Estimate of the mutation rate per nucleotide in humans

Many previous estimates of the mutation rate in humans have relied on screens of visible mutants. We investigated the rate and pattern of mutations at the nucleotide level by comparing pseudogenes in humans and chimpanzees to (i) provide an estimate of the average mutation rate per nucleotide, (ii) assess heterogeneity of mutation rate at different sites and for different types of mutations, (iii) test the hypothesis that the X chromosome has a lower mutation rate than autosomes, and (iv) estimate the deleterious mutation rate. Eighteen processed pseudogenes were sequenced, including 12 on autosomes and 6 on the X chromosome. The average mutation rate was estimated to be approximately 2.5 x 10(-8) mutations per nucleotide site or 175 mutations per diploid genome per generation. Rates of mutation for both transitions and transversions at CpG dinucleotides are one order of magnitude higher than mutation rates at other sites. Single nucleotide substitutions are 10 times more frequent than length mutations. Comparison of rates of evolution for X-linked and autosomal pseudogenes suggests that the male mutation rate is 4 times the female mutation rate, but provides no evidence for a reduction in mutation rate that is specific to the X chromosome. Using conservative calculations of the proportion of the genome subject to purifying selection, we estimate that the genomic deleterious mutation rate (U) is at least 3. This high rate is difficult to reconcile with multiplicative fitness effects of individual mutations and suggests that synergistic epistasis among harmful mutations may be common.     

Cyclical dynamics under constant selection against mutations in haploid and diploid populations with facultative selfing

We have found that constant selection against mutations can cause cyclical dynamics in a population with facultative selfing. When this happens, the distribution of the number of deleterious mutations per genotype fluctuates with the period approximately 1/sHe generations, where sHe is the coefficient of selection against a heterozygous mutation. The amplitude of oscillations of the mean population fitness often exceeds an order of magnitude. Cyclical dynamics can occur under intermediate selfing rates if selection against heterozygous mutations is weak and selection against homozygous mutations is much stronger. Cycling is possible without epistasis or with diminishing-returns epistasis, but not with synergistic epistasis. Under multiplicative selection, cycling might happen if the haploid mutation rate exceeds 1.9 in the case of selfing of haploids, and if this diploid mutation rate exceeds 4.5 in the case of selfing of diploids. We propose a heuristic explanation for cycling under facultative selfing and discuss its possible relevance.     

The rate of mutation and the homozygous and heterozygous mutational effects for competitive viability: a long-term experiment with Drosophila melanogaster

The effect of 250 generations of mutation accumulation (MA) on the second chromosome competitive viability of Drosophila melanogaster was analyzed both in homozygous and heterozygous conditions. We used full-sib MA lines, where selection hampers the accumulation of severely deleterious mutations but is ineffective against mildly deleterious ones. A large control population was simultaneously evaluated. Competitive viability scores, unaffected by the expression of mutations in heterozygosis, were obtained relative to a Cy/L(2) genotype. The rate of decline in mean DeltaM approximately 0.1% was small. However, that of increase in variance DeltaV approximately 0.08 x 10(-3) was similar to the values obtained in previous experiments when severely deleterious mutations were excluded. The corresponding estimates of the mutation rate lambda > or = 0.01 and the average effect of mutations E(s) < or = 0.08 are in good agreement with Bateman-Mukai and minimum distance estimates for noncompetitive viability obtained from the same MA lines after 105 generations. Thus, competitive and noncompetitive viability show similar mutational properties. The regression estimate of the degree of dominance for mild-to-moderate deleterious mutations was approximately 0.3, suggesting that the pertinent value for new unselected mutations should be somewhat smaller.     

The rate of spontaneous mutation for life-history traits in Caenorhabditis elegans.

Spontaneous mutations were accumulated in 100 replicate lines of Caenorhabditis elegans over a period of approximately 50 generations. Periodic assays of these lines and comparison to a frozen control suggest that the deleterious mutation rate for typical life-history characters in this species is at least 0.05 per diploid genome per generation, with the average mutational effect on the order of 14% or less in the homozygous state and the average mutational heritability approximately 0.0034. While the average mutation rate per character and the average mutational heritability for this species are somewhat lower than previous estimates for Drosophila, these differences can be reconciled to a large extent when the biological differences between these species are taken into consideration.     

