Accumulation of transposable elements in laboratory lines ... - CiteSeerX

167

Genetica 100: 167–175, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Accumulation of transposable elements in laboratory lines of Drosophila melanogaster Sergey V. Nuzhdin1 2  , Elena G. Pasyukova2 & Trudy F.C. Mackay1 ; ;

1

Genetics-7614, North Carolina State University, Raleigh, NC 27695, USA; 2 Institute of Molecular Genetics, Kurchatov sq, Moscow, Russia, 123182;  Address for correspondence: Section of Evolution and Ecology, University of California at Davis, Davis, CA 95616-5755, USA Accepted 22 April 1997

Abstract It is recognized that a stable number of transposable element (TE) copies per genome is maintained in natural populations of D. melanogaster as a result of the dynamic equilibrium between transposition to new sites and natural selection eliminating copies. The force of natural selection opposing TE multiplication is partly relaxed in inbred laboratory lines of flies. The average rate of TE transposition is from 2.6  10 4 to 5.0 10 4 per copy per generation, and the average rate of excision is at least two orders of magnitude lower; therefore inbred lines accumulate increasing numbers of copies with time. Correlations between the rate of transposition and TE copy number have been determined for copia, Doc, roo, and 412 and found to be either zero or positive. Because the rate of transposition is not a decreasing function of TE copy number, TE accumulation in inbred lines is selfaccelerating. Transpositions cause a substantial fraction of mutations in D. melanogaster, therefore the mutation rate should increase with time in laboratory lines of this species. Inferences about the properties of spontaneous mutations from studies of mutation accumulation in laboratory lines should be reevaluated, because they are based on the assumption of a constant mutation rate. Introduction The variability of individuals in natural populations originates from mutations, most of which are unconditionally deleterious, and are eliminated by natural selection (Kondrashov, 1988). Quantitative theoretical descriptions of the mutation-selection balance are based on the mutation rate and the rate of elimination of mutations given their selective disadvantage (Falconer & Mackay, 1996). However, estimating these parameters is difficult because the harmful effect of a mutation a) is usually too small to be reliably measured for an individual mutation and b) may be conditional upon the number and properties of other mutations of the organism (Kondrashov, 1988). To measure the reduction in fitness from spontaneous mutations, mutations are usually accumulated in replicate laboratory lines maintained under conditions of suppressed natural selection (Crow & Simmons, 1983; Houle et al., 1994; Mukai, 1964; Mukai et al., 1972; Ohnishi, 1977). However,

with a few exceptions (Mackay, Lyman & Jackson, 1992), the number of mutations contributing to the decline in fitness is unknown, and this complicates straightforward inferences (Keightley, 1994). Nevertheless, key properties of mutations may be inferred if we assume that the rate of mutation is not conditional on the number of mutations previously accumulated in the genome (Mukai et al., 1972). Under this assumption, the distribution of the numbers of mutations independently accumulated in the replicates of the mutation-accumulation lines follows a Poisson distribution. Here we examine whether the assumption of independence of the rate of mutations on the number of accumulated mutations is reasonable. Transposition of TEs to novel sites represents one of the largest sources of mutation: nearly 50% of spontaneous mutations with major morphological effects in D. melanogaster are caused by TEs and insertional mutations reduce fitness (Finnegan, 1992, Mackay et al., 1992). This class of mutations is especially

168 interesting because it is possible to count them directly, independently of their phenotypic effects. Moreover, the rate of TE transposition per element, that is the number of transpositions per TE copy per generation, may be measured directly (Pasyukova & Nuzhdin, 1992). Based on this measurement, one can calculate the family transposition rate, which is the number of transpositions per genome per generation for all copies of one TE family. Then the total genomic transposition rate for all copies of all families taken together is calculated by adding family transposition rates. The definitions of the transposition rate, family transposition rate, and total genomic transposition rate will be referred to throughout the text. If this is done for lines with different numbers of TE copies in the genome, the relationship between the total genomic transposition rate and TE copy number can be determined. The rate of mutation per genome per generation is the total genomic transposition rate, plus the rate of mutations from all other sources (e.g., base pair mutation, ectopic exchange). The former component is measurable, and the latter may be reasonably well estimated. Consideration of rates and effects of TE transposition will clarify the characteristics of the process of mutation accumulation in general. Below we describe the origin of several laboratory lines with a range of TE copy numbers and the direct measurement of the rates of transpositions in these lines. We will also discuss estimates of the total genomic transposition rate, and how the relationship of the rate to TE copy number modifies inferences derived from mutation-accumulation experiments assuming a constant mutation rate. Although this paper represents an overview of the literature, it is not intended as a comprehensive review. We shall mostly restrict our discussion to experiments in which the technique of in situ hybridization of labelled TE DNA to polytene salivary gland chromosomes was used to directly observe transpositions (mutations). Although this technique provides the most accurate description of the distribution of TE insertion sites (and is currently the only reliable method to directly measure the rate of transposition), this description is necessarily restricted to euchromatic regions of the chromosomes. However, mutations in these regions represent the major fraction of all genomic mutations.

