Claude Maisonhaute

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.

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.

A nested alpha-amylase gene in Drosophila ananassae.

The amylase gene family of Drosophilaananassae consists in seven copies, scattered on several chromosomal arms. We have evidenced that a member of the family, Amy35, lies within an intron of a gene homologous to the CG14696 gene of D. melanogaster. This nested arrangement seems restricted to the D. ananassae subgroup. The nested and the nest genes are encoded on opposite strands. Both are actively transcribed in the midgut at the same time, raising the possibility of interference between their mRNAs. Our data also help to elucidate the history of the Amy family, suggesting that Amy35 arose by duplication and translocation from another ancestral locus, into a formerly short intron, in an ancestor of the subgroup.

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 class of genomic copies of retrotransposon 1731 with different expression strategies, one of which might be driven by natural selection. The first class uses conventional translation frameshifting known to ensure expression of revere 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 recoginsing rare tRNA by a codon preferred by host genome, whereas the RT ORF is restored by downstream single nuclotide deletion. We suggest that natural selection has driven the switching of 1731 expression strategy from retrovirus-like to the fussion-ORF expression. This observation is in accordance with the detection in testes of fused Gag-RT polypetide encoded by 1731. The abundance of RT in testes may serve for normal development of host tissue.

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.

From transcript modulations to protein phosphorylation: a short survey of some effects of ecdysteroid

Abstract An overview of several effects of 20-OH-ecdysone on Drosophila cells shows that this hormone exerts its action through the modulation of transcription but also at the post-translational level, e.g. by protein phosphorylation.

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.

From transcript modulations to protein phosphorylation

Abstract An overview of several effects of 20-OH-ecdysone on Drosophila cells shows that this hormone exerts its action through the modulation of transcription but also at the post-translational level, e.g. by protein phosphorylation.

The drosophila melanogaster 1731 retro-transposon gag gene: Expression in an in vitro system and in drosophila cultured Kc cells

As part of our effort to characterize the structure and function of the vacuolar apparatus in plant cells we found that the vacuole membrane (tonoplast) from the meristematic cells in cauliflower (Brassica oleracea var. botrytis) heads contains uroteins of 27 kDa that are members of the major intrinsic nrotein (MIP) fimily, an ancient family of membrane channel proteins: Such proteins from the tonoplast in plant cells are referred to as TIPS (Tonoplast Intrinsic Proteins, JOHNSON K. D.. HERMAN E. M.. and CHRISPEELS M. J. (1989). ~~anr Physiol, 91. 1006-1013). In the tonoplast from Brmsica oleracea var. botrytis their homologs are tentatively called bobTIPs. It has been shown recently that y-TIP,-an isofonn that is present in the tonoplast of cells from vegetative tissues, is an aquaporin because it forms a membrane channel which is selective for water transnort (MAUREL C.. REIZER I., SCHROEDER J. I:. and CHRISPEELS M. J. (1493). EMBCJ 1.. It, 2241-2247). TIP isoforms, identified on the basis of their aminoacid and/or nucleic acid sequences, are organ-specific expressed (HOm H., HUBBART L.. REIZER J., LUDEVID D.: IIERMAN E. M.. and CHRISPEELS M. J. (1992). P/ant PhysioL, 99, 561-570) or Induced by specific physiological or stress conditions (GUERRERO F. D., JONES J. T., and MULLET J.E. (1990). PIant Mol. Biol.. 15. 11-26). However, only a few have been developmentally characterized (LUDEVID D., H&TE II., HIMELBLAUE., and CHRISPEELS M.J. (1992). Plant Physiol., 100. 1633-1639). The aim of our work was to analyze the molecular details of bobTIPs. Poly(A)+ RNA from the meristema& cells of cauliflower were translated in vitro and translation products were subjected to immunoprecipitation with a polyclonal serum containing antibodies which recognized exclusively bobTIPs. Immunoprecipitated polypeptides migrated as a single band in SDS-PAGE. Therefore, we hypothesized that the Poly(A)’ RNA purified from the cauliflower head contained the mRNA specifically required for the synthesis of bobTIPs or polypeptides immunologically related in meristematic cells. Poly(A)+ RNA were subsequently used to make a cDNA library in Igt 11. The library was screened with the polyclonal semm. Four positive clones were detected. The sizes of the inserts were determined and experiments showed that the four clones cross hybridize. All cDNAs are now being sequenced. THE END3 GENE ENCODES A PROTEIN THAT IS REQUIRED FOR