Abundance, distribution and dynamics of retrotransposable elements and transposons: similarities and differences.

Retrotransposable elements and transposons are generally both found in most eukaryotes. These two classes of elements are usually distinguished on the basis of their differing mechanisms of transposition. However, their respective frequencies, their intragenomic dynamics and distributions, and the frequencies of their horizontal transfer from one species to another can also differ. The main objective of this review is to compare these two types of elements from a new perspective, using data provided by genome sequencing projects and relating this to the theoretical and observed dynamics. It is shown that the traditional division into two classes, based on the transposition mechanisms, becomes less obvious when other factors are taken into consideration. A great diversity in distribution and dynamics within each class is observed. In contrast, the impact on and the interactions with the genome can show striking similarities between families of the two classes.

Virus-like particle formation in Drosophila melanogaster germ cells suggests a complex translational regulation of the retrotransposon cycle and new mechanisms inhibiting transposition.

Transposition of 1731, a Drosophila melanogaster LTR retrotransposon, was investigated in reproductive organs by RNA, protein and VLP distribution during its life cycle. We detected 1731 transcription in oogonia but not in spermatogonia; in all cells during oogenesis but only in primary spermatocytes; and in ovarian cytoplasm but both in nuclei and cytoplasm of primary spermatocytes. By confocal scanning, we showed that whereas Gag protein appeared in all cytoplasms during oogenesis, in testes Gag detection began in late premeiotic primary spermatocytes and increased in elongating spermatids suggesting distinct mechanisms of 1731 transcription and translation regulation. By electron microscopy, we did not detect 1731 VLPs in ovaries, suggesting a complex post-translational control blocking VLP assembly and transposition. Interestingly, in testes we discovered VLP aggregates in cystic cytoplasm of maturing partially individualized spermatids. In testes, we observed two delays in 1731 product expressions, suggesting a complex temporal control mechanism. Transcriptional/translational delay may be determined by accumulation of 1731 RNAs in primary spermatocyte nuclei. Translational/VLP assembly delay may be determined by post-transductional mechanisms controlling +1 frameshift and Pol-protein degradation. Our results indicated two differential mechanisms inhibiting 1731 transposition in Drosophila melanogaster ovaries and testes. In addition, we proposed a new mechanism for transposition control at the cell cycle level.

Selective expansion of the newly evolved genomic variants of retrotransposon 1731 in the Drosophila genomes.

The structural variants of the regulatory and coding regions of the LTR-retrotransposon 1731 are described. Two classes of genomic copies of retrotransposon 1731, with and without frameshifting strategy to express Gag and Pol proteins, were earlier revealed in the D. melanogaster genome. Copies without frameshifting are shown to be evolved from an ancient variant with frameshifting and are widespread in the genomes of the melanogaster complex species. Position of a rare codon responsible for ribosome pausing and efficient frameshifting is identified. Two structural variants of 1731 LTRs were detected in the melanogaster complex species: the predominant structural variant A1A2 of 1731 LTR in the D. melanogaster, D. simulans, and D. sechellia genomes contains duplicated and diverged copies of 28 bp in the U3 region, whereas A1 variant lacking this duplication is expanded in the D. mauritiana genome. Selective expansion of the A1A2 variant was detected in the independently established D. melanogaster cell cultures. A1A2 variant is expressed in embryos, cell culture, and testes, whereas A1 is expressed only in testes of D. melanogaster. Relief of expression of the A1A2 but not A1 variant in the ovaries as a result of mutation in the RNA interference (RNAi) spn-E gene is shown. Thus, expansion of the recently evolved genomic variants of the LTR retrotransposon 1731 possessing a new translation strategy, duplication in the U3 region, and extended profile of expression is revealed.

The GATE retrotransposon in Drosophila melanogaster: mobility in heterochromatin and aspects of its expression in germline tissues.

A full-length copy of the retrotransposon GATE was identified as an insertion in the tandemly repeated, heterochromatic, Stellate genes, which are expressed in the testis of Drosophila melanogaster. Sequencing of this heterochromatic GATE copy revealed that it is closely related to the BEL retrotransposon, a representative of the recently defined BEL-like group of LTR retrotransposons. This copy contains identical LTRs, indicating that the insertion is a recent event. By contrast, the euchromatic part of the D. melanogaster genome contains only profoundly damaged GATE copies or fragments of the transposon. The preferential localization of GATE sequences in heterochromatin was confirmed for the other species in the melanogaster subgroup. The level of GATE expression is dramatically increased in ovaries, but not in testes, of spn-E(1) homozygous flies. We speculate that spn-E is involved in the silencing of GATE via an RNA interference mechanism.

