Genetic instability of C. elegans comes naturally

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<ul><li><p>gl o</p><p>extremely difficult. Conversely, it has proved difficult to</p><p>studied MA lines of a DNA repair-deficient C. elegansstrain, and concluded that the number of mutations</p><p>allozyme variants (band-morph mutations) in large</p><p>Update TRENDS in Genetics Vol.21 No.2 February 2005sequence level (Box 1). Many experiments measuring(Figure 1). Under close inbreeding conditions, all but themost strongly deleterious mutations behave as effectivelyneutral and accumulate randomly at a rate that is equal tothe mutation rate.</p><p>It is important to distinguish between the estimates ofmutation rates derived from changes in quantitative traitsfrom those obtained by detecting changes at the DNA</p><p>cohorts of human families [6], and in replicated inbredDrosophila melanogaster lines that accumulated spon-taneous mutations at random (summarized in Ref. [1]).Estimates of point-mutation rates per base pair from thesestudies are broadly consistent with estimates based on theevolutionary divergence between closely related species atsilent sites in the genome [1].</p><p>Denver et al. [3] employed direct DNA sequencing ofresult of the random accumulation of new mutations</p><p>balancers), during which time the sublines diverge as a in eukaryotes have focused on the appearance of newinfer the distribution of mutational effects for fitness andother quantitative traits because mutations with largeeffects tend to dominate phenotypic changes. However,mutations with small effects below the experimentaldetection limit are likely to have the most influence onthe evolution of phenomena such as ageing, outbreedingand genetic recombination.</p><p>For forty years, mutation accumulation (MA) experi-ments have been the basis of our knowledge of genome-wide rates of spontaneous mutations and the sizes of theireffects. The standard design for a MA experiment is tostart with an isogenic line, subdivide the line into severalindependent MA lines, then maintain these by closeinbreeding for several generations (e.g. full sib mating,selfing or as chromosomes sheltered from selection byResearch Focus</p><p>Genetic instability of C. ele</p><p>Peter D. Keightley and Brian Charlesworth</p><p>University of Edinburgh, Institute of Evolutionary Biology, Schoo</p><p>There have been many attempts to measure thegenome-widemutation rate for spontaneousmutations,using measurements of traits in inbred lines in whichmutations have accumulated. However, these are likelyto miss many small-effect mutations that are importantfor evolutionary processes. Recently, the genome-widespontaneous mutation rate in inbred lines of Caeno-rhabditis elegans was estimated, using DNA sequenc-ing. The results imply that the mutation rate issurprisingly high, and that insertion-deletion mutationsare unexpectedly common. Phenotypic assays of thesame lines detected only a small proportion of muta-tions that were predicted to have evolutionarily signifi-cant fitness effects.</p><p>IntroductionThe greatest gap in our knowledge of the causes of evo-lution is the frequency of spontaneous mutations.Although spontaneous mutations are the primary sourceof all variation, new mutations are so infrequent thatmeasurements of mutation rates at themolecular level aredetectable from phenotypic changes was as little as 1%of the number of deleterious mutations, estimated usingDNA sequencing. Thus, in C. elegans at least, most of thefunctionally significant EMS-induced mutations (mainlytransition point mutations) and those generated bydeficiency in mismatch repair (the detailed nature ofthese is currently unknown) appear to have crypticeffects on fitness traits.</p><p>Obtaining direct estimates of the spontaneousmutation rate at the DNA level is more challengingbecause spontaneous mutations occur at a low rate at thenucleotide level. Previous attempts to estimate these ratesans comes naturally</p><p>f Biological Sciences, West Mains Road, Edinburgh, UK EH9 3JT</p><p>quantitative trait changes have been performed [1].A trait-based estimate of the mean number of non-lethalmutations affecting a fitness trait that arise per gener-ation (Ud) can be obtained by comparing the rate of changeof the mean phenotype with the rate of increase in thevariance of the means of the MA lines [2]. This estimate isonly equal to the mutation rate if all mutations have equaleffects; if there is a wide distribution of effects, Ud can begreatly underestimated. An experiment that detectedchanges at the DNA level to estimate the mutation rateper nucleotide site in the eukaryotic nuclear genome wasperformed for the first time by Denver and colleagues [3].