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Direct estimation of per nucleotide and genomic deleterious mutation rates in
Drosophila
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"LETTERS Direct estimation of per nucleotide and genomic deleterious mutation rates in Drosophila CathyHaag-Liautard 1 *,MarkDorris 1 *,XulioMaside 1 {,StevenMacaskill 1 {,DanielL.Halligan 1 ,BrianCharlesworth 1 & Peter D. Keightley 1 Spontaneous mutations are the source of genetic variation required for evolutionary change, and are therefore important for many aspects of evolutionary biology. For example, the diver- gence between taxa at neutrally evolving sites in the genome is proportional to the per nucleotide mutation rate, u (ref. 1), and thiscanbe usedtodatespeciation events byassuming a molecular clock. The overall rate of occurrence of deleterious mutations in the genome each generation (U) appears in theories of nucleotide divergence and polymorphism 2 , the evolution of sex and recom- bination 3 , and the evolutionary consequences of inbreeding 2 . However, estimates of U based on changes in allozymes 4 or DNA sequences 5 and fitness traits are discordant 6?8 . Here we directly estimate u in Drosophila melanogaster by scanning 20 million bases of DNA from three sets of mutation accumulation lines by using denaturing high-performance liquid chromatography 9 . From 37 mutation events that we detected, we obtained a mean estimateforuof8.4310 29 pergeneration.Moreover,wedetected significant heterogeneity in u among the three mutation-accu- mulation-line genotypes. By multiplying u by an estimate of the fractionofmutationsthataredeleteriousinnaturalpopulationsof Drosophila 10 , we estimate that U is 1.2 per diploid genome. This high rate suggests that selection against deleterious mutations may have a key role in explaining patterns of genetic variation in the genome, and help to maintain recombination and sexual reproduction. Recurrent deleterious mutations have been implicated in several important evolutionary phenomena. For example, interference between deleterious mutations favours the spread of mutations that increase recombination or sex in finite populations 11 . Synergistic fit- nesseffectsofmutationsmaycontributetothemaintenanceofrecom- bination and sex in large populations 3 . The positive correlation between recombination rate and nucleotide diversity in several spe- cies 12,13 may be caused by linked deleterious mutations reducing diversity in regions of low recombination 2 . Deleterious mutations are also thought to be a major contributor to inbreeding depression 2 . However,theroleofdeleteriousmutationsintheseandotherprocesses depends on the distribution of fitness effects and the number of dele- terious mutations appearing in the genome in each generation (U). Unfortunately, empirical estimates of U have been inconsistent and controversial. Two principal methods have been employed to inferU.Thefirstisbasedondifferences infitnesstraitsamongmuta- tionaccumulation(MA)lines,whichareinitiallygeneticallyuniform and are subsequently maintained at a low population size in benign conditions,sothatmostnewmutationsbehaveneutrallyandbecome fixed at random. However, this method will underestimate U because many deleterious mutations are unlikely to affect fitness detectably in the laboratory 6?8 . A second method 14 has no such a bias. U is esti- mated from the product of the mutation rate per nucleotide site per generation (u), the number of bases in the diploid genome (2G), and the fraction of sites in the genome that are subject to selective con- straints (C): U52uGC (1) Ccanbeestimatedfrombetween-speciesgenomecomparisons 10,14 . In principle, u can be estimated from the nucleotide divergence in unselected genomic