I. Transposition-selection balance in natural populations and transposition accumulation in laboratory lines: retrotransposable elements The rate of transposition per element has been estimated indirectly for natural populations or directly for laboratory lines. In the former case it was inferred from the distribution of insertion site frequencies under the assumption of a balance between TE transposition to new sites, and selective elimination of hosts with more TEs due to their decreased fitness. The authors first sampled chromosomes from natural populations and made them homozygous, and then determined the cytological insertion sites with in situ hybridization. Given available estimates of the effective population size (Ne ), the rate of transposition (u) was estimated as 10 5 10 3 for natural populations of D. melanogaster (Montgomery & Langley, 1983; LeighBrown & Moss, 1987; Charlesworth & Lapid, 1989; Charlesworth, Lapid & Canada, 1992a,b; Bi´emont et al., 1994), and D. simulans (Nuzhdin, 1995; Vieira & Bi´emont, 1996). The results of most of these experiments have been summarized and thoroughly discussed by Charlesworth, Sniegowski and Stephan (1994). It is assumed that TEs are in equilibrium between transposition and selection, with selection coefficient s against a TE copy segregating in a natural population. If elements are in equilibrium, as is indicated by the population data, s should be similar to the average transposition rate, because for TE copy number at equilibrium u = s + v, where v is the rate of excision (Charlesworth, Sniegowski & Stephan, 1994). Because v  u for retrotransposons (see section II), s = 10 5 10 3 . This estimate represents the lower bound for selection against a TE copy since it was obtained by considering inserts segregating in natural populations, i.e., those without a dramatic effect on the host fitness. The average heterozygous fitness decline per new spontaneous mutation is 0.02 (Mukai et al., 1972). There is no reason to believe that TE-induced mutations are less deleterious than average (Mackay, Lyman & Jackson, 1992). Most probably, many transpositions cause harmful mutations that decrease fitness by several percent, and thus are immediately selected out. The remaining insertions have slight indirect deleterious effects (Charlesworth, 1991) caused by ectopic recombination between TE copies (Langley et al., 1988) or by TE expression (Nuzhdin, Pasyukova & Mackay, 1996). The relative contribution of different selection forces to the maintenance of TE copy number

169 Table 1. Estimation of the average rate of TE accumulation from transposition-accumulation experiments Size of the experiment

Transposition

Excision

Accumulation rate

17,160 448,000 1,264,700

7 229 327

1 3 3

3.5 5.0 2.6

 10  10  10

4 4 4

Reference

Eggleston, Johnson-Schlitz & Engels, 1988 Harada, Yukuhiro & Mukai, 1990 Nuzhdin & Mackay, 1995

 We have excluded cases of induced transposition from P, I, and hobo elements.  Size of the experiment = (number of TE copies scored per transposition accumulation replicate line)  (number



of replicate transposition accumulation lines) (number of generations of transposition accumulation).  Accumulation rate is calculated as the number of transpositions minus the number of excisions divided by the size of the experiment.

in natural populations is discussed by Bi´emont et al. (this volume). The second and more precise approach to estimating transposition rates is to observe transpositions directly, over time. In these experiments, the positions of TEs along chromosomes were scored in individual chromosomes or in the whole homozygous genomes by the in situ hybridization technique, and rescored again after many generations of line maintenance. New insertion sites were caused by transpositions. The rates of transposition were estimated from these data assuming neutrality of transposition events (Eggleston, JohnsonSchlitz & Engels, 1988; Harada, Yukuhiro & Mukai, 1991; Nuzhdin & Mackay, 1994, 1995). This is a reasonable assumption if s against a TE copy is on average 10 5 10 2 , since TE inserts will be predominantly affected by drift but not by selection when Ne is small (i.e., the condition 4Ne s < 1 is satisfied, Kimura, 1983). Ne in the above experiments was between 1 (Mukai, 1964, 1969) and 14 (Mackay, 1992). Eggleston, Johnson-Schlitz and Engels (1988), Harada, Yukuhiro and Mukai (1991), and Nuzhdin and Mackay (1994, 1995) determined the rates of transposition for many TE families in large scale experiments. Transposition rates varied widely between TE families, for instance from 1 (Crow & Morton, 1955) for laboratory lines kept as mass cultures, and these inserts will be eliminated by selection. Thus, TE accumulation in laboratory lines should be less than 0.37 copies per generation. Muller’s ratchet may, nevertheless, be responsible for accumulation of even highly deleterious inserts (Charlesworth, Morgan & Charlesworth, 1993). For example Pasyukova, Nuzhdin and Filatov (submitted) observed progressive accumulation of copia copies over a period of nine years in lines kept as small mass cultures. The total genomic rate of transposition presented in Table 1 was inferred based on three experiments in each of which transpositions of many TE families were found. It is possible that the three experiments were started from lines with unusually active TEs, and normally the total genomic rate of transpositions is lower. However, frequent transpositions of one or many TE families have been observed in many other experiments (Table 2). Although the total genomic rate of transposition could not be estimated from these experiments because the number of scored TE families was low, the number of transposition events was low, and/or Ne was large, they provide a good argument against this hypothesis. To the best of our knowledge, no laboratory line has been found to date for which copies of at least one TE family did not transpose with an appreciable rate. TE transposition is an important source of mutations in the majority, if not all, laboratory lines of D. melanogaster.

