Developmental expression analysis of the 1731 retrotransposon reveals an enhancement of Gag–Pol frameshifting in males of Drosophila melanogaster

Gene 196 ( 1997) 83–93

Developmental expression analysis of the 1731 retrotransposon reveals an enhancement of Gag–Pol frameshifting in males of Drosophila melanogaster Abdelali Haoudi a,b, Mohammed Rachidi c, Myeong-Hee Kim a,1, Serge Champion a,2, Martin Best-Belpomme a, Claude Maisonhaute a,* a Laboratoire de Genetique Cellulaire et Moleculaire, UA 1135 CNRS, Universite´ Pierre and Marie Curie, Paris, France b Department of Health and Human Services, P.O. Box 12233, Research Triangle Park, NC 27709, USA c Institut de Biologie Mole´culaire, Institut Pasteur, Paris, France Received 13 December 1996; accepted 21 March 1997; Received by A. Bernardi

Abstract Extensive analyses of Drosophila melanogaster retrotransposon transcriptions in cultured cells or during development have been reported, but little is known about their translation during the development of the fly. Analysis of the translational products of the 1731 Drosophila melanogaster retrotransposon in Kc Drosophila cultured cells has been reported, showing the existence of primary products (Gag and Pol ) and of processed polypeptides of various sizes. Study of 1731 retrotransposon expression at both levels of transcription and translation during the development of Drosophila melanogaster, is presented. 1731 transcripts were detected by in situ hybridization and 1731 proteins were detected by immunostaining and immunoblotting in embryos and in adult gonads. 1731 transcripts and proteins were detected in the mesoderm and central nervous system during embryonic development, in nurse cells and follicle cells in adult ovaries and in primary spermatocytes in adult testes. Moreover, Western blot analysis of the 1731 proteins with anti-Gag or anti-Pol antibodies in gonads revealed that the 1731 mRNA could be translated di
erentially according to the expressing tissue: essentially, ovarian translation and/or processing of 1731 products is di
erent from that operating in testes, where the Gag–Pol fusion polyprotein is the most prominent product. Our results indicate that expression of the 1731 mobile element is regulated not only at the transcriptional level but also at the translational level, and that this regulation is di
erent in the two sexes. © 1997 Elsevier Science B.V. Keywords: Transposable element; Drosophila; Embryo; Ovary; Testis; Frameshift

1. Introduction It is well known that the genome of eukaryotes includes a variety of elements that possess the ability to move to new locations within the genome in which they * Corresponding author. Populations, Ge´ne´tique et Evolution, UPR 9034, Av. de la Terrasse, Baˆt. 13, 91198 Gif-sur-Yvette, France. Tel. +33 1 69823732; Fax: +33 1 69070421; e-mail: [email protected] 1 Present address: LRF Institute of Cancer Research, Chester Beatty Laboratory, 237 Fulham Road, London, SW3 6JB, UK. 2 Present address: Institut de Chimie Biologique, Universite´ AixMarseille I, Marseille, France. Abbreviations: BSA, bovine serum albumin; CNS, central nervous system; FITC, fluorescein isothiocyanate; LTR, long terminal repeat; ORF, open reading frame; PBS, phosphate bu
er saline; RT, reverse transcriptase; SDS, sodium dodecyl sulfate. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 78 - 11 19 ( 9 7 ) 00 20 3 -5

reside (Biessmann et al., 1992; Devine and Boeke, 1996; Zou et al., 1996). In Drosophila, mobile genetic elements constitute about 10% of the genome. They can be categorized according to their structure and the mechanism of transposition they use ( Finnegan, 1990; Spradling and Rubin, 1981 ). The most common class of eukaryotic transposable elements is the retroelements, able to code for a reverse transcriptase, the key enzyme involved in their transposition. The 1731 Drosophila retroelement belongs to the LTR ( long terminal repeat)containing retrotransposon Ty1-copia group, which is part of the superfamily of retrotransposons (Finnegan, 1990; Flavell et al., 1994). The internal sequence of 1731 consists of two long open reading frames: ORF1 and ORF2, the latter slightly overlapping the former. The ORF1 and ORF2 share structural features with those of integrated retrovi-

