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Nucleic Acids Research, Vol. 19, No. 4 913 Cloning and analysis of the mobile element gypsy from D. virilis Lev J.Mizrokhi* and Alexander M.Mazo1 Laboratory of Biochemistry, National Cancer Institute and laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA Received September 26, 1990; Revised and Accepted December 20, 1990 GenBank accession no. M38438 ABSTRACT The homologue of the Drosophila melanogaster mobile element gypsy was cloned from the distantly related species D. virilis. It has three ORFs highly homologous to those of the element from D. melanogaster. gypsy from D. virilis appears to be actively transcribed and is capable of transposition. Comparison of the untranslated regions of both elements revealed conserved sequences including those which had previously been demonstrated to be important in transcription regulation. Distribution of gypsy among the different strains of D. virilis and different species within the D. virilis group was analyzed. Possible involvement of horizontal transmission in the process of spreading and evolution of gypsy is discussed. INTRODUCTION The mobile element gypsy was originally cloned from D. melanogaster (1). It belongs to the LTR-containing class of the retroelements (2) and is responsible for a number of mutations described in D. melanogaster (3). gypsy appears to be the best studied LTR-containing mobile element in Drosophila. Its structure (4) and regulation of transcription (5) have been investigated in great detail as well as the phenomenon of suppression of its mutations (5, 6) and transposition memory (7). The distribution of gypsy within the Drosophila genus has also been investigated previously (8). It was found by Southern hybridizations with the entire gypsy DNA as a probe that genomes of some species—D. virilis, D. busckii, D. pinicola and few others—have some homologies with the D. melanogaster element. However, the extent of the homologies and the relatedness of these sequences to gypsy were unclear. In this paper we report cloning and sequencing of gypsy homologues from D. virilis. The analysis of the nucleotide and putative amino acid sequences has shown that D. virilis indeed possesses highly homologous mobile element which is capable of transposition and is actively transcribed. This element is widely distributed within different strains of D. virilis and other species • To whom correspondence should be addressed of the same group. We speculate that the existence of the highly homologous gypsy in distant species might be a result of the horizontal transmissions of this element. MATERIALS AND METHODS Drosophila strains Drosophila strains were obtained from Bowling Green (Ohio) stock center except for strain '9', which was isolated by Dr. M.Evgen'ev in a vineyard in Batumi, USSR. The cell culture of D. virilis was a gift from Dr. I.Schneider. DNA preparation, electrophoresis and hybridization DNA was prepared as described previously (7), digested with HindHI, electrophoresed in 1 % agarose and transferred to a nylon filter. Hybridization was performed in 6x SSPE, 10 X Denhardt, 100 /t/ml sheared salmon sperm DNA, 0.5% SDS for 24 h at 55°C. The filter was washed three times for 15 min in 2 x SSC, 0.5% SDS at 37°C. Library construction and screening Genomic DNA from adult D. virilis (strain '9') flies was partially digested with Sau3A, ligated and packaged into phage EMBL4. Hybridization conditions for screening were the same as described above. DNA sequencing Nucleotide sequence was determined for both strands by using the dideoxynucleotide chain-termination method (9) for double stranded DNA and Sequenase™ (United States Biochemical). For sequence comparisons, the programs from the University of Wisconsin, Genetics Computer Group (UWGCG) were used (10). RNA preparation and hybridization Poly(A) RNA from adult flies (strain '9') were purified as described (11). Northern hybridization was conducted under the same conditions as for the Southerns (see above). Washing conditions were O.lx SSC, 0.5% SDS at 50°C. 914 Nucleic Acids Research, Vol. 19, No. 4 RESULTS Structure of gypsy from D. virilis and its similarity to gypsy from D. melanogaster The D. virilis genomic library