Tissue-specificity of 412 retrotransposon expression in Drosophila simulans and D. melanogaster.

We analyse the expression of the retrotransposon 412 in the soma, testes, and ovaries in populations of Drosophila simulans and D. melanogaster, using RT-PCR and in situ hybridization. We find that expression of 412 is highly variable in the soma, confirming previous findings based on Northern blots. No 412RNA is detected in the ovaries by either in situ hybridization or RT-PCR, in any population of either species. Transcripts are, however, detected in the male germline, which show a very characteristic spatial pattern of 412 expression in primary spermatocytes. There is no relationship between expression of the 412 element in the soma and in the testes in the populations. These findings show that the expression of 412 is independently regulated in the soma and the testes, and this raises the question of the real influence of the somatic transcripts on the organism and on the transposition rate.

[Acquisition and loss of modules: the construction set of transposable elements]. / Priobretenie i poteria modulei: nabor dlia konstruirovaniia mobil'nykh élementov.

Phylogenetic analysis of transposable elements (TEs) allows us to define the relationships between the domains or gene(s) that compose them. Moreover, modules of a few amino-acids can be detected within gag, pol, env genes or within the integrase domain of retrotransposons and transposase of DNA elements. The combination of these observations clearly shows that the evolutionary history of TEs is the outcome of the acquisition and loss of modules with differing origins and histories. This raises the question of the origin of TEs: are they derived from viruses? Are they where viruses come from? Do the basic building bricks come from the prokaryotes, and can they be assembled in the eukaryotes? Are the TEs found in prokaryotes the result of the disintegration of complex elements such as retroelements? Do they evolve from the simplest to the more complex, or are they opportunistic sequences evolving by acquiring and/or losing modules which may be either important or superfluous to their fitness (i.e., their ability to transpose). These are some of the questions that are addressed and discussed in the light of the comparative structures of TEs.

Retrotransposon 1731 in Drosophila melanogaster changes retrovirus-like expression strategy in host genome.

Earlier related to parasitic elements, retrotransposons of eukaryotes have been demonstrated to participate in general cell processes such as chromosome repair and evolution of gene expression (Teng et al., 1996; McDonald, 1993). Here, we report the existence of two classes of genomic copies of retrotransposon 1731 with different expression strategies, one of which might be driven by natural selection. The first class uses conventional translational frameshifting known to ensure expression of reverse transcriptase (RT) open reading frame (ORF), depending on the efficiency of frameshifting. The bulk of genomic copies are related to the second class where the frameshift is prevented as a result of the substitution of a rare codon recognising rare tRNA by a codon preferred by host genome, whereas the RT ORF is restored by downstream single nucleotide deletion. We suggest that natural selection has driven the switching of 1731 expression strategy from retrovirus-like to the fusion-ORF expression. This observation is in accordance with the detection in testes of fused Gag-RT polypeptide encoded by 1731. The abundance of RT in testes may serve for normal development of host tissue.

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

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 differentially according to the expressing tissue: essentially, ovarian translation and/or processing of 1731 products is different 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 different in the two sexes.

The Gag polypeptides of the Drosophila 1731 retrotransposon are associated to virus-like particles and to nuclei.

1731 is a Drosophila melanogaster retrotransposon whose nucleotide sequence shows a proviral architecture with two long terminal repeats (LTRs) framing two internal Open Reading Frames (ORFs). The pol ORF2 of this mobile genetic element was demonstrated to code for an active Reverse Transcriptase (RT) and the ORF1 is expected to code for the structural Gag proteins of the virus-like particles (VLP). Using specific anti-Gag antibodies, we have characterized the 1731 Gag polypeptides expressed either in vitro or in Kc Drosophila melanogaster cultured cells. Together with the 1731 RT, the largest, likely post-translationaly-modified Gag polypeptides are gathered into cytoplasmic virus-like particles. Moreover and consistent with the nuclear localization signal present in the Gag sequence, we observed that a short 1731 Gag polypeptide is associated to the cell nuclei.

The microinjected Drosophila melanogaster 1731 retrotransposon is activated after the midblastula stage of the amphibian Pleurodeles waltl development.