II. The relationship between the rate of transposition and TE copy number: retrotransposable elements The relationship between the rate of transposition and TE copy number determines the distribution of the numbers of accumulated TE copies in independently maintained replicates of mutation/transpositionaccumulation lines. These distributions for the roo and copia families have been compared to a Poisson distribution expected under the null hypothesis of no relationship between the family rate of transpositions and TE copy number (Nuzhdin & Mackay, 1994). The distribution of the numbers of new insertions among sublines was not significantly different from random for roo (2 goodness-of-fit statistic to a Poisson distribution was 28 = 3:43), but was highly significant for copia (212 = 93:70, P < 0.001). This justifies a consideration of the relationship between the family transposition rate and the number of TE copies in the genome. The rate of transposition is usually too low to be measured directly. However, in a few cases the rate was elevated to a level that allowed its estimation in individual flies from replicates of the transpositionaccumulation lines described above (copia, Nuzhdin, Pasyukova & Mackay, 1996; copia, Pasyukova, Nuzhdin & Filatov, submitted; Doc, roo, Nuzhdin & Pasyukova, unpublished) or from natural populations of D. simulans with a wide range of the 412 TE copy numbers (Vieira & Bi´emont, 1996; see also Bi´emont et al., this volume). To estimate transposition rate, single flies are crossed to flies of a tester line, in which the rate of transposition of the TE family of interest is

171 Table 3. The relationship between transposition rate and TE copy number TE family

Number of transpositions

Relationship

Reference

copia copia Doc P roo 412

429 598 65 N/A 6 7

positive no or positive positive negative no no

Nuzhdin, Pasyukova & Mackay, 1996 Pasyukova, Nuzhdin & Filatov, unpublished Nuzhdin & Pasyukova, unpublished Bi´emont, 1994 Nuzhdin & Pasyukova, unpublished Vieira & Bi´emont, 1996

 The relationship is positive for transposition rates measured in 1994, but not for the rates measured in 1995, nor for combined data of both years.  Not available, but see Bi´emont, 1994.

negligible, and many progeny are analyzed for TE positions by in situ hybridization. The TE positions in the tester and tested flies are known or can be reconstructed (Pasyukova & Nuzhdin, 1992), so it is possible to infer de novo transpositions by comparing inserts in parents and F1 progeny. Transposition rates are estimated by dividing the number of observed transpositions by the TE copy number in the tested flies. In the six studies that measured transposition rate, positive (three studies) or no (three studies) association between the rate of transposition and TE copy number was found (Table 3). When a positive relationship was detected, the rate of transposition increased from two fold (Doc, Nuzhdin & Pasyukova, unpublished) to over a hundred (copia, Nuzhdin, Pasyukova & Mackay, 1996; Pasyukova, Nuzhdin & Filatov, submitted) when copy number increased by a factor of two. Because TE copy number is progressively increasing in mutation/transposition-accumulation lines, the family transposition rate should increase linearly with TE copy number for elements with a constant transposition rate, and greater than linearly for the TEs with a positive relationship between the rate of transposition per copy and TE copy number. Another case for which there are indications of a positive relationship between the family transposition rate and TE copy number has been reported; Prud’homme et al. (1995) estimated the activity of gypsy transpositions by the rate of reversions of ovoD1 mutation (most reversions were caused by gypsy transpositions, Mevel-Ninio, Mariol & Gans, 1989) in grand-daughters of females with different gypsy copy numbers. Although gypsy activity appeared to be positively correlated with the gypsy copy number, it was difficult to quantify the effects observed from indirect data.