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ral proviruses (Fourcade-Peronnet et al., 1988). Actually, ORF1 is able to code for the structural 1731 Gag proteins (Haoudi et al., 1995 ). ORF2 codes for an active reverse transcriptase (Champion et al., 1992) and is assumed to code for a protease, a RNaseH and an integrase. These enzymes are expected to be post-translationally deriving from the 1731 Gag–Pol fusion polyprotein produced by a ribosomal +1 frameshifting. Several retrotransposons are transcribed either throughout or during particular stages of the Drosophila life cycle ( Flavell et al., 1980; Parkhurst and Corces, 1987; Schwartz et al., 1982). Although tissue-specific transcription of Drosophila retrotransposons has appeared likely for some time (Meyerowitz and Hogness, 1982 ), few such cases are documented ( Hochstenbach et al., 1996; Huijser et al., 1988; Lankenau et al., 1994; Mozer and Benzer, 1994; Bro¨nner et al., 1995 ). As an example, the 412 retrotransposon shows a highly regulated pattern of transcription during embryonic development of Drosophila melanogaster. Initially expressed in a set of parasegmentally repeated stripes at the extended germ band stage and during germ band retraction, its expression declines except in some parasegments where high levels of 412 expression remain in mesodermal cells. Also, this element is regulated by genes involved in mesoderm specification (Brookman et al., 1992). More recently, it was reported that 17.6 retrotransposon expression occurs specifically in the lamina anlage of the developing adult visual system. This is the first demonstration of tissue-specific expression of a retrotransposon in the Drosophila nervous system, and it was suggested that regulatory sequences in the 17.6 element may be involved in the innervation-dependent expression of adjacent cellular genes (Mozer and Benzer, 1994). In testes of Drosophila hydei, transcripts of the micropia retrotransposon are found in primary spermatocytes ( Hochstenbach et al., 1996; Huijser et al., 1988; Lankenau et al., 1994). In another report, 15 families of retrotransposons (17.6, 297, 412, 1731, 3S18, blood, copia, gypsy, HMS Beagle, Kermit/flea, mdg1, mdg3, opus, roo/B104 and Springer) were shown to generate patterns of spatially and temporally regulated expression during embryogenesis. The patterns of transcript accumulation of each of these elements are almost perfectly conserved among the di
erent wild-type Drosophila melanogaster strains examined, suggesting that they carry cis-acting elements that control their spatial and temporal expression (Ding and Lipshitz, 1994). The aim of the present work was to analyse the expression patterns of 1731 at the level of transcription and translation during embryogenesis and in adult gonads. Spatial and temporal distribution of both transcripts and proteins of the 1731 retrotransposon were analysed. 1731 transcripts were detected by in situ hybridization in whole mounts of embryos, ovaries and

testes. Immunostaining or Western blot analysis of proteins of this mobile element showed that these proteins were also present in early embryos and in adult gonads. Analysis of proteins in adult gonads revealed di
erent mechanisms of 1731 protein maturation, depending on whether it occurred in ovaries or in testes; thus, in proteins extracted from testes, we detected mainly 1731 Gag–Pol proteins, while in ovaries, we detected only Gag proteins. This indicated to us that 1731 expression is controlled not only at the transcription level, but that a tissue-specific and possibly translational and/or posttranslational control could also be involved in the maturation of 1731 polypeptides. These di
erences in the expression of 1731 could explain how male and female flies control 1731 transposable element germ line expression.

2. Materials and methods 2.1. Fly strains The wild-type Drosophila melanogaster Oregon R or Canton S strains and the wild-type Drosophila virilis Cordoba strain (gift of. F. Lemeunier, Gif/Yvette) were used for the preparation of in situ hybridization or Western blotting experiments.

2.2. Antibodies and antisera The antibodies were: (1) rabbit anti-reverse transcriptase (anti-RT ) anity-purified antibodies (Champion et al., 1992); and (2 ) rabbit and guinea-pig anti-Gag antibodies prepared against the central part of the Gag protein ( Haoudi et al., 1995; Kim et al., 1993). Guinea-pig anti-Gag antisera (GPI) were used after being pre-absorbed on bacterial proteins and tested against Gag recombinant proteins; rabbit anti-Gag antibodies were anity-purified on Gag recombinant protein.