The Baker's Yeast Diploid Genome Is Remarkably Stable in Vegetative Growth and Meiosis

Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1–2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms.     

Isogenic Strain Construction and Gene Mapping in Candida Albicans

Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura(-) strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura(-) strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura(-) phenotype. These Ura(-) segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.     

Accumulation of Spontaneous Mutations in the Ciliate Tetrahymena thermophila

Knowledge of the rate and fitness effects of mutations is essential for understanding the process of evolution. Mutations are inherently difficult to study because they are rare and are frequently eliminated by natural selection. In the ciliate Tetrahymena thermophila, mutations can accumulate in the germline genome without being exposed to selection. We have conducted a mutation accumulation (MA) experiment in this species. Assuming that all mutations are deleterious and have the same effect, we estimate that the deleterious mutation rate per haploid germline genome per generation is U = 0.0047 (95% credible interval: 0.0015, 0.0125), and that germline mutations decrease fitness by s = 11% when expressed in a homozygous state (95% CI: 4.4%, 27%). We also estimate that deleterious mutations are partially recessive on average (h = 0.26; 95% CI: –0.022, 0.62) and that the rate of lethal mutations is <10% of the deleterious mutation rate. Comparisons between the observed evolutionary responses in the germline and somatic genomes and the results from individual-based simulations of MA suggest that the two genomes have similar mutational parameters. These are the first estimates of the deleterious mutation rate and fitness effects from the eukaryotic supergroup Chromalveolata and are within the range of those of other eukaryotes.     

Positive and negative selection on the human genome.

The distinction between deleterious, neutral, and adaptive mutations is a fundamental problem in the study of molecular evolution. Two significant quantities are the fraction of DNA variation in natural populations that is deleterious and destined to be eliminated and the fraction of fixed differences between species driven by positive Darwinian selection. We estimate these quantities using the large number of human genes for which there are polymorphism and divergence data. The fraction of amino acid mutations that is neutral is estimated to be 0.20 from the ratio of common amino acid (A) to synonymous (S) single nucleotide polymorphisms (SNPs) at frequencies of > or =15%. Among the 80% of amino acid mutations that are deleterious at least 20% of them are only slightly deleterious and often attain frequencies of 1-10%. We estimate that these slightly deleterious mutations comprise at least 3% of amino acid SNPs in the average individual or at least 300 per diploid genome. This estimate is not sensitive to human population history. The A/S ratio of fixed differences is greater than that of common SNPs and suggests that a large fraction of protein divergence is adaptive and driven by positive Darwinian selection.     

Rates and Genomic Consequences of Spontaneous Mutational Events in Drosophila melanogaster

Because spontaneous mutation is the source of all genetic diversity, measuring mutation rates can reveal how natural selection drives patterns of variation within and between species. We sequenced eight genomes produced by a mutation-accumulation experiment in Drosophila melanogaster. Our analysis reveals that point mutation and small indel rates vary significantly between the two different genetic backgrounds examined. We also find evidence that ∼2% of mutational events affect multiple closely spaced nucleotides. Unlike previous similar experiments, we were able to estimate genome-wide rates of large deletions and tandem duplications. These results suggest that, at least in inbred lines like those examined here, mutational pressures may result in net growth rather than contraction of the Drosophila genome. By comparing our mutation rate estimates to polymorphism data, we are able to estimate the fraction of new mutations that are eliminated by purifying selection. These results suggest that ∼99% of duplications and deletions are deleterious—making them 10 times more likely to be removed by selection than nonsynonymous mutations. Our results illuminate not only the rates of new small- and large-scale mutations, but also the selective forces that they encounter once they arise.     

