Molecular Nature of 11 Spontaneous de Novo Mutations in Drosophila ...

Copyright  2001 by the Genetics Society of America

Molecular Nature of 11 Spontaneous de Novo Mutations in Drosophila melanogaster Hsiao-Pei Yang,* Ana Y. Tanikawa* and Alexey S. Kondrashov† *Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York 14853 and †National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland 20894 Manuscript received June 23, 2000 Accepted for publication November 27, 2000 ABSTRACT To investigate the molecular nature and rate of spontaneous mutation in Drosophila melanogaster, we screened 887,000 individuals for de novo recessive loss-of-function mutations at eight loci that affect eye color. In total, 28 mutants were found in 16 independent events (13 singletons and three clusters). The molecular nature of the 13 events was analyzed. Coding exons of the locus were affected by insertions or deletions ⬎100 nucleotides long (6 events), short frameshift insertions or deletions (4 events), and replacement nucleotide substitutions (1 event). In the case of 2 mutant alleles, coding regions were not affected. Because ⵑ70% of spontaneous de novo loss-of-function mutations in Homo sapiens are due to nucleotide substitutions within coding regions, insertions and deletions appear to play a much larger role in spontaneous mutation in D. melanogaster than in H. sapiens. If so, the per nucleotide mutation rate in D. melanogaster may be lower than in H. sapiens, even if their per locus mutation rates are similar.

S

PONTANEOUS mutation is the key genetic process that supplies raw material for stabilizing (deleterious mutations) and directional (beneficial mutations) natural selection. However, we know relatively little about the molecular nature of this process, i.e., about how common different events (nucleotide substitutions, deletions, insertions, duplications, etc.) are among all spontaneous mutations, or about the value of its basic quantitative parameter, the per nucleotide per generation spontaneous mutation rate ␮ (Drake et al. 1998; Kondrashov 1998). Two approaches can be used to estimate ␮. First, one can measure the degree of divergence between homologous selectively neutral DNA sequences in related species, provided that the total number of generations from their last common ancestor is known with good precision. The best data of this kind are available for the human-chimpanzee pair, where sequence divergence, mostly due to nucleotide substitutions, between orthologous pseudogenes is ⵑ1.3%. This implies, assuming a 20-year generation time and 5 million years since the last common ancestor, that ␮ ≈ 2 ⫻ 10⫺8 (Nachman and Crowell 2000). Unfortunately, this approach cannot currently be applied to Drosophila because the numbers of generations that separate pairs of Drosophila species are known, at best, only within a factor of 3–5, due to large uncertainties regarding the absolute divergence time and the number of generations per year in nature.

Corresponding author: Hsiao-Pei Yang, Department of Ecology and Evolutionary Biology, Corson Hall, Cornell University, Ithaca, NY 14853. E-mail: [email protected] Genetics 157: 1285–1292 (March 2001)

Furthermore, since Drosophila populations are large, even weak selection can be effective, leading to selection on silent sites causing substantial codon bias (Akashi 1997). Thus, it is difficult to be sure that a particular DNA sequence within a Drosophila genome evolved at a rate equal to the mutation rate (Pritchard and Schaeffer 1997). In contrast, there is little doubt that mutations in hominoid pseudogenes were effectively neutral. Second, ␮ can be estimated from the per locus mutation rate, m. If the mutational target at a locus, i.e., the number of nucleotides whose changes will lead to a phenotypically detectable mutation, is t, ␮ ⫽ m/t. However, the size of the mutational target depends strongly on the molecular nature of mutation. If we consider loss-of-function mutations, t for insertions and deletions is close to the total number N of protein-coding nucleotides at a locus, since most of such events (except inframe insertions and deletions) lead to malfunction of the affected proteins. In contrast, t for nucleotide substitutions may be closer to N/5, since only ⵑ5% of substitutions create an in-frame stop codon, and ⬍25% of missense substitutions (and very rare synonymous substitutions) lead to total loss of function (Mohrenweiser 1994; Kondrashov 1998). Thus, studying the molecular nature of mutation is important in its own right and is also essential for estimating ␮. In humans, the majority of loss-of-function de novo spontaneous mutations are substitutions (Krawczak et al. 2000). Thus, m ⫽ 10⫺5 for a locus with 2000 coding nucleotides implies ␮ ≈ 2 ⫻ 10⫺8, in agreement with the rate of neutral evolution. Data of this kind, first