2.3. In situ hybridization The in situ hybridization experiments using whole mount embryos, ovaries and testes were performed according to Tautz and Pfeifle (1989). The NdeI–NdeI 4.3 kb 1731 DNA probe was prepared from the 1731-containing pFP6c plasmid ( Fig. 1 ) and labelled using the digoxigenin DNA labelling kit ( Boehringer Mannheim, Germany) according to the manufacturer’s instructions. Control labelling was done with vector plasmid DNA.

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Fig. 1. Physical map and structural organization of the 1731 element. (A) Physical map of 1731. The main restriction sites used are indicated. N: NdeI; B: BamHI; P: PstI; K: KpnI; X: XhoI; and E: EcoRV. The NdeI–NdeI DNA fragment was used as a probe for in situ hybridization. (B) Structural organization of 1731. Overlap between ORF1 and ORF2 corresponds to the +1 frameshift ( Fourcade-Peronnet et al., 1988). Arrowheaded line indicates the major 1731 transcript.

2.4. Immunohistochemical detection 2.4.1. On whole mount embryos Embryos were dechorionated in 50% bleach and fixed in 4% paraformaldehyde/heptane for 10 min, followed by devitellinization in heptane/methanol. Embryos were washed three times for 10 min each in phosphate bu
er saline (PBS)-0.05% Triton X-100. After blocking with 1% BSA in PBS for 1 h, embryos were incubated at 4°C overnight with GPI antiserum diluted 1:250 in PBS-1% BSA-0.05% Triton X-100. After washing three times for 15 min each in PBS, embryos were incubated in FITCconjugated secondary antibody (Sigma) diluted 1:100 in PBS-1% BSA-0.05% Triton X-100. Finally, embryos were washed in PBS and mounted in Citifluor medium (London, UK ). Observations were done by confocal laser scanning microscopy using a Biorad MRC-600 attached to an Optiphot II–Nikon microscope. 2.4.2. On whole mount ovaries and testes Ovaries and testes were dissected in PBS and fixed. Preparation of immunostaining was done according to the method described for embryos. Observations were done by confocal microscopy.

glycerol ) (Laemmli, 1970). After being boiled for 5 min, the samples were spun at 10 000 rpm for 1 min at 4°C and the supernatants were loaded on polyacrylamide– SDS gel. After electrophoresis, proteins were electroblotted (Biorad electroblotter, USA) for 3 h at 300 mA on to a nitrocellulose membrane ( BA-85, Schleicher and Schuell, Germany). The membrane was blocked in 5% nonfat dry milk in PBS for 2 h at 37°C, and then primary antibodies were added (anti-RT diluted 1:250; rabbit anti-Gag diluted 1:100 or preabsorbed GPI diluted 1:500) in 5% nonfat dry milk in PBS, at 4°C overnight. The membrane was washed in PBT (PBS-0.1% Tween 20 ) three times at room temperature, and treated with secondary HRP-conjugated antibodies (anti-guinea-pig or anti-rabbit IgG, Dako) diluted 1:2000, for 1 h at room temperature. The membrane was then washed three times in PBT and, as previously described (Haoudi et al., 1995), peroxidase activity revealed by using a sensitive chemiluminescent system (Boehringer Mannheim, Germany) according to the manufacturer’s instructions. 3. Results 3.1. Transcript distribution

2.5. Immunoblot analysis Embryos from di
erent stages and ovaries and testes from adult flies were homogenized in lysis bu
er (0.12 M Tris, pH 6.8; 10% b-mercaptoethanol; 4% SDS; 10%

3.1.1. Spatially restricted expression of 1731 transcripts during embryogenesis In order to see whether 1731 transcripts are ubiquitously expressed or confined to particular regions of the

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Fig. 2. In situ hybridization to 1731 RNAs in Drosophila melanogaster wild-type embryos and adult gonads (Oregon R strain). 1731 DNA probe: digoxygenin-labelled NdeI–NdeI fragment. (A) Cleavage stage: labelling is in the cleavage energids (arrowheads). ( B) Syncytial blastoderm stage: labelling occurrs in the central yolk nuclei (arrowheads) and syncytial blastoderm (sb). No staining is seen in pole cells (pc). (C) Cellular blastoderm