regions between a species pair 1 but is subject to uncertainty because the divergence date and generation interval are needed, and identifying neutrally evolving regions can be problem- atic. Alternatively, u can be estimated directly from the molecular divergence between MA lines. The first such estimate was based on electrophoretic mutations in D. melanogaster 4 , but only three events were detected, and electrophoretic mutations can be related only indirectlytochangesintheDNA.Morerecently,uhasbeenestimated inCaenorhabditiselegansbysequencingMA-lineDNA 5 .Fromthis,an estimate of U for codingsequenceswas obtained, whichisone to two orders of magnitude higher than an estimate from the phenotypic divergence of the MA lines, consistent with the expectation outlined above.Herewedirectlyestimateu inD.melanogasterbyscanningthe genomes of MA lines, and infer U from equation (1). We scanned 20 megabases (Mb) of DNA, comprising 277 seg- ments (amplicons) of coding, intronic and intergenic DNA (Supple- mentary Tables S1 and S2, and Supplementary Fig. S1) from 133 MA lines of three genotypes (Florida-33, Florida-39 (ref. 15) and Madrid 16,17 ), by denaturing high-performance liquid chromato- graphy (DHPLC) 9 . The efficiency of DHPLC at detecting mutations was verified by analysing synthetic positive controls containing mutations. We successfully detected 45 out of 46 controls (Supple- mentary Table S3 and Supplementary Fig. S2), which is a similar rate to that in previous reports 18,19 . Putative mutations detected by DHPLC were verified and identified by sequencing. We found evid- ence for genetic variation in the inbred progenitor of the Florida-39 lines (seeMethods and Supplementary Fig.S3 formoredetails). This manifested itself as fixed nucleotide differences between groups of MA lines for blocks of linked amplicons. Affected amplicons of these lines were excluded from the analysis. Among20,002,585basepairs(bp)screened,weobserved37muta- tions (Tables 1 and 2, and Supplementary Fig. S4), of which 3 segre- gated at a frequency of 0.5 in the line in which they occurred. The mutation detection rate was fairly uniform over the experiment (Supplementary Fig. S5). Our estimate of the single-nucleotide mutation rate per generation is 5.8310 29 (95% confidence interval *These authors contributed equally to this work. 1 Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK. {Present addresses: Grupo de Medicina Xeno�mica, Instituto de Medicina Legal, Universidade de Santiago, S. Francisco s/n, 15782 Santiago de Compostela, Spain (X.M.); Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria 3002, Australia (S.M.). Vol 445|4 January 2007|doi:10.1038/nature05388 82 Nature �2007 Publishing Group (CI) 2.1310 29 to 1.31310 28 ). This is about two-thirds of a direct estimate in C. elegans 5 . Our estimate of u for all mutation events is 8.4310 29 (95% CI 3.6310 29 to 1.6310 28 ). However, there is significant heterogeneity in u between the three genotypes (likeli- hood ratio test, 2logL512.5; P50.002). In pairwise tests, the muta- tion rate in Florida-33 is significantly higher than that in Madrid (2logL512.4; P,0.001) and nearly significantly higher than in Florida-39 (2logL53.6; P50.059). Transitions were about twice as frequent as transversions (17 versus 8, Table 2); this is higher than the roughly 1:1 ratio observed in noncoding polymorphisms in Drosophila 20 . Insertion?deletion events (indels) were a minority of the mutations (eight, excluding transposable elements (TEs)). Among these, deletions (six) were more frequent than insertions (two), which is consistent with the high deletion/insertion ratio observed in Drosophila pseudogenes 21 . However, our findings are significantly different from the results of sequencing of C. elegans MA