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obtained for the hemophilia B locus (Sommer 1995), are now available for ⬎20 human loci (Krawczak et al. 2000). In contrast, we have little data on the molecular nature of de novo spontaneous mutation in Drosophila melanogaster. Yamaguchi et al. (1994) found that, out of six lossof-function (null) mutations at the Gpdh locus obtained in a mutation-accumulation experiment, five are P-element insertions and one is a long deletion. Loss-offunction alleles segregating in wild populations of D. melanogaster can be caused by both minor and largescale events (Nitasaka et al. 1995; ten Have et al. 1995); unfortunately these authors did not discriminate between substitutions and short indels. Data on molecular evolution imply that indels (mostly deletions) constitute ⵑ20% of all mutations in Drosophila, i.e., substantially more than in mammals (Petrov et al. 1996; Pritchard and Schaeffer 1997; Petrov and Hartl 1998). Petrov and Hartl (1998) found that deletions are much more common than insertions, while Pritchard and Schaeffer (1997) claim that they are about equally common. Thus, although extensive data on m are available for many loci of D. melanogaster (Schalet 1960; Woodruff et al. 1983), they cannot currently be used to estimate ␮. Obviously, we need more data on the molecular nature of mutation in Drosophila. Here, we report the results of specific locus tests (SLTs) of several loci that affect eye color; four loci are autosomal [chromosome 2, purple (pr), cinnabar (cn), and brown (bw); chromosome 3, scarlet (st)] and four are X-linked [prune (pn), vermilion (v), garnet (g), and white (w)]. Sixteen independent de novo spontaneous mutational events were detected in wild-type D. melanogaster, and molecular analysis of 13 mutations was performed.

MATERIALS AND METHODS Flies and cultural conditions: We sampled ⵑ100 mated females from a large wild population of D. melanogaster near Ithaca, NY. Flies were bred in 2.5 ⫻ 9.0-cm vials. Each vial contained 8 ml of food (1% agar, 0.1% propionic acid, 10% brewers yeast, and 10% glucose) seeded with a few grains of live baker’s yeast. At least 150 flies can develop simultaneously in such a vial without a significant increase of mortality. Flies were kept under a 12/12 light/dark cycle, at 25⬚ and 75% humidity. Generation one (G1) parents (see below) were stored at 16⬚, and remained fertile for at least 45 days. CO2 anesthesia was used. Tester strain and balancer strain: The tester strain, V, which is homozygous for the alleles pr1, cn1, bw1, st1, and kar1, and the balancer strain, STV, which has balancers on both autosomes (SM1 and TM3) and is heterozygous for alleles pr1, cn1, bw1, st1, and kar1, were both created by introducing mutant alleles paternally into the flies that originated from the same wild population. Large population sizes, at least 1000 flies, were maintained at all intermediate stages of the strain creation. As a result, both strains are vigorous, despite carrying several marker alleles. Detection of eye color mutation by SLT: Offspring of differ-