There are biological grounds for believing that a positive correlation of the rate of transposition and TE copy number may be frequently found. For LINE elements, the concentration of the LINE transcript in the cell should be proportional to n, where n is the TE copy number (the simplest assumption of no regulation of transcription is adopted here). The concentration of the reverse transcriptase-integrase complex should therefore be proportional to n. The probability for the reverse transcriptase-integrase complex to meet the LINE transcript available for integration and initiate transposition should be proportional to n2 . Thus the family rate of the LINE transposition should be proportional to n2 , and the rate of transposition per copy to n. For retroviruslike TEs, the power of the relationship might be even greater than 2 (Nuzhdin, Pasyukova & Mackay, 1996). One may argue that TEs should have evolved to self-regulate their own transpositions. However, it has been shown theoretically that self-regulation of the rate of transposition is not a favorable evolutionary strategy for TEs. This holds true unless there is a strong deleterious effect associated directly with the TE copy whose activity is responsible for transposition (the mother copy), rather than with its progeny copies (Charlesworth & Langley, 1986). Even highly deleterious mutations that may be caused by new insertions have no or little impact on the survivorship of the mother copy (Charlesworth & Langley, 1986) when there is no linkage disequilibrium between the mother copy and progeny copies, as would occur with free recombination, and new insertions are randomly distributed on chromosomes with respect to the location of the mother copy. Although the survivorship of the mother copy is not (or is only slightly) influenced by its activity, the number of its progeny is proportional to activity. Therefore, there is likely to be selection

172 for maximal activity rather than for self-regulation to maximize the number of progeny copies per mother copy. A positive relationship between the rate of transposition and TE copy number might be characteristic of the majority of TE families. An example of self-regulation is available for the I retrotransposon, the activity of which directly decreases the fitness of flies (Bucheton et al., 1976; Pelisson & Bregliano, 1987; Pritchard et al., 1988; Vaury et al., 1993). If the rate of I transposition is not zero, but the rate of excision is, I element copy number will increase, and transposition rate will decrease with generations of mutation/transposition- accumulation. The family rate of I transposition may decrease, stay constant, or increase depending on the exact (and unknown) relationship between the rate of I transposition and I copy number. However, the rate of I transpositions is very similar to the rate of transposition of other TEs in non-dysgenic conditions (Leigh-Brown & Moss, 1987; Harada, Yukuhiro & Mukai, 1988). Unless mutation-accumulation is started from a dysgenic cross, the proportion of mutations caused solely by I element transpositions will not be high among mutations caused by all TEs. This is indirectly supported by the fact that only two of hundreds of mutations found in natural populations of D. melanogaster were caused by I transpositions (Bucheton et al., 1992). The unknown relationship between the rate of I-induced mutations and I copy number should not dominate the total relationship. We conclude that the retrotransposon total genomic transposition rate increases with generations of mutation/transposition-accumulation experiments, provided that the new mutations arising in the host do not suppress transpositions. This is a likely condition for the limited duration of mutation-accumulation experiments (see Prud’homme et al., 1995; Pasyukova, Nuzhdin & Filatov, submitted, for details). However, it is still unclear what would be the increment in the total genomic transposition rate for a given number of generations.

III. Transposition-selection balance in natural populations and laboratory lines: transposons Only limited data are available about transposon copy number dynamics in natural populations or laboratory lines. In one experiment, the number of P element copies remained stable in laboratory lines as a result of the balance between transpositions and excisions

(Bi´emont, 1994). This conclusion should be taken with caution. Some inserts may be fixed by chance, and a true excision from fixed sites is impossible, because the mechanism of transposon excision necessitates the presence of an insert-free template for repairing the double strand breaks caused by excision. It has been suggested that many transposons selfregulate their own copy number. Similar to the I element, transposon activity is associated with a direct deleterious effect on host fitness (Kidwell, Kidwell & Sved, 1977). In addition, transpositions of the P element are not random; rather, the progeny copies are frequently inserted close to the mother copy. This links harmful effects caused by new insertions to the mother copy (Tower et al., 1994). Thus, self-regulation of copy numbers should be a favorable evolutionary strategy for transposons (Brookfield, 1991, 1996). One could predict that the rate of transpositions would decline if transpositions accumulated in the laboratory lines with time. However, the transposon copy number becomes stable in laboratory lines (Bi´emont, 1994). Because the transposon copy number does not change across generations, the genomic rate of transposition will be the same whatever the relationship between copy numbers and transposition rates.

