Distribution and Conservation of Sequences

Distribution and Conservation of SequencesHomologous to the I731 Retrotransposon in Drosophila’ Catherine Montchamp-Moreau, Mphane Ronsseray, Micheline Jacques,Monique Lehmann, and Dominique AnxolaEhGre Dkpartement Dynamique du Ginome et Evolution, Institut J. Monod, CNRS-Universitk

The distribution of 1731 retrotransposon-hybridizing sequencesin the family Drosophilidae has been studied using a I731 probe from Drosophila melanogaster. Squash blot and Southern blot analyses of 42 species reveal that the I731 sequences are widespread within both the Sophophoru and Drosophila subgenera and are also present in the genera Scuptomyza and Zaprionus. Hence the 1731 retrotransposon family appears to have a long evolutionary history in the Drosophilidae genome. Differences of hybridization signal intensity suggestedthat the 1731 sequence is well conserved only in the three species most closely related to D. melanoguster (D. simulans, D. mauritiana, and D. sechellia). A survey of insertion sites in numerous different populations of the previous four speciesby in situ hybridization to polytene chromosomes has shown in all casesboth chromocentric hybridizations and a low number of sites (O-5 ) on the chromosomal arms. This number of sites is among the lowest observed

in D. melanoguster

and D. simuluns

when

1731

is compared with other retrotransposon families. In addition, we have observed species-specificpatterns of the chromocentric hybridization signal, suggestingrapid modifications of the P-heterochromatin anogaster subgroup.

components

since the radiation

of the mel-

Introduction

Two nonexclusive mechanisms of genetic transfer have been proposed to explain the observed distributions of the mobile-DNA-element families within phylogenies. Transposable elements can be transmitted vertically from a common ancestor during the process of speciation: the relationships among element sequences are those of orthology and paralogy and thus, in this case, are expected to reflect, variously, phylogenetic relationships among their hosts. Transposable elements can also undergo horizontal transfers between reproductively isolated species, either by nonsexual means, which implies an exogenous vector, or by unusual interspecific mating. In this last case, in which the hybrids are usually sterile, the transfer of mobile elements could be consecutive with rare polyspermy events. In Drosophilidae, incongruences in transposable-element distribution patterns and phylogenies have frequently been justified by invoking horizontal transfers (e.g., see Brookfield et al. 1984; Stacey et al. 1986; Mizrokhi and Mazo 1990). The distributions of copia-like retrotransposons among Drosophila species can be explained by either of these models. More than 40 families of retrotransposons have been identified, of which 11 are’well characterized to date (for reviews, see 1. Key words: 2 731, retrotransposon, Drosophilidae, P-heterochromatin. Address for correspondence and reprints: Dominique Anxolabkh&e, DCpartement Dynamique du GB nome et Evolution, Institut J. Monod, CNRS-UniversitC Paris-7, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France. Mol. Biol. Evol. 10(4):791-803. 1993. 0 1993 by The University of Chicago. 0737-4038/93/1004-0005$02.00

All rights reserved.