</p><p>Estimating the per nucleotide mutation rate inCaenorhabditis elegansTwo studies in Caenorhabditis elegans have attempted tomeasure the extent by which trait-based estimates of Udin MA experiments are underestimates. Davies et al. [4]estimated the frequency of mutations induced in function-ally important regions of the genome by the chemicalmutagen ethyl methanesulphonate (EMS), and concludedthat only w4% of these mutations were detectable on thebasis of changes in mean and variance of fitness amongmutagenised cell lines in the laboratory. Estes et al. [5]more that four million bases of DNA to study spontaneousmutations in O50 C. elegans MA lines of the N2 strain</p><p>Corresponding author: Keightley, P.D.Available online 15 December 2004</p><p></p></li><li><p>Update TRENDS in Genetics Vol.21 No.2 February 200568Inbred progenitor,generaion O </p><p>that had undergone an average of 339 generations of MA.Previous analyses of fitness in these lines had detectedonly w0.02 mutations per diploid genome per generation[7]. Even after 339 generations, the expected cumulativefrequency of mutations at the DNA level is so low thatsequencing with an accuracy of 10K6 per base pair or moreis required to obtain reliable estimates, both from thepoint of view of not missing mutations and declaring non-existent mutations. It is impressive that Denver et al.could achieve sufficient accuracy in their study by usingan initial single sequencing pass on one strand, followedby verification of the sequences of clear-cut putative</p><p>Figure 1. The design of a mutation accumulation (MA) experiment for Caenorhabditis ele</p><p>of independent MA sublines that are subsequently maintained on plates by the transfer</p><p>that arise in the lines become fixed at random, because the transfer of a single worm ea</p><p>progeny, are represented in colour in subsequent generations. At generation t, estimat</p><p>obtain a phenotype-based estimate of the genome-wide deleterious mutation rate, Ud (B</p><p>the per nucleotide mutation rate, which can be used to produce a molecular-based esti</p><p>Box 1. Estimating mutation rates in MA lines</p><p>In a mutation accumulation (MA) experiment, mutations are allowedto accumulate in inbred lines in the virtual absence of natural selection(see Figure 1). Because each line is expected to accumulate differentmutations, the lines will diverge both phenotypically and at the DNAlevel. Estimates of the mean fitness of the MA lines at generation t andof a mutation-free control population (i.e. the ancestral line that has,for example, been maintained in a cryopreserved state) can be used toinfer the change of mean fitness per generation that is due tomutationaccumulation, DM. A second parameter that can be estimated is theincrease in genetic variance in fitness among lines per generation, VM.Under a model of equal mutational effects (s), DMZUds, where Ud isthe genomic rate for mutations affecting fitness per generation, andVMZUds</p><p>2 [1,2]. Ud can therefore be estimated from UdZDM2/VM.</p><p></p><p> variants on the opposite strand and by reamplifying andresequencing a subset of the mutations that weredetected. Denver et al. reported a total of 30 mutationsin their study, of whichmore than half (17) were insertionsor deletions (indels) and the remainder were singlenucleotide mutations (SNMs). These numbers translateto mutation rates per site per generation of 1.2!10K8 and0.9!10K8, respectively.</p><p>Implications of the mutation rate estimatesHow do these estimates compare with less direct estimatesof mutation rates per nucleotide site for other eukaryotes?</p><p>TRENDS in Genetics </p><p>Generation t</p><p>gans. The progeny of an inbred progenitor at generation 0 are used to found a series</p><p>of a randomly picked single hermaphrodite progeny in each generation. Mutations</p><p>ch generation implies that selection is ineffective. The mutant individuals, and their</p><p>es of fitnesses of the sample of worms from the individual sublines can be used to</p><p>ox 1). Denver et al. [3] sequenced the DNA of samples from generation t to estimate</p><p>mate of Um (Box 1).</p><p>However, variation among mutational effects leads this method tounderestimate Ud; the greater the variability the more serious theunderestimation. A second method to obtain a molecular-basedestimate of the genome-wide deleterious mutation rate (Um) in a MAexperiment is to count mutations at the DNA level directly bysequencing or by some other mutation-detection method, and toinfer the fraction of the mutations detected that are deleterious. Aminimum estimate of the fraction of deleterious mutations is thefraction of nucleotides in the genome that, if changed, would lead toan amino acid substitution in a protein-coding gene; it is known thatthe vast majority of amino acid substitutions are deleterious. Therewill be a further contribution to Um from selectively constrained sitesin non-coding DNA.</p></li><li><p>The C. elegans SNM-rate estimate is approximately threetimes lower than an estimate for hominids based on themean nucleotide divergence at pseudogenes betweenhumans and chimpanzees (i.e.w2.5!10K8 per generation[8]). In Drosophila, molecular divergence at synonymoussites suggests a mutation rate of w2!10K9 [1], which is4.5 times less than the C. elegans estimate. As mentionedpreviously, the estimates for hominids and Drosophila arein broad agreement with the estimates obtained from theaccumulation of allozyme band morph variants. Unfortu-nately, there are no known close relatives of C. elegans</p><p>Update TRENDS in Genetics Vol.21 No.2 February 2005 69that could enable indirect estimation of the mutation ratevia molecular evolutionary divergence.