lines 5 , in which indels substantially outnumbered point muta- tions (Fisher?s exact test: P50.05) and insertions predominated among the indels (P50.02). Three events involved simultaneous indel and point mutations (Table 1); similar complex events also segregate within some Drosophila populations (P. Haddrill, personal communication). We detected only one TE insertion (of the family Cr1a), giving an insertion rate per base pair per generation of 2.7310 210 (95% CI 6.8310 212 to 1.5310 29 ), corresponding to an insertion rate per diploid of 0.06 per generation (95% CI 0.002 to 0.35). This is not significantly different from estimates obtained by extrapolatingmovementratesofactiveTEfamiliesintheMadridMA lines 17 .Mutationratesweresimilarincoding,intronicandintergenic DNA (Supplementary Table S4; likelihood ratio test of heterogeneity ofmutationrates2logL52.1,P50.35),soaneffectoftranscription- coupledrepairisnotevidentinourdata.Twolineshadtwomutation events (none had more than two), and this is not significantly differ- ent from expectation under a Poisson distribution (randomization test: P.0.5). The euchromatic Drosophila genome size, G, is about 118Mb, so our estimate of the mean diploid genomic mutation rate from all types of mutations is 2uG51.99. From a comparison of the D. mel- anogaster and D. simulans genomes, the fraction of point mutations inDrosophilathatareselectivelyeliminated,C,isestimatedtobe0.58 (ref. 10). From equation (1), assuming that point mutations and indels are equally deleterious on average, the mean genomic deleteri- ous mutation rate is U51.15 (95% CI 0.49?2.19). However, indels are more likely to be strongly deleterious than point mutations (Supplementary Fig. S6), and including this information gives a slightly higher estimate for U of 1.20 (95% CI 0.51?2.28; Table 2). Wemay have underestimated the genomic mutation rate for three reasons. First, hypermutable, repetitive regions are probably under- represented because amplicons containing them can be difficult to analyse by DHPLC. Second, we may have missed mutations because ofthelimitationsofDHPLC,althoughourdetectionrateforpositive controls was 98%. Third, C in equation (1) is likely to be an under- estimate 10 . If, however, recessive modifiers that increased the muta- tion rate hadbecome fixed in theMAline progenitors by inbreeding, we might have overestimated U for natural populations. This is a generic problem with experimental estimates of mutation rates that use inbred lines. Table 1 | Mutation events detected by DHPLC and confirmed by sequencing Amplicon Line Mutation type Context 2L-CG8965-C F33.49 complex cod CCAAGGATGTCTTRCCAAGGACCATCTT 3R-113648 M11 complex intron CATATCGTTCGCAAGRCATATCGCAGG X-13003975 M62 complex intron GATAT(A) 4 TTTGCAACTATTTARGATAT(A) 4 TATATCTTA(AT) 8 T(AT) 2 (A) 4 TAAACTATTTCA 2L-20718966 M87 del.* interg. GTAGTGTGTTT?ATGTAACCRTAAGAGTA(GT) 3 AACC 2R-CG30377 F33.45 del.{ intron TCTAATGCG?AGTCARTCTAATGTCA 2R-fus M70 del. cod AGG(CGG) 2 TGGTTGTGRAGG(CGG) 2 TTGTG 3R-7922936 F33.67 ins. intron (T) 5 AAGG(T) 9 GTGR(T) 5 AAGG(T) 9 TGTG 3R-Fru-bis F33.55 del. intron AATGACTCTGATATTRAATGACTGATATT 3R-19561997 F33.42 del. intron GGCGTGCCAAARGGCGTCCAAA X-3198685 M137 del. intron AGAG(A) 8 AGGRAGAG(A) 8 GG X-11335521 M148/M149 ins. intron TT(A) 9 CCTTGRTT(A) 9 ACCTTG 3L-22018790 F33.5 TE interg. CATATGGTATRCATAT (Cr1a) 2L-cul2-NC2 M78/M79 ts interg. AATGTATGRAATGCATG 2L-cul-2-C F33.8 ts cod CTTAAGCT RCTTGAGCT 2R-CG3136 F33.42 ts cod GCAGGTCRGCAGATC 2R-CG30377-up F39.72 ts interg. GTCTTGATRGTCTCGAT 2R-3097863-down F33.27 ts interg. TAAACGGTRTAAATGGT 3L-Bab2-C F33.8 ts cod CTGTGGGGRCTGTAGGG 3L-22018790- down F33.8 ts interg. CTAGGAAGRCTAGAAAG 3L- BcDNAGHO3694 F33.6/F33.71 ts cod GGGTCACTRGGGTTACT 3R-113648 F33.49 ts intron