was screened with the probes derived from regions corresponding to the first and the third ORFs and LTR of gypsy from D. melanogaster. Two clones containing presumably full-length copies were picked for subcloning and sequencing. The nucleotide sequence of one of the clones was determined completely. Only the untranslated regions of the other clone were sequenced. No differences were found between the two copies in the sequenced regions. The nucleotide and putative amino acid sequences of gypsy from D. virilis are presented in Fig. 1A. This element consists of 7156 bp and contains two 411 bp long LTRs. In the central part of the element we have found three ORFs whose sizes, sequences and locations correspond to those of the D. melanogaster element (Fig. IB). The very high homology between amino acid sequences of the corresponding ORFs from both species (see Fig. IB) did not permit to define any conservative motifs in addition to those found earlier (4). In contrast, comparison of the untranslated regions revealed distinct conservative sequences. Comparison of the sequences of the gypsy LTRs (Fig. 2A) disclosed two long conservative domains in the 5' part of the LTR and also a short conserved sequence 18 nucleotides upstream of the site of the transcription initiation. Sites of transcription initiation and termination were determined previously for the D. melanogaster element (12). It was also noted that the sites of initiation of the several LTR-containing mobile elements are located within or immediately after the sequence TCAGTPy {gypsy RNA begins at A and G in this sequence). We did not find the same sequence in the appropriate region of D. virilis gypsy. At the same time sequences surrounding the polyadenylation signal and the site of termination are well conserved in D. virilis gypsy. There are no other conserved sequences in the LTR downstream of the site of initiation; moreover, this region in the D. virilis element is significantly ( — 70 nucleotides) shorter (not shown). This results in the difference in the lengths of the LTRs (see Fig. IB). Two sequences important for the cycle of replication of retroelements, the tRNA binding site adjacent to the 5' LTR (Fig. 2B) and the oligopurine stretch adjacent to the 3' LTR (Fig. 2 Q are conserved in D. virilis gypsy. Conservation of these sequences indicates that the new element is capable of replication and that the same tRNA, Lvs is involved in the priming of the synthesis of the first strand. MELANOGASTER UOM0LOG? m — VIRILIS • * •Llfcl 1 451** IOIOH 451** •a 1009** 470a* I I I Bi .•:•:•«.•:•:•.•:• ORT2^ 1 479** BB OR73 A. AGTTAAOACTAACCATAAAIATATTGC. . CCTACCAATTAJLCT. kCOCTCCACGCOAJU I I I I I I I _, 47 gTATCHCHTGCTOi 49 97 UTGCCI I I I I 99 CTaAaciTccT. 147 GCTGCMCAUTGCTGAO' I I I I I I TJCCGAC OCATT..CCTCGACgCTA 11 I I I TTGTGGCGGCGCCA' I I I 123 222 I 1111111 I CCTJUTTTTTTAa 221 I 223 TJkTTOTCTTCTACTCAOTTCJUUU I II I I II Mil I I CTTO 272 II II II IAM 222 • TATACTO 26S 273 CTCCOCCTC*TTOCCOTTAAJU^TC«TOTTCITATTIAC»»TC»»» I I II II I I I I I I I I I I II I I III II I I I I 269 CTCCOa. .CAnOCCaATAAAACATATCTTTaSTAATAmCTOQU 319 313 B. 469 I I I I I I I I I I I I I I I I I I I 39S c. 6971 6727 Figure 2. Comparison of the nucleotide sequences of the LTRs (bold letters) and adjacent regions of gypsy from D. melanogaster (top line) and D. virilis (bottom line). A. Nucleotide sequences of the 5' parts of the LTRs. Tworegionsof extensive homology are shadowed; sites of initiation and termination of transcription of D. melanogaster gypsy are indicated by horizontal arrow and vertical arrowheads, respectively (12); polyadenylation signal is underlined. B. Nucleotide sequences of gypsy adjacent to the 5' LTRs; tRNA(Lyl binding site is underlined. C. Nucleotide sequences of gypsy adjacent to the 3' LTR; polypurine stretch is underlined. 