ent wild-caught females (G0) were crossed individually to unrelated G0 flies, and produced sibships of G1 flies (Figure 1). From each G1 sibship, at least 30 females were mated, in groups of 10, with strain V males (10–12 per group). Two or 3 days later, each group of females was allowed to lay eggs for 4 hr in a vial. During this time, 100–200 eggs were laid. After this, the mothers were removed and stored at 16⬚. The G2 offspring that emerged in these vials were screened for salient eye-color phenotypes due to mutations at the four autosomal loci (kar mutants could not be reliably detected) and at X-linked loci that affect eye color (only in males). Mosaic mutants were ignored. Since our design involves large G1 sibships, we can distinguish de novo clusters of mutations from preexisting heterozygosity. If an original G0 fly carried a heterozygous loss-of function allele at pr, cn, bw, st, or kar, 25% the G2 offspring of her or his G1 daughters should have a mutant phenotype. Seven such families, each with ⬎100 G2 flies with abnormal eye color found in more than one vial, were identified and removed from the analysis. These families were clearly different from the three de novo clusters that we have found (see below). If a single mutant was found in a vial, the G1 females that laid eggs in it were allowed to lay eggs again to make sure that the singleton was not actually a small cluster. Thus, we effectively studied the mutation process in G1, while screening G2. Identifying and isolating mutant alleles: Mutants detected in G2 during screening were mated individually with flies of the balancer strain, STV. Mendelian segregation within the offspring from these crosses determined the chromosome affected by a mutation and, for mutations at the four autosomal loci (pr, cn, bw, and st), identified the affected locus. Identity of the mutant locus was then confirmed by the appropriate complementation tests performed on offspring from G2 ⫻ STV cross. If a G2 fly was not a mutant at one of our four autosomal loci, only male mutants were further analyzed by the appropriate complementation tests with the following four X-linked loci: w, g, v, and pn. To isolate an autosome that carried the de novo mutant allele, we identified, using the appropriate crosses, male offspring from the G2 ⫻ STV cross that carry both SM1 and TM3 balancers and an autosome carrying a mutant allele affecting eye color only at the locus where a de novo mutation occurred. Only one such autosome was analyzed if the G2 mutant was a male (because there is no crossing over in males). Five chromosomes were analyzed if the G2 mutant was a female (in all such cases, the mutation occurred at bw, and bw is far away from pr and cn on chromosome 2), and the mutant allele different from the one present in strain V was regarded as the new one. All mutations discovered in fertile G2 individuals were homozygous viable and were, after being isolated, kept as pure strains, with the sole exception of the homozygous lethal loss-of-function mutation at pr, which was kept heterozygous with a second balancer strain (CyO). Molecular characterization of mutant alleles: Mutations found in our screen can be due to molecular events at different scales. Thus, we applied sequentially to each mutant three different techniques. 1. Cytogenetic analysis: Ectopic exchanges between transposable elements (TEs) situated in heterozygous positions around a locus at which mutations are screened can lead to cytogenetically detectable events. Slides of squashed salivary glands from five third instar larvae per mutant chromosome were prepared, following Ashburner (1989), and scored for the presence of deletions and duplications. In all cases, no major chromosomal alterations were found. 2. Southern blot analysis: Southern blotting was performed to detect mutations caused by transpositions and other rela-

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Figure 1.—Mating scheme for one family. R denotes meiosis, which forms the boundary between successive generations. S denotes the occurrence of syngamy. P denotes “perigametic interval,” the time around meiosis (from the last premeiotic DNA replication until the first DNA replication within the zygote). Fi and Mi denote females and males of the ith generation.

tively large events. Genomic DNA of ⵑ50 flies of each mutant line was extracted with SDS lysis, phenol-chloroform extraction, and ethanol precipitation (Sambrook et al. 1989). About 1 ␮g genomic DNA was restricted with one or more restriction enzymes with 6-bp recognition sequences, size-fractionated in 1% agarose gels, and blotted onto S & S Nytran neutral charge membranes by capillary transfer (Sambrook et al. 1989). The membranes were probed with 32P-random-primer-labeled DNA clones from the corresponding gene at 58⬚ overnight in hybridization solution (0.01 g/ml BSA, 1 mm EDTA pH 7.2, 0.5 m Na2HPO4 pH 7.2, and 7.5% SDS) and washed at 50⬚ for six to eight times in washing solution (40 mm Na2HPO4 pH 7.2, 1 mm EDTA pH 7.2, and 1% SDS). The hybridized membranes were exposed to X-OMAT films at ⫺80⬚ for 3–14 days. Maps of restriction sites and probed regions of the genes analyzed are shown in Figure 2. 3. Sequencing: For those mutant alleles where no large differences in the lengths of restriction fragments were detected, their coding regions were PCR amplified and sequenced. Primers for PCR reactions were designed using PRIMER3. Standard PCR conditions (100 ng genomic DNA template, 0.5 ␮g of each primer, 200 ␮m dNTPs, 1.5 mm MgCl2, 10 mm Tris-HCl pH 8.4, 50 mm KCl, 1 unit of Taq polymerase; Promega, Madison, WI) were used with thermal cycles: 95⬚ for 3 min, followed by 35 cycles of 92⬚ for 30 sec and 60⬚ for 1 min, and ending with 72⬚ for 7 min. Length of DNA amplified is within the range of 300–500 bp. The amplified DNA was purified with Ultrafree-MC centrifugal filters (Millipore, Bedford, MA) for sequencing. Both DNA strains were directly sequenced by autosequencing. The sequences were compared to wild-type alleles deposited in GenBank (accession nos. pn, Z12141; v, M34147; w, U64875; g, U31351; pr, U36232; cn, U56245; bw, M20630; and st, U39739) using basic BLAST search (version 2.0). Whenever a mismatch between the mutant sequence and the wildtype sequence was found, the gene region where the mismatch was located was PCR amplified and sequenced again to confirm the reality of the difference.

