]  In  Neurospora crassa , sequences mutated by RIP are often  methylated   de novo . [ 21 ]


RIP occurs during the sexual stage in  haploid nuclei  after fertilization but prior to  meiotic   DNA replication . [ 21 ]  In  Neurospora crassa ,  repeat sequences  of at least 400  base pairs  in length are vulnerable to RIP. Repeats with as low as 80%  nucleotide  identity may also be subject to RIP. Though the exact mechanism of repeat recognition and mutagenesis are poorly understood, RIP results in repeated sequences undergoing multiple  transition mutations .


The RIP mutations do not seem to be limited to repeated sequences. Indeed, for example, in the phytopathogenic fungus  L. maculans , RIP mutations are found in single copy regions, adjacent to the repeated elements. These regions are either non-coding regions or genes encoding small secreted proteins including avirulence genes.
The degree of RIP within these single copy regions was proportional to their proximity to repetitive elements. [ 27 ]


Rep and Kistler have speculated that the presence of highly repetitive regions containing transposons, may promote mutation of resident effector genes. [ 28 ]  So the presence of effector genes within such regions is suggested to promote their adaptation and diversification when exposed to strong selection pressure. [ 29 ]


As RIP mutation is traditionally observed to be restricted to repetitive regions and not single copy regions, Fudal  et al. [ 30 ]   suggested that leakage of RIP mutation might occur within a relatively short distance of a RIP-affected repeat. Indeed, this has been reported in  N. crassa  whereby leakage of RIP was detected in single copy sequences at least 930 bp from the boundary of neighbouring duplicated sequences. [ 31 ]
To elucidate the mechanism of detection of repeated sequences leading to RIP may allow to understand how the flanking sequences may also be affected.


RIP causes  G : C  to  A : T   transition  mutations within repeats, however, the mechanism that detects the repeated sequences is unknown. RID is the only known protein essential for RIP. It is a DNA methyltransferease-like protein, that when mutated or knocked out results in loss of RIP. [ 32 ]  Deletion of the  rid  homolog in  Aspergillus nidulans ,  dmtA , results in loss of fertility [ 33 ]  while deletion of the  rid  homolog in  Ascobolus immersens ,  masc1 , results in fertility defects and loss of  methylation induced premeiotically (MIP) . [ 34 ]


RIP is believed to have evolved as a defense mechanism against  transposable elements , which resemble  parasites  by invading and multiplying within the genome.
RIP creates multiple  missense  and  nonsense mutations  in the coding sequence. This hypermutation of G-C to A-T in repetitive sequences eliminates functional  gene products  of the sequence (if there were any to begin with). In addition, many of the C-bearing nucleotides become  methylated , thus decreasing transcription.


Because RIP is so efficient at detecting and mutating repeats, fungal biologists often use it as a tool for  mutagenesis . A second copy of a single-copy  gene  is first  transformed  into the  genome . The fungus must then  mate  and go through its sexual cycle to activate the RIP machinery. Many different mutations within the duplicated gene are obtained from even a single fertilization event so that inactivated alleles, usually due to  nonsense mutations , as well as alleles containing  missense mutations  can be obtained. [ 35 ]


The cellular reproduction process of  meiosis  was discovered by  Oscar Hertwig  in 1876.   Mitosis  was discovered several years later in 1882 by  Walther Flemming .


Hertwig studied sea urchins, and noticed that each egg contained one nucleus prior to fertilization and two nuclei after.  This discovery proved that one spermatozoon could fertilize an egg, and therefore proved the process of meiosis.  Hermann Fol continued Hertwig's research by testing the effects of injecting several spermatozoa into an egg, and found that the process did not work with more than one spermatozoon. [ 36 ]


Flemming began his research of cell division starting in 1868.  The study of cells was an increasingly popular topic in this time period.  By 1873, Schneider had already begun to describe the steps of cell division.  Flemming furthered this description in 1874 and 1875 as he explained the steps in more detail.  He also argued with Schneider's findings that the nucleus separated into rod-like structures by suggesting that the nucleus actually separated into threads that in turn separated.  Flemming concluded that cells replicate through cell division, to be more specific mitosis. [ 37 ]


Matthew Meselson  and  Franklin Stahl  are credited with the discovery of  DNA replication .   Watson  and  Crick  acknowledged that the structure of DNA did indicate that there is some form of replicating process.  However, there was not a lot of research done on this aspect of DNA until after Watson and Crick.  People considered all possible methods of determining the replication process of DNA, but none were successful until Meselson and Stahl.  Meselson and Stahl introduced a heavy isotope into some DNA and traced its distribution.  Through this experiment, Meselson and Stahl were able to prove that DNA reproduces semi-conservatively. [ 38 ]


A  polar mutation  affects expression of downstream  genes  or  operons . It can also affect the expression of the gene in which it occurs, if it occurs in a transcribed region. These mutations tend to occur early within the sequence of genes and can be  nonsense ,  frameshift , or  insertion  mutations. Polar mutations are found only in organisms containing  polycistronic mRNA . [ 1 ] [ 2 ] [ 3 ]