The entire 1731 retrotransposon of Drosophila melanogaster, tagged with the E. coli lac Z gene inserted in its gag sequence, was injected into oocytes and fertilized eggs of the urodele amphibian Pleurodeles waltl. Expression of the reporter gene indicated that the 1731 promoter (its 5'LTR) is active in the embryos and not in the oocytes. It appeared that this element is regulated as amphibian genes are at the beginning of the development, i.e. that expression was detected after the mid blastula stage and maintained up to four or five days after injection. Another construction associating the modified 1731 promoter with the CAT gene is also expressed in Pleurodeles embryos during the same period of development. This indicated that the 1731 promoter issued from a Drosophila species is activated as promoting sequences of amphibian zygotic genes are, suggesting that in the case of horizontal transfer, 1731 can be expressed into vertebrate organisms.

Translation and fates of the gag protein of 1731, a Drosophila melanogaster retrotransposon.

An entire copy of 1731, a Drosophila melanogaster retrotransposon, was tagged by fusing in frame its putative gag gene with the reporter LacZ sequence. The high transfection efficiency of Drosophila virilis cells added to the absence of 1731 in their genome allowed, by combining histochemical staining and immunological detections, the demonstration of the translation of the 1731 gag gene. The gag protein is gathered in virus-like particles. Its occurrence in nuclei is consistent with a nuclear localization signal. The expression of the sense construction was inhibited by cotransfections with its antisense homologue.

Effect of hydrogen peroxide on cytoskeletal proteins of Drosophila cells: comparison with heat shock and other stresses.

Hydrogen peroxide, which was shown to trigger the heat-shock response by activating the immediate binding of the heat-shock factor to DNA heat shock regulatory elements in the promoter of heat-shock genes of Drosophila cells, has also been reported to enhance the synthesis of actin. We show here that very short and transient H2O2 treatments, from 1 s to 2 min, are sufficient to induce an increase of actin synthesis. This increase becomes apparent 2 to 3 h after the short H2O2 treatment. It is inhibited if actinomycin D is present during the short H2O2 treatment. An increase of actin synthesis was also observed during the recovery period after two other stresses: reoxygenation after anoxia and ethanol treatment. The synthesis of two cytoskeletal proteins, tubulin and a 46-kDa insoluble protein of the intermediate filament fraction, was also slightly increased by H2O2 in Drosophila cells, but this increase was not actinomycin D-dependent. H2O2 does not provoke the translocation of the 46-kDa protein to the nuclear fraction as does heat shock. The very rapid stimulation of actin synthesis by H2O2 and the involvement of cytoskeletal elements in many stress situations suggest that actin may play a key role in the response to external stimuli.

Characterization of the reverse transcriptase of 1731, a Drosophila melanogaster retrotransposon.

The nucleotide sequence of 1731, a retrotransposon cloned from the genome of Drosophila melanogaster, reveals a structural similarity with the proviral form of the retroviruses including a pol-like gene containing a putative reverse-transcriptase(RT)-coding sequence. Diverse parts of that sequence were subcloned and expressed in Escherichia coli. It has been demonstrated that the expression of the RT-like sequence, when translated, gives rise to peptides displaying enzyme activity characteristic of a true RT enzyme. In addition, rabbit antisera directed against such recombinant proteins allowed us to detect an immunoreactive protein of around 110 kDa, which was only present in D. melanogaster cell lines, but not in cells derived from Drosophila virilis or Drosophila hydei, whose genomes do not bear the 1731 element. This protein is expected to correspond to a non-processed pol-gene translated product and cosediments with virus-like particles exhibiting RT activity.

The response of the centrosome to heat shock and related stresses in a Drosophila cell line.

The centrosome of Drosophila melanogaster cells cultured in vitro has been followed by immunofluorescence techniques with the Bx63 antibody of Frasch and Saumweber. After a heat shock, the centrosome labelling becomes very small and finally disappears after 30 min. Other heat-shock protein (hsp) inducers such as ethanol, arsenite and ecdysterone lead to the same disappearance. Moreover, the functional ability of centrosomes to nucleate microtubule assembly is inhibited by these treatments, particularly by heat shock, ethanol and ecdysterone. Two other hsp inducers, cadmium chloride and hydrogen peroxide, do not affect the centrosome seriously. With the exception of cadmium, the rapidity and the intensity of hsp induction are in good agreement with the kinetics of alteration of the organelle. We propose that a close link exists between the heat-shock response and the centrosome and that the physiological induction of hsps could be reinterpreted in terms of cell division control.