RESULTS

We screened ⵑ887,000 D. melanogaster for spontaneous de novo loss-of-function mutations that occurred in the female germ line at four autosomal loci (pr, cn, bw, and st) and four X-linked loci (pn, v, g, and w). In total, 28 mutants were found in 16 independent mutational events. Three events were clusters, of 10, 3, and 2 mutants, respectively. No mutations were found at g or w. Also, six X-linked eye color mutations at loci other than pn, v, g, or w were found. These were not analyzed because the loci involved are not yet known. Thirteen of the 16 independent mutations were isolated into pure strains and analyzed molecularly. In 11 cases, the probable cause of the mutant phenotype was found (Table 1, Figure 3).

DISCUSSION

Dealing with clusters of mutations: In multicellular organisms, a mutation can occur during any of many cellular generations that constitute a single organismal generation. As a result, a mutation can lead to a cluster of mutants (if it occurred well before gametogenesis within a parent of the individual screened), a singleton, or a mosaic (postzygotic) mutant (Figure 1; Russell and Russell 1996; Thompson et al. 1998; Russell 1999). Dealing with singletons is straightforward. Mosaics are hard to use for quantitative studies because they may be cryptic or difficult to detect (Schalet 1960, 1986). Eye-color mosaics, in particular, are manifested phenotypically only for loci that have autonomous ex-

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Figure 2.—Molecular structures of genes at the eight loci. Boxes indicate exons, and the solid and open boxes indicate the coding and nontranslated regions, respectively. Sizes of these genes are relative to the scale on the top. For genes analyzed by Southern blots, including cn, bw, pn, and v, locations of restriction sites analyzed and the probed regions are also shown in the bottom (shaded boxes) of each gene. More detailed sequence information can be found in references: pr, Kim et al. (1996); cn, Warren et al. (1996); bw, Martin-Morris et al. (1993); st, Tearle et al. (1989); pn, Teng et al. (1991); v, Searles et al. (1990); g, Lloyd (1998); w, O’Hare et al. (1984).

pression (bw and w among our eight loci) and only if a sizeable fraction of the eye surface happens to be affected. Thus, we ignored mosaics and concentrated on mutations that occurred during G1 (Woodruff and Thompson 1992; Drake et al. 1998). Any SLT must be designed in such a way that a cluster of de novo mutations can reliably be distinguished from preexisting heterozygosity. This is easy if a cluster is small, provided that enough offspring from each G1 individual are analyzed. However, mutations occurring just before the perigametic interval, which is the time between the last premeiotic DNA replication and the first DNA replication within the zygote (P in Figure 1), which are quite common (Russell and Russell 1996; Russell 1999), produce large clusters, such that up to 50% of gametes produced by the affected G1 individual carry a mutation. If such large clusters of de novo mutations are misclassi-

fied as preexisting heterozygotes, m can be underestimated by as much as a factor of 5 (Selby 1998a,b). Fortunately, even large clusters of de novo mutations can easily be distinguished from preexisting heterozygosity if sibships of 20–30 or more G1 individuals are analyzed. Thus, it is surprising that our SLT appears to be the first one to use this sibship-based design. Molecular nature of mutations: Among the 13 events analyzed, 5 mutations, all at bw, were insertions of lengths from 3100 to 4500 nucleotides, 1 was a deletion of 2000 nucleotides, 4 were short frameshift insertions or deletions in coding exons, and only 1 event, a cluster of 10 cn mutants, was probably due to a replacement nucleotide substitution (Table 1). Five long insertions were probably caused by transposable elements, although this is not certain. The frequency of insertions and deletions among lossof-function mutations must be 5–10 times higher than

vermilion

Prune

Scarlet

brown

Purple Cinnabar

Locus

1 2

1

U3

V1 V2

1 1

U1 U2

1 1 1 1 3 1

10

C4

B1 B2 B3 B4 B5 S1

1 1 1 1

No. of mutants constituting an event

P1 C1 C2 C3

Name of strain

Homozygous mutation is lethal, as are all pr loss-of-function alleles GenBank accession no. AF317317 GenBank accession no. AF317318 Mutant fly weak and sterile; the mutation was probably a major chromosomal alteration Not known which of these three replacements, if any, caused the loss of function GenBank accession no. AF317319