3GOY ,  3Q6Z ,  3Q71 ,  3SE2 ,  3SMI ,  3SMJ ,  3VFQ ,  4ABK ,  4ABL ,  4D86 ,  4F1L ,  4F1Q ,  4PY4


Q460N5 Q8N546


NP_060024 NP_060024.2


Poly(ADP-ribose) polymerase family member 14  is a  protein  that, in  humans , is encoded by the PARP14  gene .
 [ 5 ]


Poly(ADP-ribosyl)ation is an immediate  DNA  damage-dependent  post-translational modification  of  histones  and other  nuclear proteins  that contributes to the survival of injured proliferating cells. PARP14 belongs to the superfamily of  enzymes  that perform this modification (Ame et al., 2004 [PubMed 15273990]).


A  polyglutamine tract  or  polyQ tract  is a portion of a  protein  consisting of a sequence of several  glutamine  units. A tract typically consists of about 10 to a few hundred such units.


A multitude of  genes , in various eukaryotic species (including humans), contain a number of repetitions of the  nucleotide  triplet  C A G  or  C A A . When the gene is  translated  into a protein, each of these triplets gives rise to a glutamine unit, resulting in a polyglutamine tract. Different  alleles  of such a gene often have different numbers of triplets since the highly repetitive sequence is prone to contraction and expansion.


Several inheritable  neurodegenerative disorders , the  polyglutamine diseases , occur if a  mutation  causes a polyglutamine tract in a specific gene to become too long. Important examples of polyglutamine diseases are  spinocerebellar ataxia  and  Huntington's disease .   Trinucleotide repeat expansion  occurring in a parental  germline  cell can lead to children that are more affected or display an earlier onset and greater severity of the condition.  Trinucleotide repeat expansion  is considered to be a consequence of  slipped strand mispairing  either during  DNA replication  or  DNA repair  synthesis. [ 1 ]  It is believed that cells cannot properly dispose of proteins with overlong polyglutamine tracts, which over time leads to damage in  nerve cells . The longer the polyglutamine tract, the earlier in life these diseases tend to appear. [ 2 ]


Nucleotide sequences encoding a lengthy polyQ tract were first noted in the gene encoding the  Notch receptor . [ 3 ]  Variation of the length of this  Notch  polyQ tract, as caused by triplet repeat instability, was later found to cause  developmental  defects. [ 4 ]  The significance of similarly expanded tracts in humans became evident when polyQ tracts were found to underlie  Huntington's disease  and several  spinocerebellar ataxias . In general, several  neurodegenerative disorders  were found to involve  nucleotide repeat expansions  in  protein  coding sequences.


Polymorphism in microRNA Target Site  ( PolymiRTS ) is a database of naturally occurring  DNA variations  in putative  microRNA  target sites. [ 1 ]


A  postzygotic mutation  (or  post-zygotic mutation ) is a change in an organism's  genome  that is acquired during its lifespan, instead of being inherited from its parent(s) through  fusion  of two  haploid   gametes . Mutations that occur after the zygote has formed can be caused by a variety of sources that fall under two classes: spontaneous mutations and induced mutations. How detrimental a mutation is to an organism is dependent on what the mutation is, where it occurred in the genome and when it occurred. [ 1 ]


Postzygotic changes to a genome can be caused by small mutations that affect a single base pair, or large mutations that affect entire chromosomes and are divided into two classes, spontaneous mutations and induced mutations. [ 2 ]


Most spontaneous mutations are the result of naturally occurring lesions to DNA and errors during DNA replication without direct exposure to an agent. [ 2 ]  A few common spontaneous mutations are:


Induced mutations are any lesions in DNA caused by an agent or mutagen.  Mutagens  often demonstrate mutational specificity, meaning they cause predictable changes in the DNA sequence. [ 5 ]  A few common mutagens that induce mutations are:


A large determinant of the severity of consequences caused by postzygotic mutations is when and where they occur. As a result, consequences can range from being negligible to incredibly detrimental. [ 7 ]


When an individual has inherited an abnormality it is usually present in all of their cells. However some mutations like DNA code change,  epigenetic  alterations and chromosomal abnormalities, can occur later in development. This would result in one progeny  cell line  to be normal while the other cell line(s) to be abnormal. As a result, the individual is considered to be a mosaic of normal and abnormal cells. [ 7 ]


Mosaicism  is the occurrence of two or more cell lines with different  genotypes  within a single individual. It is different from  chimerism  which is the fusion of two zygotes, causing a new single zygote with two genotypes. [ 7 ]