IV. Mutation accumulation in laboratory lines It is interesting to compare the total genomic rate of transposition at early and late generations of mutation/transposition-accumulation experiments since retrotransposons accumulate with time in laboratory lines, and the transposition rate for many elements is not a decreasing function of the TE copy number (Table 3). We will assume that the duration of the experiment is 100 generations, initially 103 copies of all families of TEs are present in the genome, and 37 transpositions (Table 1) accumulated during the experiment. If the rate of transposition is independent of TE copy number, then TE copy number grows according to the function n(t) = n(0)exp(ut) (Charlesworth & Charlesworth, 1983), where t is the number of generations. If one assumes that all TE copies transpose with the same rate, then u can be estimated as 3.6  10 4 from n(0) = 1000, and n(100) = 1037. At generation 0 the total genomic transposition rate is 0.36, and at generation 100 the rate is 0.373; approximately 4% higher. Only about one fifth of all TE families are transpositionally active in laboratory lines, and are responsible

173 for transposition accumulation (Table 2). Assuming n(0) = 200 and n(100) = 237, u is 1.70  10 3 for copies of active families. Under this more realistic assumption, the total genomic transposition rate from active copies would increase from 0.34 to 0.40, or by 19%. One may now take into account the observation that the rate of transposition increases when TE copy number is increased. As an example, we will assume a 10 fold increment when copy number doubles (see section II). Since the copy number at generation 100 is 1.19 times higher than at generation 0, the rate of transposition is (1.19 1)  10 or 1.9 times higher. The total genomic transposition rate of transposition at generation 100 is 1.19  1.9, or 2.3 times the total genomic rate of transposition at generation 0. The above calculations show that under reasonable assumptions, the total genomic transposition rate may vary by a factor as large as 1.2–2.3 between early and late generations of mutation accumulation experiments. Further, TE transposition is an important source of spontaneous mutations in D. melanogaster (Finnegan, 1992): the estimate of the total genomic rate of transposition, 0.37, is of the same order as the nucleotide spontaneous mutation rate. If there are 1.6  10 8 base-pair mutations per year (Sharp & Li, 1989) and 1.7  108 base pairs in Drosophila genome (Ashburner, 1989), there are in total 2.7 mutations per year. With 10 generations of flies per year, the rate of base pair mutations is approximately 0.27 per generation. If in early generations of mutation/transpositionaccumulation experiments the total genomic transposition rate and mutation rate from all other sources are comparable, in later generations TE-induced mutations may account for up to two thirds of the total number of mutations, and the total mutation rate may be about 1.7 times higher than in earlier generations. We have considered the question of how the disturbance of the multiplication-selection balance that is characteristic for selfish DNA in natural populations may lead to an increment of mutation rate, and consequently to faster deterioration of the host fitness. Because the estimates of mutation pressure in natural populations were inferred from analysis of the lines that were kept in the laboratory for hundreds of generations before the experiments, these estimates are probably biased upward. Many other vertically inherited parasites, such as the Sigma virus (Fleuriet, 1976), and the Wolbakia (Hoffman, Turelli & Simmons, 1986) should also become more abundant in laboratory lines and cause a

decrement in fitness. For example, flies contaminated and not contaminated with Wolbakia have similar productivities when lines extracted from nature are initially kept under laboratory conditions, but their productivities may be different by 20% in a few generations (Hoffman, Turrelli & Harshman, 1990). A priori, it is impossible to predict the amount and the rate of fitness decline associated with multiplication of different selfish elements. Our intention here has been to direct attention towards the necessity for taking into account the effect of parasite multiplication as a cause of the selfaccelerated fitness decline when the selection pressure is relaxed in the laboratory. We would like to illustrate this statement using a simple example. Synergistic epistasis between deleterious mutations has important implications for a variety of evolutionary questions (Kondrashov, 1988). However, the only reliable information available on the magnitude of the possible synergistic effects of deleterious mutations (Crow, 1970) comes from the comparison of the rate of viability decline in late versus early generations of Mukai’s (1969) long-term mutation accumulation experiment. Regressing viability on the number of generations gives the estimates of linear and squared terms and their standard errors: viability = b(1)x + b(2)x2 , as b(1) = 7:7  10 4 (1.6  10 3 ) and b(2) = 1:2  10 4 (2.4  10 5 ), where x is the number of generations. The rate of viability decline is b(1) + 2b(2)x, or 30 times higher in generation 100 compared to generation 0. However, this ratio has a very high standard error since the numerator b(1) has a large error variance. If one recalculates it using the lower confidence limits for b(1) and b(2) (2.3  10 3 and 9.5  10 5 , respectively) then the ratio becomes 5. It is unknown what fraction of the faster rate of viability decline in later generations of mutation accumulation experiments is explained by multiplication of selfish elements, and what fraction is due to synergistic epistasis of deleterious mutations.