GTCGAAGGGRGTCGAGGGG 3R-19599719 F39.67 ts intron ATGGGGCGRATGAGGCG 3R-19615776 F39.65 ts cod ATTTCCTTTGRATTTCCCTTG 3R-CG8968 F33.69 ts intron CATCGCTTRCATCACTT 3R-21787667- down F33.49 ts interg. CTTGCGCTRCTTACGCT X-11331631-down M31 ts interg. GTATATATGCRGTATACATGC X-CG15745 F33.69 ts cod TGCCCGGAGRTGCCCAGAG X-CG15745 M75 ts cod CGGAACGAGRCGGAATGAG X-CG32495 F39.67 ts cod CACCGAGGRCACCAAGG 2L-CG2955-NC M73 tv interg. CAAT(T) 5 AAAGRCAAA(T) 5 AAAG 2L-215156 F33.17/F33.70 tv interg. CCGAAAGTCRCCGAAACTC 2R-3097863 M137 tv intron CGACTCAARCGACGCAA 2R-CG14748 M11 tv cod GCGGACGRGCGGTCG 3L-CG32050 F33.17 /F33.70 tv cod CACAAGATRCACACGAT 3R-419892 F39.11 tv interg. GCACAACRGCAGAAC 3R-21787667 M140 tv interg. GCATTTTGTRGCATGTTGT X-hiw M100 tv cod CAACTTGARCAACTGGA Abbreviations: cod, coding; interg., intergenic; del., deletion; ins., insertion; ts, transition, tv, transversion. *30-bp deletion. { 65-bp deletion. Three mutations were segregating at a frequency of 0.5 within their respective MA lines: 2L-CG8965-C, 3L-22018790-down and X-CG32495. Mutations are indicated in bold. NATURE | Vol 445 | 4 January 2007 LETTERS 83 Nature �2007 Publishing Group Our findings have several implications. First, we found significant genetic variation in the mutation rate between genotypes. Genetic variationinthemutationratehasbeenreportedinD.melanogaster 22 , and in the rate by which fitness declines due to MA in rhabditid nematodes 23 . Second, our estimate of the nucleotide site mutation rateisabout5-fold(95%confidencelimits2-foldand12-fold)higher than a phylogenetic estimate from synonymous site divergence 24 , assuming that wild flies undergo ten generations per year. This could bepartlyduetoinaccurateestimatesofspeciesdivergencetimesorto differencesingenerationtimesbetweenlaboratoryfliesandwildflies. Combined with the recent inference of pervasive selection against new mutations in Drosophila 10,25 , our estimate for u indicates that U probably exceeds one event per diploid genome per generation in Drosophila andisunlikely tobelessthan 0.5.Thisiscomparable with an estimate in C. elegans based on direct sequencing (0.96 for coding sequences only 5 ). However, genomic deleterious mutation rates estimated from the divergence of fitness traits in MA lines strongly disagree between these species; these are about 0.01 in C. elegans 23 and up to about 1.0inDrosophila 6?8,26 .Thedistributionoffitnesseffectsofdeleterious mutations in Drosophila is likely to be highly leptokurtic 27 ,soitis unexpected that some Drosophila MA experiments should yield sim- ilar phenotypic 6 and DNA-based (our study) estimates of U. The reasons for this discrepancy remain obscure 2,7,8,23,26 . Last, our results have implications for the evolutionary maintenance of sex and recombination. Non-zero rates of recombination can be maintained by both Hill?Robertson interference 11 and synergistic epistasis 28 , with genomic deleterious mutation rates as low as 0.5 (our lower confidence limit). However, our estimate of U51.2 seems too low for deterministic selection against deleterious mutations to allow the maintenance of sexual reproduction with a twofold cost, although themechanismmightworkifUwereashighasourupperconfidence limit 28 . Additional factors that slow the spread of asexual mutants, such as population structure 29 , might help to maintain sex in species with suitable population structure, even with U as low as 0.5. METHODS Mutation accumulation lines. We analysed D. melanogaster MA lines of three genotypes (Florida-33, Florida-39 and Madrid). Progenitors of Florida-33 and Florida-39 were derived independently from a common base population by brother?sister mating for 40 generations, then MA lines were maintained by full-sib mating until generation 90 (ref. 15), and