7kb 482bp 41 lbp / H Figure 1. General comparison of the elements from D. \irilis and D. melanogaster. The amino acid sequence of the putative proteins encoded by the three ORFs is shown below the nuclcotide sequence, the percentages of homology are indicated for identical amino acids. The region homologous to the region containing the enhancer in gypsy from D. melanogaster is underlined; the LTRs are double underlined. For the homology between LTRs see text and Figure 2. Abbreviations: Pr, protease; RT, reverse transcriptase; End, endonuclease. Figure 3. The gypsy element is transcribed in D. virilis. Northern Hot containing 5 mg of poly(A)-containing RNA from D. melanogaster and D. virilis adult flics was hybridized with a randomly labeled internal fragment of gypsy from D. virilis (EcoRI-Hindm fragment, nucleotides 4197-5975 on the Fig. 1A). Nucleic Acids Research, Vol. 19, No. 4 915 Analysis of the region located between 5' LTR and ORF1 revealed conservation of the sequence that was shown earlier to act as an enhancer of D. melanogaster gypsy (5) and to bind the product of the suppressor gene su(Hw) (5, 6). In gypsy from both species, this domain is comprised of the motif 5'PyPuT/cTGCATAc/TPyPy which is repeated 12 times (8 in some copies of D. melanogaster gypsy, see ref. 5). It seems very likely that this region acts also as an enhancer in D. virilis and binds a product of the same gene, su(Hw). A poly(A) block is also present in both species upstream of the enhancer. The conservation suggests that it is functional; perhaps it imparts some flexibility to the DNA necessary for the functioning of the enhancer. The sequence between the poly(A) block and the enhancer, which provides negative regulation of transcription in gypsy from D. melanogaster and probably interacts with the product of the su(f) gene (5), is substituted in D. virilis gypsy DC HI LU H 5kb -4kb -3kb 2kb 1.6kb AM TX NO VIRILIS PHYLAD LU VI LT MO T MONTANA PHYLAD VIRILIS GROUP Figure 4. Hybridization of the D. virilis gypsy DNA to genomic DNAs from different strains and cultured cells of D. virilis and from other species of D. virilis group (top) and the genealogical tree of the D. virilis species group (bottom). The 74Obp Hindlll-Aval fragment (nucleotides 5975-6712 on the Fig. 1A) was used as a probe. Because this fragment is not confined between internal Hindlll sites, it gives different bands for different copies of the element when hybridized to Hindm digests of genomic DNA. The following strains from Bowling Green collection were used (stock number and origin are in parentheses): VIR1-VIR4, D. virilis C9\ Batumi, USSR; 1051.0, California; 1051.9, Japan; 1051.51, Chile); VIR C, D. virilis cell culture (gift from Dr. I.Schneider); LUM, D. lummei (1011.1, Moscow, USSR); LIT, D. littoralis (1001.0, Switzerland), MEL, D. melanogaster (Oregon R). The tree was drown in accordance with (13); abbreviations are: AM, D. americana; TX, D. texana; NO, D. novamexicana; LU, D. lummei; VI, D. virilis; LT, D. littoralis; MO, D. momana. by the motif GTAAA repeated three times. Whether this domain binds some protein(s) and regulates gypsy transcription in D. virilis is unknown. Transcription of gypsy in D. virilis Northern blots with poly(A) RNA from D. virilis and D. melanogaster were probed with DNA of the D. virilis element (Fig. 3). The results of this experiment show that gypsy is actively transcribed in at least adult D. virilis flies. This implies that gypsy from D. virilis possesses all sequences essential for its transcription. Distribution of gypsy in the D. virilis strains and in other species of the D. virilis species group We analyzed the distribution of gypsy within different strains of D. virilis from various geographical regions. Southern hybridization of genomic DNAs from these strains with a DNA probe from D. virilis gypsy showed that this element is present in approximately the same numbers of copies in all the strains and the cultured cells tested. (Fig. 4). The experiment was designed such that elements from different chromosomal locations should give different bands on the autoradiograph (see legend for Fig. 4). Thus, one can conclude from the results presented in Fig. 4 that there are variations in the location of gypsy in different strains. The same observation has also been made from in situ hybridization experiments (M. Evgen'ev, personal communication). The presence of gypsy in other species of the D. virilis species group was also analyzed. For this experiment we chose D. lummei, which is, so far as is known, the species closest to D. virilis, and D. littoralis, also from D. virilis species group, but