Early activation of heat shock genes in H2O2-treated Drosophila cells.

Drosophila cells of a diploid clone derived from line Kc were treated with 1 mM H2O2 for 1 to 20 minutes. Dot blot and Northern blot analysis of RNAs extracted from control and treated cells showed that the transcriptional activation of the 6 heat-shock genes tested was early, and maximal within 5 minutes of H2O2 treatment. Analysis of the kinetics of induction of the heat-shock proteins (hsps) after an exposure to H2O2 of 2 or 5 minutes, followed by removal, suggests that this brief treatment was sufficient to trigger the synthesis of all the hsps, which was maximal 1.5 to 3h after this short H2O2 treatment.

Functional analysis of the long terminal repeats of Drosophila 1731 retrotransposon: promoter function and steroid regulation.

1731 is a Drosophila retrotransposon whose transcripts decrease in Drosophila cells after treatment by the steroid hormone 20-hydroxyecdysone (20-OH). Several constructions have been made where the bacterial chloramphenicol acetyltransferase (CAT) gene is put under the control of either the 5' or the 3' long terminal repeats (LTRs) of 1731. CAT activity assays in transfected Drosophila cells show that either the 5' or the 3'LTR constitutes a unidirectional promoter. Analysis of partially deleted LTR suggests the presence of so-called silencer and activator regions in these LTRs. Moreover, the first 260 bp of the LTR are sufficient to provoke 20-OH inhibition whereas the first 58 bp are necessary for hormonal responsiveness. These 58 bp contain sequences showing similarities with the targets of trans-acting factors such as Octal-c and NFkB.

Hydrogen peroxide (H2O2) induces actin and some heat-shock proteins in Drosophila cells.

Drosophila cells of a clone derived from line Kc were treated with various concentrations of hydrogen peroxide (H2O2). The concentration of 10 mM was lethal, whereas concentrations of 1-100 microM did not affect cell viability, rate of multiplication or protein synthesis. The intermediate concentration of 1 mM H2O2 was used to study the response of the cells to an oxidative stress. We observed a transitory decrease of the global protein synthesis, which was accompanied by changes in the polypeptide pattern. There was a 2.5-fold increase of the synthesis of the heat-shock proteins 70-68 and 23. The most prominent response was a 6.5-fold increase of actin synthesis 3 h after a 1 mM H2O2 treatment. When aminotriazole (an inhibitor of catalase) was added in association with H2O2, the increase of actin synthesis became 8.5-fold. Experiments in which catalase was added at various times after H2O2 showed that a 10-min treatment with H2O2 was sufficient to induce actin and heat-shock protein synthesis 3 h later. H2O2 was shown to induce the transcriptional activation of an actin gene and of the heat-shock protein genes 70 and 23 within minutes. These results are coherent with the hypothesis that the byproducts of O2 reduction (the superoxide ion and hydrogen peroxide) could be inducers of the heat-shock response. Whether the increase of actin synthesis is a stress-related response, and the mode of action of H2O2 are discussed.

Stable transformation of Drosophila Kc cells to antibiotic resistance with the bacterial neomycin resistance gene.

By transfection with a plasmid containing the APH(3') gene under control of the HSV I thymidine kinase promoter, independent series of stably transformed Drosophila cells were established and grown for more than one and a half years under highly selective pressure (2 mg G 418/ml). Analysis of transformed Drosophila cell DNAs shows that the APH(3') gene was integrated into the genome. Neomycin phosphotransferase is constitutively expressed in transformed cells. This efficient selective system by a dominant marker makes it possible to introduce, by cotransfection, any DNA sequence of interest into the genome of cultured Drosophila cells.

Heat shock proteins are induced by cadmium in Drosophila cells.

Drosophila cells were treated with increasing concentrations of CdCl2 (10 microM-1 mM). The toxicity of cadmium, as observed by cellular death and the ability of the cells to survive after removal of CdCl2, depended on concentration and duration of treatment. The overall synthesis of protein, measured by incorporation of [35S]methionine, decreased. It fell to 66% of the controls after 24 h of exposition to 50 microM CdCl2 and to 29% after 48 h. We showed that cadmium induced the synthesis of 'heat shock proteins' (hsps), which started after 6 h and was maximal after 24 h of 50-100 microM CdCl2 treatment.