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embryo, we examined the spatial patterns of 1731 expression by in situ hybridization to whole mount embryos. For these experiments the 4.3 kb NdeI–NdeI DNA fragment containing the entire 1731 sequence, i.e., LTR sequence and internal sequence, was used as a molecular probe ( Fig. 1). The results are presented in Fig. 2 and show that 1731 expression displayed temporally and spatially restricted patterns during embryogenesis. 1731 transcripts were detected during stages 3–4 (Campos-Ortega and Hartenstein, 1985). At these stages, 1731 transcripts were localized in the yolk nuclear and cytoplasmic islands (energids) (Fig. 2A); within the syncytial blastoderm 1731 transcripts appeared evenly distributed and pole cells appeared devoid of any staining ( Fig. 2B). During formation of the cellular blastoderm, at stage 5, 1731 transcripts were observed transiently and mainly in three areas: in the anterior part, in the central part as a large band and in the posterior part of the embryo ( Fig. 2C). At germ band extension stage, 1731 transcripts were again shown to be accumulating in the posterior midgut, mesoderm and future brain of the developing embryo ( Fig. 2D). During later stages of development, 1731 transcripts were mainly located in the central nervous system (CNS) and observed in midgut and presumed future malpighian tubes (Fig. 2E, F ); apparently little or no 1731 transcripts were detectable in the endoderm and lateral parts of the embryo. These observations showed that 1731 transcripts accumulated mainly in the mesoderm of early embryos and in the CNS of late embryos. 3.1.2. Distribution of 1731 transcripts in adult gonads In order to see whether 1731 expression occurred in the germ line of the adult fly, we examined the distribution of 1731 transcripts by in situ hybridization to whole mounts of testes and ovaries, using the 4.3 kb 1731 DNA probe. 3.1.2.1. 1731 RNAs distribution in ovaries. Analysis of whole mounts revealed the presence of 1731 transcripts in ovaries. In egg chambers younger than stage 6 ( King, 1970 ) cells seemed equally weakly labelled, however by stage 10 labelling appeared restricted to the cytoplasm of the nurse cells ( Fig. 2G). The brown staining

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observed in the oocyte is background staining produced by an endogenous enzymatic activity, as seen in a control experiment. 3.1.2.2. 1731 RNAs distribution in testes. In situ hybridization of the 1731 probe revealed the presence of 1731 transcripts along the testis in a very characteristic and reproducible pattern: RNAs were detected both near the apex and in the first half gyre of the testis. The very small apical region was not labelled, indicating that 1731 is not expressed in spermatogonia. Careful microscopic observation indicated that the labelled cells were arranged in a very characteristic speckled pattern, which is typical of di
erentiation of primary spermatocytes in the Drosophila melanogaster testis ( Fuller, 1993; Lindsley and Tokuyasu, 1981). Further stages of di
erentiation from spermatocytes II to spermatozoa, were not stained. This 1731 labelling, restricted to the nuclei of primary spermatocytes, suggests a precise control of the transcription of this retrotransposon ( Fig. 2H). 3.2. Distribution of 1731 proteins 3.2.1. Spatial distribution of 1731 proteins during embryogenesis Embryos were stained using antibodies directed against the Gag product of 1731 (expected to be the most abundant 1731 polypeptide), in order to examine 1731 protein expression pattern. Confocal microscopy showed that anti-Gag antibodies recognized 1731 Gag-related polypeptides in embryos from the early cleavage stage: intravitelline cytoplasmic energids and the periplasmic area appeared to be labelled (Fig. 3A). At the cellular blastoderm stage, the 1731 Gag proteins were mainly located in the blastoderm cell layer in an ubiquitous manner (Fig. 3B). At a higher magnification, it appeared that the cytoplasm of the blastoderm cells was stained, while nuclei and the yolk area were not (Fig. 3C ). At the extended germ band stage, only the yolk part of the embryo appeared unlabelled (Fig. 3D). After germ band extension, 1731 proteins were mainly located in the mesoderm, the future stomodeum, the midgut, and the forming CNS (Fig. 3E). In a later stage, optical sectioning of the embryo revealed the presence of 1731 proteins in the