Rates and fitness consequences of new mutations in humans

The human mutation rate per nucleotide site per generation (μ) can be estimated from data on mutation rates at loci causing Mendelian genetic disease, by comparing putatively neutrally evolving nucleotide sequences between humans and chimpanzees and by comparing the genome sequences of relatives. Direct estimates from genome sequencing of relatives suggest that μ is about 1.1 × 10(-8), which is about twofold lower than estimates based on the human-chimp divergence. This implies that an average of ~70 new mutations arise in the human diploid genome per generation. Most of these mutations are paternal in origin, but the male:female mutation rate ratio is currently uncertain and might vary even among individuals within a population. On the basis of a method proposed by Kondrashov and Crow, the genome-wide deleterious mutation rate (U) can be estimated from the product of the number of nucleotide sites in the genome, μ, and the mean selective constraint per site. Although the presence of many weakly selected mutations in human noncoding DNA makes this approach somewhat problematic, estimates are U ≈ 2.2 for the whole diploid genome per generation and 0.35 for mutations that change an amino acid of a protein-coding gene. A genome-wide deleterious mutation rate of 2.2 seems higher than humans could tolerate if natural selection is "hard," but could be tolerated if selection acts on relative fitness differences between individuals or if there is synergistic epistasis. I argue that in the foreseeable future, an accumulation of new deleterious mutations is unlikely to lead to a detectable decline in fitness of human populations.     

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.     

QUANTITATIVE VARIATION IN FINITE PARTHENOGENETIC POPULATIONS: WHAT STOPS MULLER'S RATCHET IN THE ABSENCE OF RECOMBINATION?

Finite parthenogenetic populations with high genomic mutation rates accumulate deleterious mutations if back mutations are rare. This mechanism, known as Muller's ratchet, can explain the rarity of parthenogenetic species among so called higher organisms. However, estimates of genomic mutation rates for deleterious alleles and their average effect in the diploid condition in Drosophila suggest that Muller's ratchet should eliminate parthenogenetic insect populations within several hundred generations, provided all mutations are unconditionally deleterious. This fact is inconsistent with the existence of obligatory parthenogenetic insect species. In this paper an analysis of the extent to which compensatory mutations can counter Muller's ratchet is presented. Compensatory mutations are defined as all mutations that compensate for the phenotypic effects of a deleterious mutation. In the case of quantitative traits under stabilizing selection, the rate of compensatory mutations is easily predicted. It is shown that there is a strong analogy between the Muller's ratchet model of Felsenstein (1974) and the quantitative genetic model considered here, except for the frequency of compensatory mutations. If the intensity of stabilizing selection is too small or the mutation rate too high, the optimal genotype becomes extinct and the population mean drifts from the optimum but still reaches a stationary distribution. This distance is essentially the same as predicted for sexually reproducing populations under the same circumstances. Hence, at least in the short run, compensatory mutations for quantitative characters are as effective as recombination in halting the decline of mean fitness otherwise caused by Muller's ratchet. However, it is questionable whether compensatory mutations can prevent Muller's ratchet in the long run because there might be a limit to the capacity of the genome to provide compensatory mutations without eliminating deleterious mutations at least during occasional episodes of sex.     

The rate of adaptation in asexuals

I study the population genetics of adaptation in asexuals. I show that the rate of adaptive substitution in an asexual species or nonrecombining chromosome region is a bell-shaped function of the mutation rate: at some point, increasing the mutation rate decreases the rate of substitution. Curiously, the mutation rate that maximizes the rate of adaptation depends solely on the strength of selection against deleterious mutations. In particular, adaptation is fastest when the genomic rate of mutation, U, equals the harmonic mean of selection coefficients against deleterious mutations, where we assume that selection for favorable alleles is milder than that against deleterious ones. This simple result is independent of the shape of the distribution of effects among favorable and deleterious mutations, population size, and the action of clonal interference. In the course of this work, I derive an approximation to the probability of fixation of a favorable mutation in an asexual genome or nonrecombining chromosome region in which both favorable and deleterious mutations occur.     