by a mixture of full-sib and half-sib mating until generation 187, on average (D. Houle, personal commun- ication). The Madrid progenitor was established by chromosome extraction 16 . MA lines were maintained by full-sib or double first-cousin mating until gen- eration 47, then by full-sib mating until generation 262 (refs 16, 17). DNA was extracted from pools of 25 individuals per line. Mutation detection by DHPLC. We randomly selected 77 nucleotide positions from the euchromatic genome sequence of D. melanogaster (Release 4.3 for chromosome 4, otherwise Release 3.1). A coding and a non-coding amplicon, each of 650?750bp, were chosen close to each position. At an additional 56 random positions we selected either a coding or a non-coding amplicon. Finally, we selected 67 non-coding amplicons flanking suspected mutations (see below). For each amplicon, 5ng of template was amplified by PCR with AmpliTaq Gold (Applied Biosystems), and the length and quality of products were verified on 1% agarose gels. Significantly weaker products than the others were excluded, because detection of variants at a frequency of less than 10% in a pooledsampleisunreliable.ThesequencesofPCRproductsofthesameMA-line genotype were compared by DHPLC 9 . Products were mixed in groups of four (labelled?vials?),themixturesweredenaturedandreannealed,andthefragments were separated on a Transgenomic Wave 3500A DHPLC instrument with a DNASep column at two to five temperatures with elution gradients chosen according to the sequence of the amplicon. In the absence of a mutation, vials gave similar elution profiles. If a line carried a mutation, the difference in reten- tiontimebetweenheteroduplexesandhomoduplexesresultedinitsvialshowing a wider profile or a double peak (Supplementary Fig. S4). Positive controls. We assessed the detection rate of mutations by using positive controls for 46 amplicons, generated with the use of the relatively high mis- incorporation rate of traditional Taq polymerase. We amplified MA-line geno- mic DNA with a non-proofreading polymerase, and cloned and sequenced PCR products.Positiveandnegativecontrolswereselectedamongthecloneswithone and zero mutations, respectively, compared with the wild-type sequence (Supplementary Table S3). Controls were then amplified by PCR along with the MA lines. Mixtures were produced between the positive control product and products from three MA lines of the same genotype, the negative and positive control products, and products of the negative control and the three MA lines of the same genotype. These were analysed by DHPLC along with the MA-line vials of that amplicon. Most positive controls are transitions (Supplementary Table S3), which are more difficult to detect by DHPLC than indels or transversions 30 , making our positive control panel conservative. Characterization of mutations. Whenever DHPLC elution profiles showed differences, the four lines of that vial were reamplified by PCR and directly sequenced in both directions. Some mutations were found to be segregating within a line; the strategy we used to investigate these is described in Supplementary Table S4. Polymorphicsitesnotrepresentingnewmutations.Polymorphismspresentat the start of the MA phase are expected to become fixed in different lines, and polymorphism is likely to affect a chromosomal region. We detected several regions having such characteristics in Florida-39, but not in Florida-33 or Madrid (Supplementary Fig. S3). Lines showing polymorphism in Florida-39 were excludedfrom the dataon affectedamplicons. Furthermore, to distinguish between genuine mutations and polymorphism blocks, noncoding amplicons closely linked to either side of putative mutationswereanalysed.This procedure makes it improbable that a polymorphism