from a different philad (see bottom of Fig. 4 and ref. 13). We found that elements highly homologous to D. virilis gypsy are present in D. lummei but with an apparently lower copy number. D. littoralis probably also possesses a few copies of gypsy. DISCUSSION We have cloned and sequenced homologues of D. melanogaster (subgenus Sophophora) gypsy from D. virilis, the species which belongs to the other subgenus, namely Drosophila. Comparison of the nucleotide sequences of the elements from both species shows that they are highly homologous and that certain domains (both upstream and downstream of the initiation site) some of which were proven earlier to be involved in the transcriptional regulation of gypsy in D. melanogaster are present in the new element.The gypsy family of mobile elements is broadly distributed in D. virilis and its relatives where it appears to be active in terms of transcription and transpositional capability (Fig. 3,4). This is so far the first example of the active homologue of the D. melanogaster mobile element in the different subgenus of the genus Drosophila. The finding of the intermediates of the reverse transcription of gypsy and other LTR-containing mobile elements has shown that the step of the reverse transcription is included in its cycle of replication (12). The reverse transcription by the enzymes from different sources is highly inaccurate (see ref. 14 and refs. within). Thus, it is assumed that an active mobile element evolves much more rapidly than the 'usual' gene. This point of view is supported by sequence comparisons of the different groups of mobile elements (15). Although the elements of the 'gypsy group' 916 Nucleic Acids Research, Vol. 19, No. 4 are among the slowly diverging elements, it was found that the rate of divergency of their most conserved enzyme reverse transcriptase is significantly higher than that of the 'usual' genes. The percent of the identity of the amino acid sequences of different genes cloned from D. melanogaster and D. virilis vary from 60% to more than 80% (16-23). Our finding of the active gypsy element in D. virilis with the degree of amino acid identity comparable to that of the 'usual' genes suggests that this element may have appeared in this species by horizontal transmission and has not yet changed significantly according to the predicted rate of divergence for an active element. Recently we obtained evidence for the horizontal transmission of another Drosophila retroposon jockey (24) between distant Drosophila species (25). This is also shown to be the case for the P-element (26) which is apparently not a retrotransposon (for rev. see ref. 27). In both cases direct evidence was obtained due to the absence of these mobile elements in related species along with its presence in more distant species. Our search for the homologues of gypsy in the other species of D. virilis group revealed its presence in these species (Fig.4); this does not allow to prove directly the fact of the transmission of gypsy into D. virilis. At the same time this does not exclude the possibility of the horizontal transmission but rather shows that it might have occurred before the irradiation of the D. virilis species group. Previous studies (8) indicate that homologous to gypsy sequences (as determined by Southern hybridizations) are present in higher number of species than P-element (26) and jockey (25) within the genus Drosophila. Authors argue that the observed patchy distribution of gypsy might be the result of the multiple transmissions of this element between different species. If so, it should not be surprising that LTR-containing mobile elements which are remarkably similar in structure to retroviruses would have the ability to transmit between different species more frequently than other mobile elements for which this ability has already been proven. ACKNOWLEDGMENTS Authors thank Maxine Singer for advice and help throughout and Julie McMillan for critical reading of the manuscript. L.J.M. thanks V.Corces for support while working in his lab. REFERENCES 1. Bender.W., Akam,M., Karch.F., Beachy.P.A., Peifer.M., Spierer.P., Lewis.E.B. and Hogness,D.S.(1983) Science 221, 23-29. 2. 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