Direct estimate of the mutation rate and the distribution of fitness effects in the yeast Saccharomyces cerevisiae

Estimates of the rate and frequency distribution of deleterious effects were obtained for the first time by direct scoring and characterization of individual mutations. This was achieved by applying tetrad analysis to a large number of yeast clones. The genomic rate of spontaneous mutation deleterious to a basic fitness-related trait, that of growth rate, was U = 1.1 x 10(-3) per diploid cell division. Extrapolated to the fruit fly and humans, the per generation rate would be 0.074 and 0.92, respectively. This is likely to be an underestimate because single mutations with selection coefficients s < 0.01 could not be detected. The distribution of s > or = 0.01 was studied both for spontaneous and induced mutations. The latter were induced by ethyl methanesulfonate (EMS) or resulted from defective mismatch repair. Lethal changes accounted for approximately 30-40% of the scored mutations. The mean s of nonlethal mutations was fairly high, but most frequently its value was between 0.01 and 0.05. Although the rate and distribution of very small effects could not be determined, the joint share of such mutations in decreasing average fitness was probably no larger than approximately 1%.     

Genomewide two-generation screens for recessive mutations by ES cell mutagenesis

Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genomewide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)–mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.     

Mutation-selection balance and the evolutionary advantage of sex and recombination

Mutation-selection balance in a multi-locus system is investigated theoretically, using a modification of Bulmer's infinitesimal model of selection on a normally-distributed quantitative character, taking the number of mutations per individual (n) to represent the character value. The logarithm of the fitness of an individual with n mutations is assumed to be a quadratic, decreasing function of n. The equilibrium properties of infinitely large asexual populations, random-mating populations lacking genetic recombination, and random-mating populations with arbitrary recombination frequencies are investigated. With 'synergistic' epistasis on the scale of log fitness, such that log fitness declines more steeply as n increases, it is shown that equilibrium mean fitness is least for asexual populations. In sexual populations, mean fitness increases with the number of chromosomes and with the map length per chromosome. With 'diminishing returns' epistasis, such that log fitness declines less steeply as n increases, mean fitness behaves in the opposite way. Selection on asexual variants and genes affecting the rate of genetic recombination in random-mating populations was also studied. With synergistic epistasis, zero recombination always appears to be disfavoured, but free recombination is disfavoured when the mutation rate per genome is sufficiently small, leading to evolutionary stability of maps of intermediate length. With synergistic epistasis, an asexual mutant is unlikely to invade a sexual population if the mutation rate per diploid genome greatly exceeds unity. Recombination is selectively disadvantageous when there is diminishing returns epistasis. These results are compared with the results of previous theoretical studies of this problem, and with experimental data.     

Estimation of deleterious genomic mutation parameters in natural populations by accounting for variable mutation effects across loci

The genomes of all organisms are subject to continuous bombardment of deleterious genomic mutations (DGM). Our ability to accurately estimate various parameters of DGM has profound significance in population and evolutionary genetics. The Deng-Lynch method can estimate the parameters of DGM in natural selfing and outcrossing populations. This method assumes constant fitness effects of DGM and hence is biased under variable fitness effects of DGM. Here, we develop a statistical method to estimate DGM parameters by considering variable mutation effects across loci. Under variable mutation effects, the mean fitness and genetic variance for fitness of parental and progeny generations across selfing/outcrossing in outcrossing/selfing populations and the covariance between mean fitness of parents and that of their progeny are functions of DGM parameters: the genomic mutation rate U, average homozygous effect s, average dominance coefficient h, and covariance of selection and dominance coefficients cov(h, s). The DGM parameters can be estimated by the algorithms we developed herein, which may yield improved estimation of DGM parameters over the Deng-Lynch method as demonstrated by our simulation studies. Importantly, this method is the first one to characterize cov(h, s) for DGM.     

Novel ITGB4 mutations in lethal and nonlethal variants of epidermolysis bullosa with pyloric atresia: missense versus nonsense.