Acknowledgements This work was supported by NIH grants GM 45344 and GM 45146 to TFCM and by Russian Fund of Basic Research grant #94-04-11423-a to EGP. The participation of SVN in the transposable element workshop in Gif-sur-Yvette was financed by Kluwer Academic Publishers, for which we would like to thank Drs. Capy and McDonald. We also thanks Drs. Bi´emont and

174 Vieira for providing us with their unpublished materials, and Christian Bi´emont for helpful comments.

References Ashburner, M., 1989. Drosophila: a laboratory handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Bi´emont, C., F. Lemeunier, M. P. Garcia Guerreiro, J. F. Brookfield et al., 1994. Population dynamics of the copia, mdg1, mdg3, gypsy, and P transposable elements in a natural population of D. melanogaster. Genet. Res. 63: 197–212. Bi´emont, C., 1992. Population genetics of transposable elements. A Drosophila point of view. Genetics 86: 67–84. Bi´emont, C., 1994. Dynamic equilibrium between insertion and excision of P elements in highly inbred lines from an M0 strain of Drosophila melanogaster. J. Mol. Evol. 39: 466–472. Bi´emont, C., A. Aouar & C. Arnault, 1987. Genome reshuffling of the copia element in an inbred line of Drosophila melanogaster. Nature 329: 742–744. Birchler, J.A. & J.C. Hiebert, 1989. Interaction of the Enhancer of white-apricot with transposable element alleles at the white locus in Drosophila melanogaster. Genetics 122: 129–138. Brookfield, J.F.Y., 1991. Models of repression of transposition in PM hybrid dysgenesis by P cytotype and by zygotically encoded repressor proteins. Genetics 128: 471–486. Brookfield, J.F.Y., 1996. Models of the spread of non-autonomous selfish transposable elements when transposition and fitness are coupled. Genet. Res. 67: 199–211. Bucheton, A., J.-M. Lavige, G. Picard & P. L’Heritier, 1976. Nonmendelian female strerility in Drosophila melanogaster: quantitative variation in the efficiency of inducer and reactive strains. Heredity 36: 305–314. Bucheton, A., C. Vaury, M.-C. Chaboissier, P. Abad et al., 1992. I elements and the Drosophila genome. Genetica 86: 175–190. Charlesworth, B., 1991. Transposable elements in natural populations with a mixture of selected and neutral insertion sites. Genet. Res. 57: 127–135. Charlesworth, B. & C.H. Langley, 1986. The evolution of selfregulated transposition of transposable elements. Genetics 112: 359–383. Charlesworth, B. & D. Charlesworth, 1983. The population dynamics of transposable elements. Genet. Res. 42: 1–27. Charlesworth, B. & A. Lapid, 1989. A study of ten transposable elements on X chromosomes from a population of Drosophila melanogaster. Genet. Res. 54: 113–125. Charlesworth, B., A. Lapid & D. Canada, 1992a. The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. I Element frequencies and distribution. Genet. Res. 60: 103–114. Charlesworth, B., A. Lapid & D. Canada, 1992b. The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. II Inferences on the nature of selection against elements. Genet. Res. 60: 115–130. Charlesworth, B., P. Sniegowski & W. Stephan, 1994. The evolutionary dynamics of repetitive DNA in eukariotes. Nature 371: 215–220. Charlesworth, D., M.T. Morgan & B. Charlesworth, 1993. Mutation accumulation in finite outbreeding and inbreeding populations. Genet. Res. 61: 39–57.