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Bingham and Zachar 1989; Echalier 1989). Evolutionary studies of copia, 412, and gypsy have revealed that sequences homologous to these three transoposons are widely spread in all major Drosophila radiations but display substantial differences both in species distribution and in copy number between species (Martin et al. 1983; Stacey et al. 1986). This is consistent with the hypothesis of an ancient origin of these sequences, early in the history of the Drosophilidae family, followed by vertical transmission and stochastic loss events. However, the distribution of copia and gypsy homologous sequences, as well as the conservation of gypsy, suggests that horizontal transmissions may also have occurred (Stacey et al. 1986). On the other hand, sequences homologous to the retrotransposon 297 appear to be restricted to the melanogaster group. This result suggests a more recent origin for this element, without apparent transfer to species outside the group (Martin et al. 1983 ). Among the LINElike elements, evidence for horizontal transmission events has also been provided by the distribution of the jockey element in D. funebris and sibling species (Mizrokhi and Mazo 1990). The copia-like element, 1731, is a D. melanogaster genomic repetitive sequence: its transposition has been observed in cell lines, and its expression has been shown to be modulated by the insect ecdysteroid hormone, 20-hydroxyecdysone (ecdysterone) (Peronnet et al. 1986; Fourcade-Peronnet et al. 1988; Becker et al. 199 1). Perhaps this retrotransposon-host-gene interaction could be of evolutionary importance in the establishment of genes’ sensitivity to ecdysterone. Regulation by a steroid hormone has also been shown for the provirus of the mouse mammary tumor virus and has been correlated to the presence, in the long-terminal repeats (LTR), of specific hexanucleotide sequences that are possibly target sites for the hormone-receptor complex; the regulatory regions of several steroid-modulated eukaryotic genes possessconsensus sequences with these specific hexanucleotides (Beat0 et al. 1987). The occurrence in the LTR of 1731 of four similar hexanucleotides (Fourcade-Peronnet et al. 1988) gives us the opportunity to (a) study a possible coevolution between these copia-like sequences and other genes, whose expression is also controlled by ecdysterone in Drosophila, and also (b) to test the hypothesis that whole-retrotransposon insertion (or when at least a single LTR remains after incomplete excision) can confer hormonal sensitivity to the adjacent genes. The first step of this study is to establish the evolutionary history of I731 homologous sequences in the Drosophilidae family. We present here the first data on its phylogenic distribution. Material

and Methods

The species stocks under analysis originated from our laboratory or were obtained from the Laboratoire de Biologie et Genetique Evolutives, CNRS, Gif sur Yvette, France, or from the National Drosophila Species Resource Center, Bowling Green, Ohio. Stock references are given in table 1. Squash Blot Method Entire flies were squashed’ onto Biodyn@ A filters (Pall) (Anxolabehere et al. 1985; Tchen et al. 1985). Filters were hybridized with the NdeI internal fragment of I731 from the subclone DNA pFP5c ( Peronnet et al. 1986 ) . Hybridization was done in 2 X standard saline citrate (SSC), 1 X Denhardt’s solution, 25 mM KH2P04, pH 7.5, 0.1% sodium dodecyl sulfate, 10 mM ethylenediaminetetraacetate, 10% dextran sulfate, and 150 ug sheared and denatured salmon sperm DNA/ ml. The filters were washed three times, for -20 min each time, under high-stringency conditions

Table 1 Distribution

of 1731 Homologous

Origin” PANA PANA G1163.1 G1228 GI188.1 GI115 G1128.2 GI154.1 G1242.1 G1243.1 G1227.5 G1227.6 G1227.1 G1223.13 G1125.3 G1188.6 G1242.3 G1168.5 G1195.2 G1183.1 G1165.12 PADOBl PADGUl PADSUl PADMIl BG0141.0 BG0181.0 BG0151.0 G1156.4 BG0721.0 PADTRl BG0771.0 PADNEl BG0911.0 BG0831.0 BG0871.0 BG0841.0 PADVIl G1219.2 PADIMl

Subgenus . ..

Sophophora

Sequences among Species Studied Group

melanogaster

Subgroup

melanogaster

. . . ,. .. . .. .. .. ..

ananassae takahashit elegans eugracilis montium

. .. ... . .. .. . .

. . . . . . . . .

obscura

. . . . .

obscura

aftnis

. .

willistoni

willistoni

saltans

saltans cordata sturtevanti elliptica virilis hydei immigrans

.. . . . .

... . . .. .

Drosophila

virilis repleta immigrans

Species

melanogaster simulans mauritiana sechellia orena yakuba teissieri erecta ananassae malerkotliana takahashii elegans eugracilis malagassya tsacasi bakoue bocqueti greeni vulcana davidi kikkawai obscura guanche subobscura miranda afinis bifasciata algonquin willistoni capricorni tropicalis paulistorum nebulosa saltans neocordata sturtevanti emarginata virilis hydei immigrans

Signal b

++++++ ++++++ ++++++ ++++++ +++ ++ ++ ++ ++ ++ ++ EW +++ f ++ ++ + EW + + ++ ++ ++ EW +++ ++ EW ++ +++ ++ + ++ ++ ++ + + ++ NR NR +

Genus PAZ01 PASPT

. .. .