Comments

Four replacement substitutions causing three amino acid replacements: a. Nucleotide 3979, G → T; Arg → Met b and c. Nucleotide 4542, T → A and nucleotide 4543, C → A; Val → Glu d. Nucleotide 5468, G → C: Ala → Pro Insertion of ⵑ4500 nucleotides Insertion of ⵑ4100 nucleotides Insertion of ⵑ3600 nucleotides Insertion of ⵑ3100 nucleotides Insertion of ⵑ4500 nucleotides Not analyzed Mutant developed slowly and was sterile; presumably the mutation was a major chromosomal event Insertion of 4 nucleotides (AAAG) after nucleotide 1504 GenBank accession no. AF317313 No insertions, deletions, or replacement substitutions in Perhaps a regulatory mutation the coding regions GenBank accession no. AF317314 No insertions, deletions, or replacement substitutions in Perhaps a regulatory mutation the coding regions GenBank accession no. AF317315 Insertion of 4 nucleotides (GTGC) after nucleotide 1921 GenBank accession no. AF317316 Not analyzed Experimental error

Deletion of ⵑ2000 nucleotides Insertion of 1 nucleotide (C), after nucleotide 5004 Deletion of 40 nucleotides, from nucleotide 4771–4810 Not analyzed

Molecular nature of the event

Spontaneous de novo mutations found in the experiment

TABLE 1

Molecular Nature of New Mutations 1289

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Figure 3.—Southern blot analysis of bw mutant alleles. Samples of genomic DNA from each strain were digested with EcoRI (a), and with EcoRI and HindIII (b). Probed fragments and their expected sizes in wild-type (wt) alleles are indicated. (Lanes 1–7 in a correspond to wt, B1, B2, B3, B4, B5, and bw1 and lanes 1–6 in b correspond to wt B5, B4, B1, B2, B3, and bw1 respectively. Thin lines on the left are marker sizes.)

among all de novo mutations, because almost 100% of insertions and deletions, but only ⵑ10–20% of substitutions, within the coding regions inactivate a protein (Kondrashov 1998). Thus, our data imply that insertions and deletions, both major and short, constitute at least ⵑ20% of all spontaneous mutations in Drosophila, which agrees well with estimates by Petrov and Hartl (1998; 1999) based on patterns of DNA sequence evolution. Among loss-of-function mutations segregating in wild D. melanogaster populations, insertions and deletions are

also common (Nitasaka et al. 1995; ten Have et al. 1995). However, data on autosomal recessive loss-offunction alleles, both in Drosophila and in Homo, may not accurately reflect the properties of de novo mutation because many generations elapse between the origin of a mutation and its detection, which allows selection to act on heterozygotes. Our data also agree with early results by Mukai and Cockerham (1977), Voelker et al. (1980), and Harada et al. (1993), who found that the mutation rate toward loss-of-function (null) alleles of several proteins is ⵑ10 times higher than toward active alleles with changed electrophoretic mobility. This is to be expected if insertions and deletions are common. Indeed, all six null mutants found at the Gpdh locus during a mutationaccumulation experiment were major insertions (five) or deletions (one; Yamaguchi et al. 1994). The molecular nature of spontaneous mutation in humans is rather different. Replacement and nonsense nucleotide substitutions in coding regions cause ⵑ70% of X-linked recessive (Sommer 1995; Giannelli et al. 1998; Lemahieu et al. 1999; Tuffery-Giraud et al. 1999) and autosomal dominant (haplo-insufficient) de novo loss-of-function mutations (Krantz et al. 1998; Prosser and van Heyningen 1998; Clough et al. 1999; Lohmann 1999; Mayer et al. 1999; Niida et al. 1999; Westerman et al. 1999; Krawczak et al. 2000). For individual loci, however, the fraction of substitutions varies from ⬎90% (hemophilia B, Sommer 1995; Giannelli et al. 1998) to ⬍50% (Alagille syndrome, Krantz et al. 1998). Thus, insertions and deletions should account for only ⵑ5% of all spontaneous human mutations, which is confirmed by data on DNA sequence evolution (Nachman and Crowell 2000). This difference may be at least partially due to methylation of mammalian DNA at CpG dinucleotides, which drastically increases substitution rates at many such nucleotides. As a result, ⬎30% of human nucleotide substitutions occur at CpG hotspots (Shiang et al. 1994; Sommer 1995). In contrast, DNA in Drosophila is not methylated (see Lyko et al. 1999), and CpG dinucleotides apparently mutate at a normal rate. Per locus mutation rate m: Because mutations at the four X-linked loci could be detected only in male offspring, our estimate of the per locus rate for independent mutational events is k ⫽ 16/B ⫽ 3.0 ⫻ 10⫺6, where B is the total number of loci screened. Since B ⫽ 4 ⫻ 887,000 ⫹ 4 ⫻ 443,500 ⫽ 5,322,000, and the total number of mutants found was 28 (Table 1), our estimate of the per locus mutation rate is m ⫽ 28/B ⫽ 5.3 ⫻ 10⫺6. The number of rare independent events has a Poisson distribution, so that the 95% confidence interval for k is 1.7–4.9 ⫻ 10⫺6. The confidence limits for m cannot be calculated precisely, due to insufficient data on the fraction of clusters among all mutational events and on the distribution of cluster size. Assuming that between 50 and 75% of all spontaneous mutations occur in clus-