Crow, J.F., 1970. Genetic loads and the cost of natural selection, pp. 128–177 in Mathematical Topics in Population Genetics, edited by K. Kojima. Berlin: Springer-Verlag. Crow, J.F. & M.J. Simmons, 1983. The mutation load in Drosophila, pp. 1–35 in The Genetics and Biology of Drosophila, Vol. 3c, edited by M. Ashburner, H.L. Carson and J.N. Thompson, Jr. Academic Press, London. Crow, J.F. & N.E. Morton, 1955. Measurement of gene frequency drift in small populations. Evolution 9: 202–214. Csink, A.K., R. Linsk & J.A. Birchler, 1994. Mosaic suppressor, a gene in Drosophila that modifies retrotransposon expression and interacts with zeste. Genetics 136: 573–583. Csink, A.K. & J.F. McDonald, 1990. copia expression is variable among natural populations. Genetics 126: 375–382. Eggleston, W.B., D.M. Johnson-Schlitz & W.R. Engels, 1988. P-M hybrid dysgenesis does not mobilize other transposable element families in Drosophila melanogaster. Nature 331: 368–370. Falconer, D.S. & T.F.C. Mackay, 1996. Introduction to Quantitative Genetics. Addison Wesley Longman. Finnegan, D.J., 1992. Transposable elements, pp. 1096–1107 in The genome of Drosophila melanogaster, edited by D.L. Lindsley and G. Zimm. New York: Academic Press. Fleuriet, A., 1976. Presence of the hereditary Rhabdovirus sigma and polymorphism for a gene for resistance to this virus in natural populations of Drosophila melanogaster. Evolution 30: 735–739. Georgiev, P.G., S.L. Kiselev, O.B. Simonova & T.I. Gerasimova, 1990. A novel transposition system in Drosophila melanogaster depending on the Stalker mobile genetic element. EMBO J. 9: 2037–2044. Glushkova, I.V., E.S. Belyaeva & V.A. Gvozdev, 1991. Maintenance of copy number of retrotransposon mdg3 in Drosophila melanogaster. Genetika 27: 404–410. Harada, K., K. Yukuhiro & T. Mukai, 1990. Transposition rates of movable genetic elements in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 87: 3248–3252. Hoffman, A.A., M. Turelli & L.G. Harshman, 1990. Factors affecting the distribution of cytoplasmic incompatibility in Drosophila simulans. Genetics 126: 933–948. Hoffman, A.A., M. Turelli & G.M. Simmons, 1986. Unidirectional incompatibility between populations of Drosophila simulans. Evolution 40: 692–701. Houle, D., K.A. Hughes, D.K. Hoffmaster, J. Ihara, S. Assimacopoulos, D. Canada & B. Charlesworth, 1994. The effects of spontaneous mutation on quantitative traits. I. Variances and covariances of life history traits. Genetics 188: 773–785. Kaplan, N., T. Darden & C.H. Langley, 1985. Evolution and extinction of transposable elements in mendelian populations. Genetics 109: 459–480. Keightley, P.D., 1994. The distribution of mutation effects on viability in Drosophila melanogaster. Genetics 138: 1315–1322. Kidwell, M.G., J.F. Kidwell & J.A. Sved, 1977. Hybrid dysgenesis in Drosophila melanogaster: a syndrome of aberrant traits including mutation, sterility, and male recombination. Genetics 86: 813– 833. Kim, A., E.S. Belyaeva & M.M. Aslanian, 1990. Autonomous transposition of gypsy mobile elements and genetic instability in Drosophila melanogaster. Mol. Gen. Genet. 224: 303–308. Kim A.I., N.V. Lyubomirskaya, E.S. Belyaeva, N.G. Shostack & Yu.V. Ilyuin, 1994. The introduction of a transpositionally active copy of retotransposon gypsy into a stable strain of Drosophila melanogaster causes genetic instability. Mol. Gen. Genet. 242: 472–477. Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge University Press.

175 Kondrashov, A.S., 1988. deleterious mutations and the evolution of sexual reproduction. Nature 336: 435–440. Langley, C.H., E.A. Montgomery, R. Hudson, N. Kaplan & B. Charlesworth, 1988. On the role of unequal exchange in the containment of transposable element copy number. Genet. Res. 52: 223–235. Leigh-Brown, A.J. & J.E. Moss, 1987. Transposition of the I element and copia in a natural population of Drosophila melanogaster. Genet. Res. 49: 121–128. Mackay, T.F.C., R.F. Lyman, M.S. Jackson, M.S. Terzian & W.G. Hill, 1992. Polygenic mutation in Drosophila melanogaster: estimates from divergence among inbred strains. Evolution 46: 300– 316. Mackay, T.F.C., R.F. Lyman & M.S. Jackson, 1992. Effects of P element insertions on quantitative traits in Drosophila melanogaster. Genetics 130: 315–332. McDonald, J.F., D.J. Strand, M.R. Brown, S.M. Paskewitz, A.K. Csink & S.H. Voss, 1988. Evidence of host-mediated regulation of retroviral element expression at the posttranscriptional level, pp. 219–234 in Eukaryotic Transposable Elements as Mutagenic Agents, edited by E.M. Lambert, J.F. McDonald and I.B. Weinstein. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Montgomery, E.A. & C.H. Langley, 1983. Transposable elements in Mendelian populations. II. Distribution of three copia-like elements in a natural population of Drosophila melanogaster. Genetics 104: 473-483. Maruyama, K. & D. Hartl, 1991. Evolution of the transposable element mariner in Drosophila species. Genetics 128: 319–329. Mevel-Ninio, M., M.C. Mariol & M. Gans, 1989. Mobilization of the gypsy and copia retrotransposons in Drosophila melanogaster induces reversion of the ovoD dominant female-sterile mutations: molecular analysis of revertant alleles. EMBO J. 8: 1549–1558. Mukai, T., 1964. The genetic structure of natural populations of Drosophila melanogaster. I. Spontaneous mutation rate of polygenes controlling viability. Genetics 50: 1–19. Mukai, T., 1969. The genetic structure of natural populations of Drosophila melanogaster. VII. Synergistic interactions of spontaneous mutant polygenes affecting viability. Genetics 61: 749– 761. Mukai, T., S.I. Ghiguso, L.E. Mettler & J.F. Crow, 1972. Mutation rate and dominance of genes affecting viability in Drosophila melanogaster. Genetics 72: 335–355. Nuzhdin, S.V., 1995. The distribution of transposable elements on X chromosomes from a natural population of Drosophila simulans. Genet. Res. 66: 159–166. Nuzhdin, S.V. & T.F.C. Mackay, 1994. Direct determination of retrotransposon transposition rates in Drosophila melanogaster. Genet. Res. 63: 139–144. Nuzhdin, S.V. & T.F.C. Mackay, 1995. The genomic rate of transposable element movement in D. melanogaster. Mol. Biol. Evol. 12: 180–181. Nuzhdin, S.V., E.G. Pasyukova & T.F.C. Mackay, 1996. Positive association between copia transposition rate and copy number in Drosophila melanogaster. Proc. R. Soc. Lond. B 263: 823–831.