stage: 1731 RNAs are detected in the anterior region on the dorsal side, in a large stripe in the middle of the embryo and in the posterior part of the embryo (arrowheads). The middle stripe is wider on the ventral side than on the dorsal side. (D) Germ band extension stage: notice a strong expression in the developing mesoderm (ms), the future brain ( br) and the posterior midgut invagination (pmg). ( E, F ) Condensation of central nervous system (CNS) stage: transcripts can be detected in the ventral nervous system (vns) and brain ( br). ( E, F) are respectively dorsal and ventral views of the embryo. Staining is also observed in the internal part of the embryo. mp, future malpighian tubules. (G) Whole mount ovaries. Egg chambers are labelled from very early to late stages. In the stage 10 egg chamber, accumulation of transcripts is observed mainly in the cytoplasm of nurse cells (nc ) while no labelling appears in the oocyte (oc ) or in follicular cells (fc). The red–brown color observed in the oocyte was also observed in control experiments and therefore due to background staining. ( H ) Whole mount adult testes. The staining follows the sense of maturation of germ cells, from the apex of the sperm tube where the spermatogonia (sg) reside to the median part where groups of spermatocytes, spermatids and developing spermatozoa are closely intermingled. The labelling appeared limited to the nuclei of spermatocytes I (spI ).

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Fig. 3. Localization of 1731 Gag proteins during embryonic development using GPI anti-Gag antiserum pre-absorbed on bacterial proteins. Confocal microscopy photographs are shown. Scale bars correspond to 100 mm, except for (C ) where it corresponds to 10 mm. (A) Early stage. The staining is detected mainly around the internal nuclei of cleaving energids and in the periplasm of the embryo. ( B) Cellular blastoderm stage. The staining is seen in the cytoplasm of the preblastoderm; no staining in nuclei. (C ) Cellular blastoderm stage: enlarged view of the blastoderm in ( B) (white rectangle). Gag-immunorelated staining is limited to the cytoplasmic part of the preblastoderm. (D) Germ band extension stage. The staining occurs strongly in the extended germ band and is absent in the internal, vitellin-containing part of the embryo. ( E) Extended germ band stage: 1731 Gag-staining is observed in the posterior midgut (pmg), in the ventral nervous system and in the anterior part of the embryo containing the forming brain (br). (F ) Condensation of central nervous system. The staining is distributed in the ventral ganglion (vg) of CNS and in midgut (mg).

CNS and in other internal organs, particularly in the midgut ( Fig. 3F). 3.2.2. Tissue-specific expression of 1731 proteins in adult gonads 1731 proteins were detected in both ovaries and testes. In ovaries, 1731 Gag proteins were mainly present in

the cytoplasm of nurse cells: at stage 10 of ovary development, the cytoplasm of the nurse cells was labelled, follicles cells were also labelled. In contrast, the optical section showed that the oocyte and nurse cell nuclei were totally unlabelled ( Fig. 4A). In testis, 1731 Gag proteins were found in all the organs except a gap in the immunostaining located in

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Fig. 4. Localization of 1731 Gag proteins in adult ovaries and testes of a wild-type Drosophila melanogaster strain by immunostaining on whole mounts using GPI anti-Gag antibodies. ( A) In stage 10, egg chamber shows staining mainly in the cytoplasm of nurse cells (NC ) and in follicular cells ( FC ). Cytoplasm of the oocyte (O) is free of 1731 Gag-related protein. ( B) The 1731 Gag staining is revealed along the sperm tube with highest intensity midway along the length of the testis. In the center part of the testis tube no staining is detected.

the anterior, spermatocyte-containing part. In contrast to the speckled appearance of the transcripts, the Gag proteins appeared distributed evenly, but a greater fluorescence in the two-thirds posterior part of the testis ( Fig. 4B) suggested that Gag proteins accumulated mostly there. 3.3. Immunoblot analysis By immunoblot analysis of total protein extracted from embryos at di
erent stages, we identified a 1731 Gag-related 35×103 M protein which was present r during the cleavage and syncytial stages ( 0–2 h), blastoderm to early gastrula stage (2–4 h), late gastrula stage (4–6 h) and in later stages (6–16 h) ( Fig. 5A, lanes 1–4 ). Apparently, there was little or no variation in the quantity of this protein during embryonic development. No Gag-related 1731 proteins could be detected in late third larval stage (wandering stage) either in total proteins extract ( Fig. 5A, lane 5 ) or in larval testis extracts ( Fig. 5C, lane 2 ). This might be linked to the observation that, as shown in Drosophila melanogaster cultured cells ( Ziarczyk and Best-Belpomme, 1991), 1731 expression is inhibited by the steroid hormone 20-hydroxyecdysone present in the late third larval stage. In protein extracts from adult ovaries, we detected a Gag-related 35×103 M protein (Fig. 5A, lane 6 ) but no 1731 Pol-related r protein was recognized by anti-RT antibodies (Fig. 5B, lane 4 ).