Zapriounus Scaptomyza

ornatus pallida

f +

* Strains originate from our laboratory (PA), from Bowling Green (BG), and from Gif sur Yvette (GI). b Signal intensity observed on squash blots refers to fig. 1. EW = extremely weak signal: and NR = not repeatable signal from fly to fly (weak, if present).

794

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(2 X SSC and 0.1% SDS at 65°C). The reproducibility of the scoring of signal intensity was confirmed by several different independent observers. Southern Blots Additional strains were used: D. melanogaster from Nasr’Allah (NA) and La Marsa (MA) (Tunisia, 1983 and 1987, respectively) and Brjansk (BJ) (USSR, 1987 ); and D. simulans from Nasr’Allah (NA) (Tunisia, 1987)) Noumea (NO) (Nouvelle Caledonie, 1989)) and Ranamafana (RA) (Madagascar, 1987). For each strain, genomic DNA was extracted from a group of 50 females by a method derived from the work of Junakovic et al. ( 1984). Approximately 4 pg of genomic DNA was digested with BamHI and Sal1 or with Hind111 and was electrophoresed in a 0.7% agarose gel. Transfers and hybridizations were performed on Biodyn@ A filters, according to the manufacturer’s instructions. Complete digestion of DNA extracts used to perform the Southern blots was checked for each digest by hybridizing replicate blots with a unique sequence (an internal Hind111 fragment of the ADH gene of D. melanogaster). Filters were washed by following the same conditions as were used for the squash blots. In Situ Hybridization

of Polytene Chromosomes

Drosophila melanogaster strains used were mass-mated populations established with at least 50 individuals collected in the wild. They were maintained in the laboratory by mass culture at 18 “C in half-pint bottles ( 200 couples each generation ) . We studied 30 larvae from 10 natural populations: Katsunuma (Japan, 1985; three larvae), Golubac (Yougoslavia, 1986; four larvae), Samarkand (USSR, 1986; four larvae), Brjansk (USSR, 1987; five larvae), La Meilleraye (France, 1988; five larvae), La Marsa (Tunisia, 1987; five larvae), Tashkent (USSR, 198 1; one larva), Makokou (Gabon, 198 1; one larva), Kamenica-Valjevo (Yugoslavia, 1986; one larva), and Edinburgh ( 1988, one larva ) . Drosophila simulans polytene chromosomes were analyzed with isofemale lines from Nasr’Allah (Tunisia, 1987; 11 lines), California ( 1989; 10 lines), Ranamafana (Madagascar, 1987; 10 lines), Bretagne (France, 1989; 9 lines), and Saint Denis (La Reunion island, 1989; 10 lines). We studied 11 isofemale lines of D. mauritiana collected in 1985 from four different locations in the Mauritius islands, and we also studied 4 isofemale lines of D. sechellia collected in 1985 on Praslin island. Slides were treated and hybridized according to the method of Pardue and Gall ( 1975), as revised by Strobe1 et al. ( 1979), with the 3H-labeled plasmid N42 containing the NdeI internal fragment from a complete I731 element of D. melanogaster (Ziarczyk et al. 1989). This fragment corresponds to one LTR and the complete internal sequence (fig. 2) and does not contain any other Drosophila sequences. Slides were stained with Giemsa.

Results Presence of I731 Homologous Sequences Detected by Squash Blots We searched for 1731 homologous sequences in 40 Drosophila species, most of them belonging to the Sophophora subgenus, and also in two species of the genera Zaprionus and Scaptomyza. Squash blots with species of the melanogaster group are presented in figure 1. The results corresponding to all species tested are summarized in table 1. I731 Homologous sequences have been detected in all the species studied; however, the signal was barely detectable and possibly results from an artifact for D. elegans, D. greeni (see fig. 1), D. subobscura, and D. bifasciata (data not shown). The strongest signals were observed with D. melanogaster and its three sibling species