</p><p>The high proportion of indel mutations observed byDenver et al. is also surprising because indel polymorph-isms are about a quarter as frequent as single nucleotidepolymorphisms in non-coding sequences in C. eleganspopulations [9]. One possible explanation is that someSNMs were missed by Denver and colleagues directsequencing strategy (e.g. because indels are easier toidentify in sequence traces than SNMs). A secondpossibility is that indels aremore strongly selected againstthan SNMs; therefore, indels segregate in populations atlower frequencies than single nucleotide polymorphisms.This would particularly apply to species with largeeffective population sizes in which natural selection ismost effective (Box 2). The population size of C. elegans isclearly large, but less is known about the effectivepopulation size (Ne) of breeding individuals. Low poly-morphism rates among worldwide samples of C. elegans[10,11] seem to imply low Ne values. Using the meansilent-site diversity for three nuclear loci reported byGraustein et al. [10] and Denver and colleagues estimateof the mutation rate for single nucleotides, the effectivepopulation size ofC. elegans, is estimated to be only 15 600if the diversity is equated to its expected equilibrium value[12]. This suggests that there is currently little scope foreffective selection on weakly selected mutations.</p><p>By multiplying the per nucleotide mutation rateestimate by the number of nucleotides in the genomethat lead to amino acid changes if mutated (which arealmost always deleterious in natural conditions), Denveret al. estimated that the deleterious mutation rate perhaploid genome (Um) in protein coding genes isw0.5, andthat there must be a further contribution from mutationsin functionally constrained non-coding DNA sites. Themolecular mutation rate estimate is therefore nearly twoorders of magnitude higher than the phenotypic</p><p>Box 2. The effectiveness of natural selection against</p><p>deleterious mutations depends on population size</p><p>A measure of the selective disadvantage of a new deleteriousmutation is its selection coefficient (s). However, the effectiveness ofnatural selection against such a mutations depends on Nes, theproduct of the effective size of the population (Ne) in which themutation segregates and s. The effective population size is related tothe number of parents that contribute to the next generation, and canbe substantially lower than the actual population size. As Nesapproaches zero, the fixation probability of a deleterious mutationapproaches that of a neutral mutation (i.e. 1/2Ne in a diploid);</p><p>mutations for which Nes!1 are often termed effectively neutral.</p><p>www.sciencedirect.comestimates. The study of Denver et al, together with thetwo previous comparisons of phenotypic and molecularresults, therefore, strongly suggests that the vast majorityof new mutations that are deleterious have extremelysmall effects in laboratory conditions. How does this relateto data on genomic mutation rates and the distribution ofmutational effects for fitness in other species? By far thegreatest amount of work on measuring spontaneousmutational parameters in eukaryotes has been carriedout inDrosophila, by eitherMA on chromosomes shelteredagainst balancers [2,1316] or in lines maintained by fullsib mating in which all but the most strongly deleteriousmutations accumulate at random [17,18]. Estimates forUd based on phenotypic measures vary greatly betweenexperiments. The highest estimates for Ud approach one.If the worm results are extrapolated to flies, this impliesthat the true value for Ud in flies for all deleteriousmutations, irrespective of their phenotypic effects, liessomewhere between ten and 100. These values seemimplausibly high, however, given the information we haveon band morph mutation rates in Drosophila and rates ofmolecular divergence at silent sites between species.</p><p>Can the fly and worm results be reconciled? Onepossibility is that the great variation among phenotypicestimates forUd in flies is due to variation in the mutationrate, brought about by variability among genotypes in therate of movement of transposable elements (TEs) [16,19].Given that TE insertions in flies tend to have averagefitness effects of the order of a few percent [16], theiractivity could account for the highUd values in some of thefly experiments, whereas the mutation rate caused by lessdrastic changes in the DNA could in fact be lower than inC. elegans. TE activity is essentially absent from the N2strain of C. elegans.</p><p>The high frequency of indel mutations in C. elegansobserved by Denver et al. would be expected to causefrequent lethal mutations, because most indels in a codingsequence would disrupt gene function. RNA interference(RNAi) experiments suggest that knockouts of at least 7%of C. elegans genes result in lethality, and RNAi detects ahigh proportion of known...</p></li></ul>