Analysis of adult testes extract delivered a completely di
erent result. Using anti-Gag antibodies we detected both a 40 and a 150×103 M Gag-related protein r (Fig. 5C, lane 1 ). Surprisingly, anti-Gag antibodies revealed the high molecular weight product as the major band, whereas the low molecular weight product appeared as a less abundant peptide. This was in opposition to the commonly accepted notion that the Gag to Pol molar ratio is high, the Gag protein being produced in larger quantity. When the same extract was probed with purified 1731 anti-RT antibodies, the same 150×103 M protein and no Pol-processed polypeptides r were revealed ( Fig. 5B, lane 1 ). The specificity of the antibodies for Drosophila melanogaster 1731 proteins was controlled on D. virilis extracts: as expected, when taking a fly devoid of 1731-related DNA sequence, no 1731-RT immunorelated peptides were detected (Fig. 5B, lanes 2, 3 ). From our previous results (Haoudi et al., 1995), we interpret the 40×103 M small peptide r as a Gag product while the 150×103 Mr peptide as the Gag–Pol polypeptide. Apparently this large 1731 product was not processed in testes: only overexposed films displayed faint bands corresponding to processed Gag–Pol or Pol polypeptides found in virus-like particles extracted from Drosophila melanogaster Kc0 cultured cells (not shown). All this suggests that mechanisms involved in translation, processing and/or maturation of 1731 proteins are tissue-dependent (ovaries or testes).

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4. Discussion 4.1. Control of 1731 transcription in embryos

Fig. 5. Western blot analyses of protein extracts of Drosophila melanogaster. Embryos or tissues were solubilized in Laemmli bu
er. Following electrophoresis and electrotransfer to a nitrocellulose membrane the proteins extracts were probed with di
erent antibodies (see Materials and Methods). ( A) Detection of 1731 Gag-related proteins in D. melanogaster embryos, third instar larvae and ovary extracts. Lanes 1–4: 0–2, 2–4, 4–6 and 6–16 h old embryos respectively; lane 5: third instar larva (wandering stage): total protein extract; lane 6: ovary extract. The membrane was probed with the GPI anti-Gag antibodies (dilution was approx. 1:500 ); peroxidase-conjugated antiguinea-pig antibody (Dako) as secondary antibody was diluted 1/2000. ( B) Detection of 1731 RT-related proteins in D. melanogaster and D. virilis ovary and testis extracts. Lanes 1, 4: D. melanogaster; lanes 2, 3: D. virilis. Testis extracts: lanes 1, 2; ovary extracts: lanes 3, 4. Anity-purified rabbit anti-RT ( 10) was used as primary antibody ( 1:250) and reveals a large band in D. melanogaster testis extract. (C ) Detection of 1731 Gag-related proteins in D. melanogaster adult and larval testis extracts. Lane 1: adult testes; lane 2: larvae testes (third instar). Anity-purified rabbit anti-Gag was used as primary antibody ( 1:100) and revealed two bands ( lane 1 ): the larger one corresponds to that seen in ( B) and the smaller corresponds to a previously described Gag protein form ( Haoudi et al., 1995). The faint band with 130×103 M is probably a degraded form of the largest one. No immur norelated band is detected in larval extract ( lane 2).