I731

Retrotransposon

797

in Drosophila

indicates also that the low level of hybridization signal intensity of these last species, detected by both squash and Southern blots, is not due to a low copy number. To investigate the degree of conservation of the I731 homologous sequences detected in the species studied, genomic Southern blots were performed with BamHISal1 digests, using the BamHI-Sal1 restriction fragments from the canonical 1731 element of D. melanogaster as a probe. Results are shown in figure 3. Drosophila melanogaster samples showed a major hybridization band of 3.15 kb, which corresponds to the internal BamHI- Sal I fragment of the canonical I731 element. On average, 12 other bands, most of them corresponding to fragments of larger size, probably result from degenerate elements that have lost the BamHI and/or the Sal1 restriction sites. The 3.15kb major fragment appeared to be conserved in D. simulans, but it was not observed in the other species. In addition, - 10 bands of larger size were obtained in D. simulans. In both D. simulans and D. melanogaster, bands from 1731 variants exhibit a common size between populations, a result that is consistent with those obtained with Hind111 digests. Such a result is also consistent with those of Vaury et al. ( 1989 ), who have found interstrain similarities in the hybridization pattern for defective I731 elements within these two species. Chromosomal

Distribution

of I731 Homologous

Sequences

An extensive study of the chromosomal location of I731 sites by in situ hybridization was performed in the four sibling species belonging to the melanogaster subgroup, which had exhibited strong hybridization signals with squash blots and Southern blots. We also hybridized polytene chromosomes from other species of the kb

1

2

3

4

5

6

7

8

9

lo

11

12

10

Bamtil

1731

Sall A~LTR

3)

probe I lkb

SalI.

FIG. 3.--Southern blots of DNA Species and strains are numbered

from species of the melunoguster subgroup, as in fig. 2. Exposure time was 3 h.

digested

by BumHI

and

798 Montchamp-Moreau

et al.

melanogaster subgroup, as well as from two more distant species, D. willistoni and D. guanche, belonging to the subgenus Sophophora. Two interesting features are ( 1) the number of sites observed on chromosomal arms and ( 2) the signal distribution over the chromocenter. In D. melanogaster, O-3 sites on the chromosomal arms were found per genome (mean 1.8 /genome), whereas 5-50 sites have been reported for various transposableelement families in this species, including copia-like retrotransposons (Ilyin et al. 1977; Finnegan et al. 1978; Strobe1 et al. 1979; Ananiev et al. 1984; Bucheton et al. 1984; Biemont and Terzian 1986). Moreover, data from the study of the chromosomal distribution of sites of 10 transposable-element families on 14 X chromosomes from the wild (Charlesworth and Lapid 1989 ) reinforce the result that the I731 family has one of the lowest numbers of sites per genome. Indeed, for the 10 families studied, the mean number of sites on the X chromosome was found to be greater than those observed, in the present study (0.1 ), for 1731. In addition, a deduced approximate number of sites per genome for the 10 families can be calculated by multiplying the number of sites on the X chromosome by 5 (this being the number of main chromosomal arms). Again, the number of sites for each of the 10 families was greater than the number reported here for 1731. Among the 30 larvae studied, a hybridization site in 42AB was observed for 12 larvae originating from 9 of the 10 populations studied. Similarly, a label in 39DE was observed for 13 larvae from 6 populations, and a label in 82BC was found in 8 larvae from 4 populations. Altogether these three sites represent 65% of the labels detected on the chromosomal arms. The 39DE and 42AB sites (which represent 50% of the labels) were reported to correspond to intercalary heterochromatin (Zhimulev et al. 1982). Other sites vary in location, within and between strains. In D. simulans, O-5 sites were found with a mean number ( 1.7/genome) close to that of D. melanogaster. The chromosomal locations of the hybridization signal are variable between and within populations: among 42 different locations observed in the 50 independent larvae studied, only 4 were found in more than 2 larvae. One of them is the 39DE site, which corresponds, as in D. melanogaster, to a chromosomal breakpoint (Inoue 1988) and thus probably to intercalary heterochromatin in this species. This site was found in 17 larvae from four of the five populations studied. In D. mauritiana, only two different locations were observed: 102AB on the base of chromosome 4, and 93D on the 3R arm. Both sites were observed in the 11 isofemale lines studied but were of variable intensity between larvae. In D. sechellia, no hybridization was observed on chromosomal arms within the four independent isofemale lines tested, except for a faint signal repeatedly found in 42B on the 2R arm. The hybridization signal is not homogeneously distributed over the chromocenter in these four species of the melanogaster subgroup, and its localization differs between species. For a given species, the reproducibility of the observations was repeatedly checked with several slides from independent lines. Results were also checked by different observers. To avoid a bias due to the experimental conditions of in situ hybridization, we used several different batches for a given species. Moreover, to allow interspecies comparisons, slides from the different species compared were hybridized in the same batch with the same probe preparation. In D. melanogaster (fig. 4A), the pericentromeric heterochromatin in both chromosome 2 and chromosome 3 hybridizes markedly, with the second chromosome exhibiting the strongest signal. In addition, the X chromosome and chromosome 4 occasionally show slight labels on their proximal part. In D. simulans, signals were