Ohnishi, O., 1977. Spontaneous and ethyl methanesulfonate-induced mutations controlling viability in Drosophila melanogaster. II. Homozygous effects of polygenic mutations. Genetics 87: 547– 556. Pasyukova, E.G., E.S. Belyaeva, L.E. Ilyinskaya & V.A. Gvozdev, 1988. Outcross-dependent transpositions of copia-like mobile genetic elements in chromosomes of an inbred Drosophila melanogaster stock. Mol. Gen. Genet. 212: 281–286. Pasyukova, E.G. & S.V. Nuzhdin, 1992. Mobilization of retrotransposon copia in the genome of Drosophila melanogaster. Genetika 28: 5–18. Pasyukova, E.G. & S.V. Nuzhdin, 1993. Doc and copia instability in an isogenic Drosophila melanogaster stock. Mol. Gen. Genet. 240: 302–306. Pasyukova, E.G., S.V. Nuzhdin & D.A. Filatov. Factors affecting the transposition rate of the copia retrotransposon in Drosophila melanogaster. Submitted. P´elisson, A. & J.C. Br´egliano, 1987. Evidence for rapid limitation of the I element copy number in a genome submitted to several generations of I-R hybrid dysgenesis in Drosophila melanogaster. Mol. Gen. Genet. 207: 306–313. P´elisson, A., S.U. Song, N. Prud’homme, P. Smith, A. Bucheton et al., 1994. Gypsy transposition correlates with the production of a retroviralenvelope-like protein under the tissue-specific control of Drosophila flamenco gene. EMBO J. 13: 4401–4411. Pritchard, M.A., J.M. Dura, A. P´elisson, A. Bucheton & D.J. Finnegan, 1988. A cloned I factor is fully functional in Drosophila melanogaster; a possible mechanism for transposition. Mol. Gen. Genet. 214: 533–540. Prud’homme, N., M. Gans, M. Masson, C. Terzian & A. Bucheton, 1995. Flamenco, a gene controlling the gypsy retrovirus of Drosophila melanogaster. Genetics 139: 697–711. Ronsseray, S.M. Lehmann & D. Anxolab´eh`ere, 1991. The maternally inherited regulation of P elements in Drosophila melanogaster can be elicited by two P copies at cytological site 1A on the X chromosome. Genetics 129: 501–512. Sharp, P.M. & W.-H. Li, 1989. On the rate of DNA sequence evolution in Drosophila. J. Mol. Evol. 28: 398–402. Tower, J., G.H. Karpen, N. Craig & A. C. Spradling, 1994. Preferential transposition of Drosophila P-elements to nearby chromosomal sites. Genetics 188: 347–359. Vaury, C., A. Pelisson, P. Abad & A. Bucheton, 1993. Properties of transgenic strains of Drosophila melanogaster containing I transposable elements from Drosophila teissieri. Genet. Res. 61: 81–90. Vieira, C. & C. Bi´emont, 1994. Geographical variation in insertion site number of retrotransposon 412 in Drosophila simulans. J. Mol. Evol. 42: 443–447. Vieira, C. & C. Bi´emont, 1996. Transposition rate of the 412 retrotransposable element is independent of copy number in natural populations of Drosophila simulans. Mol. Biol. Evol. (in the press).