Transcriptional regulation analysis of Drosophila retrotransposons is well documented and shown to operate through LTRs or internal sequences which are targets for stage and/or tissue-specific transcriptional factors (Arkhipova and Ilyin, 1991; Brookman et al., 1992; Bro¨nner et al., 1995; Cavarec et al., 1994; Mozer and Benzer, 1994 ). Functional analyses of 1731 5∞LTR had shown the presence of several DNA motifs as potential targets for transcriptional regulators (Lacoste and Fourcade-Peronnet, 1995; Ziarczyk et al., 1989; Ziarczyk and Best-Belpomme, 1991 ). Particularly, p11, a nuclear protein binding to the U3 region of the 5∞LTR and acting as a transrepressor has been characterized (Lacoste et al., 1995). Analysis of 1731 expression during embryogenesis reveals a spatio-temporal control of its transcriptional activity. 1731 transcripts appear transiently in the early embryo, with a specific pattern which is di
erent from that observed for other retrotransposons ( Ding and Lipshitz, 1994 ). The expression of 1731 suggests a set of transcriptional factors which are operative only during specific stages and in particular areas of the embryo (Fig. 2C, E, F ). Interestingly, the p11 protein is localized in cytoplasmic areas of early embryos up to the gastrula stage and found in nuclei in late stages, suggesting that this factor is operational after the embryo enters gastrulation. Moreover, the restriction of 1731 expression to mesodermal and neurogenic tissues associated with the absence of 1731 RNAs in ectodermal fields might depend on the presence of its transrepressor (p11) in the nuclei of ectodermal cells (Lacoste, 1995 ). 4.2. Transcripts in the gonads 4.2.1. Ovary 1731 RNAs were observed in egg chambers before stage 6 of oogenesis as components of the cytoplasm. No di
erence in labelling was observed in future nurse cells compared with the future oocyte, showing that 1731 accumulates in every cell at this stage. In contrast, by stage 10, 1731 transcripts were found mainly into the cytoplasm of nurse cells, indicating that transcription of the element is positively regulated in these cells and that 1731 RNAs accumulate and are not transferred into the oocyte at least during this stage (Fig. 2G). Nonetheless, detection of 1731 transcripts in early embryos ( Fig. 2A) indicates that 1731 RNA transfer from nurse cells to the oocyte in late stages cannot be ruled out. 4.2.2. Testis 1731 transcripts were detected in a very limited area of testes, which includes primary spermatocytes as

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described by Lindsley and Tokuyasu (1981 ). Transcripts were not detected in cells engaged in the meiotic processes and in spermatogonia. The presence of 1731 transcripts in primary spermatocytes indicates a strong positive regulation of this element. This is reminiscent of the previously described transcriptional activation of gypsy, micropia and Fex retroelements in the primary spermatocytes of Drosophila ( Hochstenbach et al., 1996; Huijser et al., 1988; Kerber et al., 1996; Lankenau et al., 1994 ). As for the copies of gypsy element contained in the Y-chromosome male fertility gene of D. hydei ( Hochstenbach et al., 1996 ), 1731 transcription activation in testes could rely on the general activation of heterochromatin genes at the onset of meiosis. In such tissue context, it can be expected that heterochromatic copies from di
erent retrotransposons should be activated in spermatocytes I. As with many premeiotically transcribed genes ( Hochstenbach et al., 1996), 1731 transcripts appear to be rapidly degraded at the end of the meiotic prophase, suggesting that translation is limited to the meiosis period. 4.3. Translational control 4.3.1. Embryos–ovaries In the amphibian embryo, we have shown that 1731 chimeric RNAs can be translated as a Gag-bGalactosidase fusion protein at the mid-blastula stage ( Kim et al., 1994 ) and not during earlier stages. Consistent with that observation and the presence of 1731 RNAs at the late blastoderm stage, the 1731 protein could have mainly a zygotic origin. It is likely that the presence of the Gag protein in cleavage energids and in periplasm could result from a de novo 1731 RNA translation, but the RNA and the proteins in the early embryo may be maternal as well. Only study of 1731 RNA and protein transport from nurse cells to the oocyte might give a clear answer. In embryos and ovaries, only the 35×103 M Gagr immunorelated protein was recognized by Western blotting ( Fig. 5A), while apparently there was no epitope for anti-RT antibodies in embryos (data not shown) or in ovaries (Fig. 5B). This suggested that the 1731 transcripts were either truncated or incompletely translated. The latter hypothesis seemed likely because the polypeptide size corresponded to that of 1731 Gag polypeptides either found in cultured cell extracts (approx. 40×103 M ) or synthetized in vitro ( 32×103 M ) (Haoudi r r et al., 1995). Incomplete translation could result either from the absence of a frameshift event in ovaries, or another mechanism restricting the 1731 mRNA translation, as described for the micropia transposable element, in which a short antisense RNA corresponding to the RT region could inactivate that portion of the message (Lankenau et al., 1994 ). Alternatively, an ecient prote-