would be misclassified as a mutation Table 2 | Results of scanning the Drosophila genome for new mutations Mutation type Mutation events detected Madrid Florida-33 Florida-39 Total Complex events 20.502.5 Insertions 11 0 Deletions 33 6 TEs 01 1 Transitions 39.53.51 Transversions 52 18 Total events 14 17 4.535.5 Mutation rate parameter Mutation rate estimates Madrid Florida-33 Florida-39 Overall u (C1I)310 9 2.0 (0.7?4.4) 5.6 (1.9?12.5) 02.6 (0.6?9.2) u (SNM)310 9 2.7 (1.2?5.4) 11.7 (5.9?20.6) 6.8 (2.1?16.6) 5.8 (2.1?13.1) u (total)310 9 4.8 (2.6?8.0) 17.2 (10.0?27.6) 6.8 (2.1?16.6) 8.4 (3.6?16.0) U 0.66 (0.36?1.11) 2.56 (1.49?4.10) 0.94 (0.28?2.28) 1.20 (0.51?2.28) Totals of 11,207,503 bp, 5,272,760 bp and 3,522,322 bp of Madrid, Florida-33 and Florida-39 DNA were scanned, respectively. u (C1I) is the mutation rate per site for complex and indel events, including TEs. u (SNM) is the mutation rate for single nucleotide mutation events (transitions and transversions). Ranges in parentheses are 95% confidence intervals. The overall estimates of mutation rates are averages, weighted by the average number of lines of each genotype successfully amplified per amplicon. We calculated confidence intervals for the overall mutation rates by maximum likelihood, under the assumption that each genotype?s mutation rate is sampled from a log-normal distribution, with Poisson error on mutation numbers within genotypes. We calculated profile likelihoods as a function of the mean of the mutation rate distribution and obtained approximate confidence intervals on the basis of drops of 2 log likelihood units from the maximum likelihoods. LETTERS NATURE | Vol 445 | 4 January 2007 84 Nature �2007 Publishing Group (Supplementary Fig. S3). In four cases, pairs of lines shared identical mutations (Table1).Inparticular,theMadridlinesinvolvedwereconsecutivelynumbered, and one of the Florida-33 events concerned lines sharing two mutations. These events presumably reflect breeding contamination between two MA lines 17 .We counted shared mutations only once, and reduced the total number of lines by 0.5 for each contaminant. Received 9 August; accepted 30 October 2006. 1. Kimura, M. TheNeutralTheoryofMolecularEvolution (Cambridge University Press, Cambridge, 1983). 2. Charlesworth, B. & Charlesworth, D. Some evolutionary consequences of deleterious mutations. Genetica 102?103, 3?19 (1998). 3. Kondrashov, A. S. Deleterious mutations and the evolution of sexual reproduction. Nature 336, 435?440 (1988). 4. Mukai, T. & Cockerham, C. C. Spontaneous mutation rates at enzyme loci in Drosophila melanogaster. Proc. Natl Acad. Sci. USA 74, 2514?2517 (1977). 5. Denver, D. R., Morris, K., Lynch, M. & Thomas, W. K. High mutation rate and predominance of insertions in the Caenorhabditis elegans nuclear genome. Nature 430, 679?682 (2004). 6. Crow, J. F. & Simmons, M. J. in The Genetics and Biology of Drosophila Vol. 3C (eds Ashburner, M., Carson, H. L. & Thompson, J. N.) 1?35 (Academic, London, 1983). 7. Keightley, P. D. & Eyre-Walker, A. Terumi Mukai and the riddle of deleterious mutation rates. Genetics 153, 515?523 (1999). 8. Lynch, M. et al. Perspective: Spontaneous deleterious mutation. Evolution 53, 645?663 (1999). 9. Oefner, P. J. & Huber, C. G. A decade of high-resolution liquid chromatography of nucleic acids on styrene divinylbenzene copolymers. J. Chromatogr. B 782, 27?55 (2002). 10. Halligan, D. L. & Keightley, P. D. Ubiquitous selective constraints in the Drosophila genome revealed by a genome-wide interspecies comparison. Genome Res. 16, 875?884 (2006). 11. Keightley, P. D. & Otto, S. P. Interference among deleterious mutations favours sex and recombination in finite populations. Nature 443, 89?92 (2006). 