FIG. 4.-In situ hybridization of I732 probe (N&I internal restriction fragment; see fig. 2) to polytene chromosomes of Drosophila (exposure time 15 d). A, D. melanogaster. B, D. simulans. C, D. mauritiana. D, D. sechellia.

800 Montchamp-Moreau

et al.

detected in the pericentromeric heterochromatin from chromosome 3 (fig. 4B), whereas faint hybridization of pericentric heterochromatin was occasionally observed on chromosomes 4 and X but, unexpectedly, never on the second chromosome. The hybridization pattern of the chromocenter in D. mauritiana (fig. 4C) appears closer to that of D. melanogaster than to that of D. simulans, with a strong signal on the pericentromeric regions of the second chromosome, whereas the pericentromeric heterodhromatin regions from chromosomes X and 3 are weakly labeled, if at all. In D. sechellia (fig. 4D), both chromosome 2 and chromosome 3 are strongly hybridized, whereas chromosomes X and 4 exhibit a faint signal. In other species of the melanogaster subgroup (i.e., D. teissieri and D. erecta), as well as in D. willistoni and D. guanche (data not shown), the exposure time necessary to detect a signal was - 10 wk (i.e., five times as long as that used for D. melanogaster sibling species). In D. teissieri and D. erecta, we detected only a marked pericentromeric hybridization. In the more distant species, D. willistoni, in addition to a faint signal on the whole chromocenter, we identified at least one hybridization site on chromosomal arms. No signal was detected in the same experimental conditions with D. guanche. Discussion Phylogenic Distribution Sequences hybridizable to 1731 appear widely spread through the Sophophora subgenus and are also present in the subgenus Drosophila, as well as in the Scaptomyza and Zaprionus genera. These different lineages have diverged early in the family’s evolution, -60 Mya, according to immunological data ( Beverley and Wilson 1984); this is consistent with an ancient origin of the I731 family, Hybridization signals are generally of decreasing intensity when species are increasingly distant from D. melanogaster, a result that supports the vertical transmission of these elements. Southern blots of D. takahashii, D. afinis, D. Kikkawai, and D. eugracilis suggest that the dispersed-element status of I731 has been conserved in the different lineages. They also suggest that the signal weakness in these species results from sequence divergence rather than from a decrease in the copy number, because the number of Hind111 bands is not that much lower than that observed for D. melanogaster or D. simulans. Only D. melanogaster and its three closest relatives, whose estimated divergence time is 2.5 Mya (Lachaise et al. 1988), exhibited strong hybridization signals in both squash and Southern blots. The signals observed with the other members of the melanogaster subgroup- D. orena, D. teissieri, D. yakuba, and D. erecta-which separated from the D. melanogaster ancestor 6-l 5 Mya (Lachaise et al. 1988; McDonald and Kreitman 199 1)) are markedly fainter and of intensity comparable to those obtained with much more distantly related species. The main, 3.15kb band, produced by the BamHI/SalI digests, which corresponds to complete and thus potentially functional 1731 elements, appears conserved only in D. simulans. Moreover, in situ observations suggest that 1731 is mobile in D. simulans and D. melanogaster, ‘whereassuch a mobility is not suggested for D. mauritiana and D. sechellia, because the chromosomal locations are constant among unrelated lines. In both of the latter species, I731 hybridization is restricted to the chromocenter, except for some very proximal euchromatic bands and for rare faintly labeled site, as expected for defective and degenerated elements, or for copies inserted into nonpolytenized intercalary heterochromatin. This agrees with the hypothesis that old defective transposable elements are mainly immobilized in P-heterochromatin (Vaury et al. 1989). Thus, the differences, in Southern blot band pattern, between D. maur-