Molecular Nature of New Mutations

ters (Thompson et al. 1998), we can therefore tentatively conclude that 2.0 ⫻ 10⫺6 ⬍ m ⬍ 15.0 ⫻ 10⫺6. This estimate is in agreement with those obtained previously for both D. melanogaster (3–5 ⫻ 10⫺6; Mukai and Cockerham 1977; Voelker et al. 1980; Woodruff et al. 1983; Harada et al. 1993) and mammals (Drake et al. 1998). Per nucleotide mutation rate ␮: Despite similar values of m for loss-of-function mutations in Drosophila and in humans (Drost and Lee 1995), ␮ in Drosophila may be substantially smaller than in humans because the mutational target size for indels, which are more common in Drosophila, is 5–10 times larger than for substitutions. Because the average length of the coding regions of the eight loci used in our screening is 1620 nucleotides, our estimate for the component of ␮ due to insertions and deletions is ␮indel ⫽ (1.2 ⵑ 10.0) ⫻ 10⫺9. Not a single nonsense substitution was found in 887,000 flies screened at the four autosomal loci in which we calculate that there are 526 possible substitutions that would create an in-frame stop codon, nor in 443,500 flies screened at the four X-linked loci in which we calculate 775 possible substitutions that could create an in-frame stop codon. Thus, assuming that all nucleotide substitutions occur with the same rate, no nonsense substitutions happened within the target equivalent of (526 ⫻ 887,000 ⫹ 775 ⫻ 443,500)/3 ⫽ 2.7 ⫻ 108 nucleotides (division over 3 is because a nucleotide can be substituted in three ways). If we ignore clustering, this implies that ␮sub ⬍ 1.2 ⫻ 10⫺8 with 95% confidence. In contrast to mammals, there is no evidence that the spontaneous mutation in Drosophila is male biased, and there are approximately equal numbers of cell divisions in the female and male germ lines (Bauer and Aquadro 1997). Screening of ⵑ100,000 flies for mutations in the male germ line did not provide any evidence for elevated mutation rate (data not reported). Thus, we probably did not underestimate k and m by our procedure. Out of ⵑ100 spontaneous de novo mutations that are estimated to occur in a human genome every generation, at least 2–3 are probably deleterious (EyreWalker and Keightley 1999). In contrast, the genomic deleterious mutation rate U in Drosophila is poorly known, although it is probably below that in humans (Kondrashov 1998). Drosophila, being sexual, may falsify the mutational deterministic hypothesis for the maintenance of sexual reproduction if it has U ⬍ 0.5– ⵑ1.0 (Kondrashov 1988). Assuming that, as in Caenorhabditis elegans (Shabalina and Kondrashov 1999), ⵑ30% of 3 ⫻ 108 nucleotides in the D. melanogaster diploid genome are controlled by selection, we can conclude that U ⬍ 0.5–1.0 and, therefore, this hypothesis must be rejected for this species, if ␮ ⬍ 0.5–1.0 ⫻ 10⫺8. We thank S. A. Shabalina, F. A. Kondrashov and V. A. Kondrashov for helping with mutant screening; S. V. Nuzhdin for guidance in the

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molecular part of the work; R. MacIntyre and C. Webb for useful suggestions, the anonymous reviewer who suggested that CpG methylation in mammals may be responsible for differences between Drosophila and humans; and the following people for providing gene clones: W. Warren (cn), L. Searles (v), P. Kim (bw), and K. O’Hare (w). This work was supported by a Fellowship for Study Abroad from the Republic of China Government to H.-P. Yang and a National Science Foundation grant DEB-9815621 to Sergey V. Nuzhdin.

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