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ase activity in ovaries might quickly eliminate the Pol part of 1731 Gag–Pol polyprotein. Whatever the control mechanism, the absence of 1731 Pol-encoded products suggests that 1731 should be unable to transpose in ovaries. 4.3.2. Translation in testis Protein distribution in testis appeared strongly di
erent from the RNA localization, with greater labelling in the posterior two-thirds of the testis (Fig. 4B). Given the gradual modifications of cells along the testis, the simplest explanation likely is that proteins are produced in the spermatocyte area and accumulate in spermatids and maturating sperm cells. In testes, contrary to expectations drawn from the 1731 sequence and previous observations on the 1731 proteins in Drosophila cultured cells (Champion et al., 1992; Haoudi et al., 1995; Kim et al., 1993 ), we observed only one large protein recognized by 1731 anti-RT antibodies (Fig. 5B). This protein is around the 150×103 M size expected for 1731 Gag–Pol polyr protein. Anti-Gag antibodies recognized the same large band and only a faint small band at approx. 40×103 M , the size previously observed for the 1731 Gag r protein in Drosophila Kc cells. Apparent di
erences in size for 1731 Gag products synthetized in ovaries (35×103 M ) and testes (40×103 M ) could be explained r r by di
erent post-translational modifications in two di
erent tissues. The translation of 1731 RNAs in testis did not produce Gag–Pol and Gag polypeptides in a Pol/Gag ratio of approx. 10%. Typically, the fusion protein is processed into the Gag and 1731-encoded enzymes, producing several protein bands (150, 110, 92, 64 and 35×103 M ) recognized by anti-RT antibodies r in virus-like particle extracts (our unpublished data). Rather, only the fusion protein ( 150×103 M ) seemed r present in Drosophila testes. This observation can be explained by the existence of an enhancement of the frameshift and by the absence of processing of the synthetized protein. Experimental enhancement of frameshift during yeast Ty1–RNA translation has led to a modified Gag to Pol molar ratio and consequently to a strong decrease in the level of Ty1 transposition (Farabaugh, 1995; Kawakami et al., 1993; Kirchner and Sandmeyer, 1993). Apparently, in Drosophila, testisspecific 1731 translation makes Gag–Pol the major product, suggesting that the 1731 virus-like cycle cannot be completed. The mechanisms capable of modifying the translation of 1731 RNA are unknown. Interestingly, translational frameshifting is involved in the regulation of mammalian ornithine decarboxylase antizyme synthesis and in Ty1 transposition regulation (Balasundaram et al., 1994a,b; Farabaugh, 1995; Hayashi et al., 1996); in both cases, and as for 1731 translation, a +1 frameshift is involved. It can be speculated that similar mechanisms are

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involved in the enhancement of the 1731+1 frameshift occurring in Drosophila testes. Alternatively, the failure to process the polyprotein could rely on the absence of Gag; in such case, the expected protease either was not processed and/or its proteolytic activity was inhibited in this tissue. As in ovaries, but through a totally di
erent mechanism, the main consequence for the 1731 element would be the inability to complete the virus-like cycle in testes and to transpose. Thus, negative control on 1731 transposition in Drosophila melanogaster testes could be performed at the translational and/or posttranslational level. In conclusion, our study of the 1731 retrotransposon expression at the level of both transcription and translation products provides new information on how control of transposition can operate during development. As far as we know it is the first demonstration of tissue-specific translational or post-translational control of retrotransposon products in Drosophila. Moreover, analyses of 1731 proteins in ovaries and testes indicate that the same transposon is controlled through two di
erent translational strategies according to the tissue of expression, but driving the same general e
ect: protection against insertional mutagenesis by avoiding 1731 transposition. Conversely, it can be speculated that any transposition could result from a defect in such controls, leading to new insertion(s) of the element and possibly creating new mutation(s) in the fly.

Acknowledgement Thanks are extended to M. Gegouzo for preparing and rearing fly stocks, to C. Brandonne and D. Rouille´ for technical assistance and to A. Debec for advice on the immunostaining methods. We also are grateful to N. Tchurikov for stimulating discussions during his stay in our lab ( Financial support from INTAS No. 94-634-Brussels Belgium) and to J. M. Mason for comments on the manuscript. This work was supported by the Centre National de la Recherche Scientifique ( UA 1135) and by grants to M.B.B. from the Association pour la Recherche contre le Cancer ( ARC ), the Fondation pour la Recherche Me´dicale (FRM ). A.H. was supported by a fellowship from the Ministe`re de l’Education Nationale (Morocco). Added thanks are addressed to referees for their suggestions and remarks to improve our manuscript.

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