I731 Retrotransposon in Drosophila

itiana lines (fig. 2B) could be attributed to restriction-site polymorphism ation between degenerated pericentromeric copies.

Chromosomal

Distribution

80 I

and/ or vari-

of I731 Homologous Sequences in the

rnelanogaster Subgroup

Usually, when a transposable element- e.g., the LINE-like elements I or the retrotransposon copia -hybridizes with the chromocenter, the signal is evenly distributed over it (Strobe1 et al. 1979; Bucheton et al. 1984). Here there are labeling differences between chromocentric regions. Moreover, the label patterns appear similar within species but different between them. Thus, the P-heterochromatin distribution of 1731 homologous sequences is probably species specific. Similar intraspecies similarities, as well as interspecies differences, have been reported for the localization of highly repeated sequences of various satellite DNA sequences in the a-heterochromatin of Drosophila (reviewed by Lohe and Roberts 1988), but the origin and the possible role of these differences are still completely unknown. This common characteristic of the chromosomal distribution of both types of sequences suggests that (a) the middle repeated sequences that characterize the P-heterochromatin and (b) the highly repeated sequences of the a-heterochromatin might follow some common evolutionary processes that lead to interspecies divergences but to an interspecies conservation of their localization and of their level of repetition. There is a similarly low per-genome number of I731 hybridization sites on the chromosomal arms of D. melanogaster and D. simulans ( 1.8 and 1.7, respectively). This contrasts with those numbers reported for various D. melanogaster transposableelement families including copia-like retrotransposons (Ilyin et al. 1977; Finnegan et al. 1978; Strobe1 et al. 1979; Ananiev et al. 1984; Bucheton et al. 1984; Biemont and Terzian 1986; Charlesworth and Lapid 1989). When we take into account that a notable proportion of sites detected on the chromosomal arms are located at intercalary heterochromatin sites and that transposable elements inserted in intercalary heterochromatin are thought to be defective and therefore immobile ( Vaury et al. 1989), then the copy number of putatively mobile I731 could be still lower than the number of sites observed on the chromosomal arms. Two of the three high-frequency sites observed for I731 in D. melanogaster are located in intercalary heterochromatin. This might be a general feature of transposable elements that tend to be located in these chromosomal regions (Ananiev et al. 1978). For example, the 39DE site, which is the main high-frequency site in both D. melanogaster and D. simulans, was also found to contain defective I elements in D. melanogaster ( Vaury et al. 1989 ) . Furthermore, the 42AB region, which is the other highfrequency site in D. melanogaster (and which is the only site in D. sechellia), was, in D. melanogaster, repeatedly found to be a high-frequency site for transposons of many families (Biemont 1992). On the other hand, the high frequency of occupation of some sites by I731 may be due to the immobilization of I731 sequences at these sites. Alternatively, the high frequency may be due to some specific property of I731 in relation to its transcriptional repression by ecdysterone (Becker et al. 199 1) . The EcR gene, which encodes an ecdysterone receptor, has been mapped in 42B (Koelle et al. 199 1) and has been described as being itself sensitive to ecdysterone. To explain the high frequency of insertion of the I731 element in 42AB, it could be hypothesized that there is a selective advantage based on some kind of interaction between the ecdysterone sensitivity of the EcR gene and those of the 1731 sequences inserted in the neighboring region.