The  loss of chromosome Y  (LOY) in  blood cells  is the most common human postzygotic mutation. It is highly associated with age, being detectable in at least 10% of blood cells for 14% and 57% of males around 70 and 94 years of age, respectively. [ 8 ] [ 9 ]  Men with LOY have a higher all-cause mortality and cancer mortality compared with unaffected males. [ 10 ]  Additionally, LOY is associated with greater risk for Alzheimer's disease and cardiovascular disease. [ 11 ]  Smoking increases the risk of inducing LOY more than three times and has a dose-dependent effect on LOY-status. [ 12 ]


Trisomy 21 ( Down syndrome ) is one of the most prevalent chromosomal abnormalities amongst live births. Of all trisomy 21 pregnancies, approximately 80% end in spontaneous abortions or still-births. 1–5% of people diagnosed with having Down Syndrome are actually in fact "high-grade" trisomy 21 mosaics. The rest of trisomy 21 mosaics are marked as "low-grade" mosaics, meaning the chromosomal mutation occurs in less than 3–5% of respective tissue. While high-grade trisomy 21 mosaics, demonstrate similar features to full Down Syndrome, low-grade mosaics have a tendency to show milder features; however, the effects are quite variable depending on the distribution of the trisomic cells. [ 13 ]


Somatic mutations are the result of a change in the genetic structure after fertilization. This type of mutation also involves cells outside of the reproductive group and thus is not transmitted to future descendants. [ 14 ] [ 15 ]


Germ-line mutations  are the result of a change in the genetic structure of  germ cells . These mutations are able to be transmitted to the offspring and give rise to a constitutional mutation. Constitutional mutations is a mutation that when present in one cell, is also present in all other cells associated with the organism. [ 15 ]


A  premutation  is a situation in which there are an excess number of  repeats  in a  gene  that is at risk of increasing in length during  reproduction  but which does not cause disease in the person with the excess number of repeats. [ 1 ]   Fragile X syndrome , a  trinucleotide repeat disorder , is a condition in which premutations may be present in the parents of affected people. [ 2 ]   Huntington's disease  is another example of a trinucleotide repeat disorder in which individuals with a certain excess of repeats (27–39) of the CAG  codon  in the  Huntingtin  gene may not have the disease but are more likely to have a child with enough repeats (40+) to cause the disease. [ 3 ]


Protein-truncating variants  ( PTVs ) are  genetic variants  predicted to shorten the  coding sequence of genes , [ 1 ]  through ways like a  stop-gain mutation . [ 2 ] [ 3 ] [ 4 ] [ 5 ]  PTV is sometime categorized under the umbrella term frameshift or truncating variants (FTVs), which includes both PTVs and DNA variants caused by  frameshift mutation .


It was believed that protein-truncating variants are not associated with human diseases. [ 2 ]  Recent studies have implied the involvement of PTVs in  autism spectrum disorder . [ 6 ]


Pyrimidine dimers  represent  molecular lesions  originating from  thymine  or  cytosine  bases within  DNA , resulting from  photochemical reactions . [ 1 ] [ 2 ]  These lesions, commonly linked to  direct DNA damage , [ 3 ]  are induced by  ultraviolet light  (UV), particularly  UVC , result in the formation of  covalent bonds  between adjacent nitrogenous bases along the  nucleotide  chain near their carbon–carbon double bonds, [ 4 ]  the photo-coupled dimers are  fluorescent . [ 5 ]  Such  dimerization , which can also occur in  double-stranded RNA  (dsRNA) involving  uracil  or  cytosine , leads to the creation of  cyclobutane pyrimidine dimers  (CPDs) and  6–4 photoproducts . These pre- mutagenic  lesions modify the  DNA helix  structure, resulting in abnormal non-canonical  base pairing  and, consequently, adjacent thymines or cytosines in DNA will form a  cyclobutane  ring when joined together and cause a distortion in the DNA. This distortion prevents  DNA replication  and  transcription  mechanisms beyond the dimerization site. [ 6 ]


While up to 100 such reactions per second may transpire in a  skin cell  exposed to sunlight resulting in  DNA damage , they are typically rectified promptly through  DNA repair , such as through  photolyase  reactivation or  nucleotide excision repair , with the latter being prevalent in humans. Conversely, certain bacteria utilize photolyase, powered by sunlight, to repair pyrimidine dimer-induced DNA damage. Unrepaired lesions may lead to erroneous nucleotide incorporation by  polymerase  machinery. Overwhelming DNA damage can precipitate  mutations  within an organism's  genome , potentially culminating in  cancer  cell formation. [ 7 ]  Unrectified lesions may also interfere with polymerase function, induce transcription or  replication errors , or halt replication. Notably, pyrimidine dimers contribute to  sunburn  and  melanin  production, and are a primary factor in  melanoma  development in humans.


Pyrimidine dimers encompass several types, each with distinct structures and implications for DNA integrity. [ citation needed ]


Cyclobutane pyrimidine dimer (CPD) is a dimer which features a four-membered ring formed by the fusion of two double-bonded carbons from adjacent pyrimidines. CPDs disrupt the formation of the  base pair  during  DNA replication , potentially leading to  mutations . [ 8 ] [ 9 ] [ 10 ]