Michèle Capri

Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms—in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3′ untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.

The yemanuclein-α: a new Drosophila DNA binding protein specific for the oocyte nucleus

Abstract The Drosophila yG 4.5 gene (now called yemanuclein-α gene), which maps at 98F, is a member of the yema gene cluster isolated in a search for differentially expressed maternal genes. The yemanuclein-α transcript (formerly yT 4.5) is specifically expressed in the female germ cells at early oogenic stages and displays a graded distribution along the antero-posterior axis of the oocyte. These provocative features are reminiscent of that of K10, bicoid and Bicaudal-D gene transcripts and lead us to hypothesize that the yemanuclein-α gene plays a key role in egg organization. We show in the present work that the yemanuclein-α is a nuclear protein highly specific for the oocyte nucleus. The sequence analysis of the 5696 bp EcoRI fragment containing the yemanuclein-α gene, and of 5 overlapping cDNAs, reveals a 3006 nucleotides long open reading frame (ORF) flanked by long untranslated 5′ and 3′ sequences. This ORF predicts a 109,215 kDa protein which is basic (pHi: 8.57), and serine rich (12.08%). It contains a 40 amino acid acidic domain in the first third of the protein with a potential α-helix organization; this domain has some similarity with the nucleolin acidic domain. Parts of the yemanuclein-α sequence are likely to form secondary structures known to interact with DNA. We demonstrate the DNA binding activity of the yemanuclein-α by affinity chromatography experiments. Our data indicate that the yemanuclein-α shares some of the features which are characteristic of genuine transcriptional activators.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3′UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)240 insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)240.4, the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)240.4 expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

A single mutation results in diploid gamete formation and parthenogenesis in a Drosophila yemanuclein-alpha meiosis I defective mutant

BackgroundSexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes) and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model.ResultsWe describe the identification and the characterization of yem1 , the first yem-alpha mutant allele (V478E), which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem1 mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization.ConclusionsWe focus here on the analysis of the meiotic defects exhibited by yem1 oocytes that could account for the formation of diploid gametes. Our results suggest that yem1 affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division.This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction.

Subcellular localization of some anthracycline derivatives in Ehrlich ascites tumor cells

Abstract With the whole set of the tested anthracyclines, a bimodal localization, nuclear and lysosomal, is observed. But the percentage of the drug which is stored in the nuclei is different according to the drug. With some derivatives which have a high therapeutic efficiency, 82–87 % of the drug is recovered in the nuclei. In a second group whatever the biological activity of the drug only 49–52 % of the drug accumulates in the nuclei.

Isolation of developmentally regulated genes in Drosophila melanogaster

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia‐like B104 repetitive sequence described by Scherr et al. [29]. We also isolated 2 maternally‐expressed genes, not previously identified in either classical genetic or similarly molecular‐based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.

Expression in the central nervous system of a subset of the yema maternally acting genes during Drosophila embryogenesis. Post-embryonic expression extends to imaginal discs and spermatocytes

The yema gene region of Drosophila melanogaster is a cluster of maternally acting genes isolated in differential screens. At least ten transcripts are encoded by the yema gene region; most of them are produced by independent transcription units (eight different transcription units). Using RNA dot-blot analysis and in situ hybridization to tissue sections, we have realized a comprehensive survey of the temporal and spatial expression of the yema transcripts. All these transcripts are maternally expressed. Five of them display a strict maternal expression. They are found exclusively in the female germ line (nurse cells and oocyte). These transcripts are still present in the embryo as maternal information. However, a subset of the yema genes also shows an embryonic and a post-embryonic expression. Interestingly, this expression is essentially restricted to the central nervous system (CNS) throughout the fly development, to the larval and pupal imaginal discs and to a subset of cells in the male gonad, the spermatocytes. Strikingly, these expression sites mainly contain proliferating and/or differentiating cells.

Deoxyribonucleic acid of Cancer pagurus: IV. Elution behaviour on hydroxyapatite chromatographic column

Fractionation of native DNA on hydroxyapatite columns depends, when flat and continuous gradients are used, on the base composition, GC-rich fractions being eluted in the first fractions. Crab satellite DNA behaves abnormally : the first eluted fractions are enriched in poly d(A-T).d(A-T) instead of GC as usual. It amy be suggested that these differences in the behaviour could be attributed to the fact that the secondary structure of crab DNA satellite is different from the secondary structure of the main DNA component.