1731 Retrotransposonin Drosophila INOUE, Y. 1988. Chromosomal mutation in Drosophila

melanogaster

and Drosophila

803 simulans.

Mutat. Res. 197:85-90. JUNAKOVIC, N., R. CANEVA, and P. BALLARIO. 1984. Genomic distribution of copia-like elements in laboratory stocks of Drosophila melanogaster. Chromosoma 90:378-382. KOELLE, M. R., W. S. TALBOT, W. A. SEGRAVES, M. T. BENDER, P. CHERBAS, and D. S. H~GNESS. 199 1. The Drosophila EcR gene encodes an ecdysone receptor, a new member

of a steroid receptor superfamily. Cell 67:59-77. LACHAISE, D., M. L. CARIOU, J. R. DAVID, F. LEMEUNIER, L. TSACAS, and M. ASHBURNER. 1988. Historical biogeography of the Drosophila melanogaster speciessubgroup. Pp. 159225 in M. K. HECHT, B. WALLACE, and G. T. PRANCE, eds. Evolutionary biology. Vol. 22.

Plenum Press,New York and London. LOHE, A., and P. ROBERTS. 1988. Evolution of satellite DNA sequencein Drosophila. Pp. 148186 in R. S. VERNA, ed. Heterochromatin: molecular and structure aspects. Cambridge University Press,Cambridge. MCDONALD, J. H., and M. KREITMAN. 1991. Adaptative protein evolution at the Adh locus in Drosophila. Nature 351:652-654. MARTIN, G., D. WIERNASZ, and P. SCHEDL. 1983. Evolution of Drosophila repetitive dispersed DNA. J. Mol. Evol. 19:203-213. MIZROKHI, L. J., and A. M. MAZO. 1990. Evidence for horizontal transmission of the mobile element jockey between distant Drosophila species.Proc. Natl. Acad. Sci. USA 87:92 169220. PARDUE, M. L., and J. G. GALL. 1975. Nucleic acid hybridization to the DNA of cytological preparations. Pp. 1- 16 in D. M. PRESCOT, ed. Methods in cell biology. Academic Press,New York. PERONNET, F., J. L. BECKER, J. BECKER, L. D’AURIOL, F. GALIBERT, and M. BEST-BELPOMME. 1986. 1731, a new retrotransposon with hormone modulated expression. Nucleic Acids Res. 14:90 17-9033. STACEY, S. N., R. A. LANSMAN, H. W. BROCK, and T. A. GRIGLIATI. 1986. Distribution and conservation of mobile elements in the genus Drosophila. Mol. Biol. Evol. 3:522-534. STROBEL, E., P. DUNSMUIR, and G. M. RUBIN. 1979. Polymorphism in the chromosomal locations of elements of the 412, copia and 297 dispersedrepeated gene families in Drosophila. Cell 17:429-439. TCHEN, P., D. ANXOLAB~H~RE, D. NOUAUD, and G. PERIQUET. 1985. Hybridization on squashed flies: a method to detect gene sequencesin individual Drosophila. Anal. Biochem. 150:4 15-420. VAURY, C., A. BUCHETON, and A. PELISSON. 1989. The /3 heterochromatic sequencesflanking

the I elements are themselves defective transposable elements. Chromosoma 98:2 15-224. I. F., V. F. SEMESHIN, V. A. KULICHKOV, and E. S. BELYAEVA. 1982. Intercalary heterochromatin in Drosophila. I. Localization and general characteristics. Chromosoma

ZHIMULEV,

87: 197-228. ZIARCZYK, P., F. FOURCADE-PERONNET, S. SIMONARD, C. MAISONHAUTE, and M. BEST-BELPOMME. 1989. Functional analysis of the long terminal repeats of Drosophila I731 retro-

transposon: promoter function and steroid regulation. Nucleic Acids Res. 17:863 l-8644. MARTIN KREITMAN, reviewing editor Received July 17, 1992; revision received February 22, 1993 Accepted February 22, 1993