12. Presgraves, D. C. Recombination enhances protein adaptation in Drosophila melanogaster. Curr. Biol. 15, 1651?1656 (2005). 13. Nachman, M. W. Single nucleotide polymorphisms and recombination rate in humans. Trends Genet. 17, 481?485 (2001). 14. Kondrashov, A. S. & Crow, J. F. A molecular approach to estimating the human deleterious mutation rate. Hum. Mutat. 2, 229?234 (1993). 15. Houle, D. & Nuzhdin, S. V. Mutation accumulation and the effect of copia insertions in Drosophila melanogaster. Genet. Res. 83, 7?18 (2004). 16. Fernandez, J. & Lo�pez-Fanjul, C. Spontaneous mutational variances and covariances for fitness-related traits in Drosophila melanogaster. Genetics 143, 829?837 (1996). 17. Maside, X., Bartolome, C., Assimacopoulos, S. & Charlesworth, B. Rates of movement and distribution of transposable elements in Drosophila melanogaster: in situ hybridization vs Southern blotting data. Genet. Res. 78, 121?136 (2001). 18. Dobson-Stone, C. et al. 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Tamura, K., Subramanian, S. & Kumar, S. Temporal patterns of fruit fly (Drosophila) evolution revealed by mutation clocks. Mol. Biol. Evol. 21, 36?44 (2004). 25. Andolfatto, P. Adaptive evolution of non-coding DNA in Drosophila. Nature 437, 1149?1152 (2005). 26. Fry, J. D. On the rate and linearity of viability declines in Drosophila mutation- accumulation experiments: Genomic mutation rates and synergistic epistasis revisited. Genetics 166, 797?806 (2004). 27. Loewe, L. & Charlesworth, B. Inferring the distribution of mutational effects on fitness in Drosophila. Biol. Lett. 2, 426?430 (2006). 28. Charlesworth, B. Mutation selection balance and the evolutionary advantage of sex and recombination. Genet. Res. 55, 199?221 (1990). 29. Salathe�, M., Schmid-Hempel, P. & Bonhoeffer, S. Mutation accumulation in space and the maintenance of sexual reproduction. Ecol. Lett. 9, 941?946 (2006). 30. Ravnik-Glavac?, M., Atkinson, A., Glavac?, D. & Dean, M. DHPLC screening of cystic fibrosis gene mutations. Hum. Mutat. 19, 374?383 (2002). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank D. Houle and C. Lo�pez-Fanjul for providing samples of MA lines, P. Andolfatto for suggesting the use of PCR errors as positive controls, F. Oliver for help with DNA sequencing, and D. Charlesworth, J. Crow, J. Drake, A. Eyre-Walker, C. Haag, D. Houle and M. Lynch for comments on the manuscript. We are grateful to the Wellcome Trust for funding by a Functional Genomics Development Initiative grant. Author Contributions S.M., C.H.-L. and M.D. performed the DHPLC analysis. M.D. cloned and sequenced putative variants. X.M. cloned and sequenced positive controls. D.L.H. analysed selective constraints on indel mutations. B.C. advised on Drosophila genetics and interpreting the data. P.D.K. conceived and designed the project. C.H.-L. and P.D.K. analysed the data and wrote the paper. All authors revised the draft manuscript. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to P.D.K. (keightley.drosmutrate@spambob.net). NATURE | Vol 445 | 4 January 2007 LETTERS 85 Nature �2007 Publishing Group CORRIGENDUM doi:10.1038/nature06946 Direct estimation of per nucleotide and genomic deleterious mutation rates in Drosophila Cathy Haag-Liautard, Mark Dorris, Xulio Maside, Steven Macaskill, Daniel L. Halligan, David Houle 1 , Brian Charlesworth & Peter D. Keightley 1 Department of Biological Science, Florida State University, Tallahassee, Florida 32306- 1100, USA. Nature 445, 82?85 (2007) In this Letter, David Houle was omitted from the author list. David Houle was responsible for producing the Florida mutation accu- mulation lines that were analysed in the experiment. CORRECTIONS & AMENDMENTS NATUREjVol 453j1 May 2008 128 Nature Publishing Group�2008 "
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