Epidermolysis bullosa with pyloric atresia (EB-PA), an autosomal recessive genodermatosis, manifests with neonatal cutaneous blistering associated with congenital pyloric atresia. The disease is frequently lethal, but nonlethal cases have also been reported. Expression of the alpha6 beta4 integrin is altered at the dermal-epidermal basement-membrane zone; recently, mutations in the corresponding genes (ITGA6 and ITGB4) have been disclosed in a limited number of patients, premature termination codons in both alleles being characteristic of lethal variants. In this study, we have examined the molecular basis of EB-PA in five families, two of them with lethal and three of them with nonlethal variants of the disease. Mutation analysis disclosed novel lesions in both ITGB4 alleles of each proband. One of the patients with lethal EB-PA was a compound heterozygote for premature termination-codon mutations (C738X/4791delCA), whereas the other patient with a lethal variant was homozygous for a missense mutation involving a cysteine residue (C61Y). The three nonlethal cases had missense mutations in both alleles (C562R/C562R, R1281W/R252C, and R1281W/R1281W). Immunofluorescence staining of skin in two of the nonlethal patients and in one of the lethal cases was positive, yet attenuated, for alpha6 and beta4 integrins. These results confirm that ITGB4 mutations underlie EB-PA and show that missense mutations may lead to nonlethal phenotypes.     

The Distribution of Mutation Effects on Viability in Drosophila Melanogaster

Parameters of continuous distributions of effects and rates of spontaneous mutation for relative viability in Drosophila are estimated by maximum likelihood from data of two published experiments on accumulation of mutations on protected second chromosomes. A model of equal mutant effects gives a poor fit to the data of the two experiments; higher likelihoods are obtained with leptokurtic distributions or for models in which there is more than one class of mutation effect. Minimum estimates of mutation rates (events per generation) at polygenes affecting viability on chromosome 2 are 0.14 and 0.068, but estimates are strongly confounded with other parameters in the model. Separate information on rates of molecular divergence between Drosophila species and from rates of movement of transposable elements is used to infer the overall genomic mutation rate in Drosophila, and the viability data are analyzed with mutation rate as a known parameter. If, for example, a mutation rate for chromosome 2 of 0.4 is assumed, maximum likelihood estimates of mean mutant effect on relative viability are 0.4% and 1%, but the majority of mutations have very much smaller effects than these values as distributions are highly leptokurtic. The methodology is applied to estimate viability effects of single P element insertional mutations. The mean effect per insertion is found to be higher, and their distribution is found to be less leptokurtic than for spontaneous mutations. The equilibrium genetic variance of viability predicted by a mutation-selection balance model with parameters estimated from the mutation accumulation experiments is similar to laboratory estimates of genetic variance of viability from natural populations of Drosophila.     

Initiation of sporulation in Saccharomyces cerevisiae. Mutations causing derepressed sporulation and G1 arrest in the cell division cycle

Mutants of Saccharomyces cerevisiae that are derepressed for meiosis and spore formation have been isolated and characterized genetically. All are the result of single, recessive nuclear mutations that fall into four linkage groups. Three of these groups are represented by spd1, spd3 and spd4 mutations, which in homozygous diploids confer poor growth and extensive sporulation on a range of non-fermentable media. Haploids carrying any of these mutations are arrested under these conditions in the G1 phase of the cell division cycle as large unbudded cells. The alleles of the spd2 mutation complemented all other mutations but were very closely linked to the spd1 locus. The fourth linkage group was represented by a mutation conferring temperature-sensitive growth and derepressed sporulation on homozygous diploids grown between 25 degrees C and 30 degrees C on media containing galactose or glycerol, but not glucose, as energy source. Above 30 degrees C this mutant lysed on all media. The mutation it carried failed to complement available cdc25 mutations. These data bring to five the number of loci at which mutation can lead to derepressed sporulation (spd1, spd3, spd4, cdc25 and cdc35). The spd1 locus has been mapped 13.9 cM to the left of the centromere on chromosome XV, adjacent to the SUP3 gene. Diploid strains homozygous for spd mutations are genetically unstable, giving rise to asporogenous mutants at high frequency, usually as the result of a second mutation unlinked to the spd mutation. Diploids homozygous for these mutations, and for spd mutations, show an altered regulation of the formulation of at least three polypeptides normally subject to carbon source repression.