A repetitive probe for FISH analysis of bovine interphase nuclei

The purpose of this study was to generate repetitive DNA sequence probes for the analysis of interphase nuclei by fluorescent in situ hybridisation (FISH). Such probes are useful for the diagnosis of chromosomal abnormalities in bovine preimplanted embryos. Of the seven probes (E1A, E4A, Ba, H1A, W18, W22, W5) that were generated and partially sequenced, five corresponded to previously described Bos taurus repetitive DNA (E1A, E4A, Ba, W18, W5), one probe (W22) shared no homology with other DNA sequences and one (H1A) displayed a significant homology with Rattus norvegicus mRNA for secretin receptor transmembrane domain 3. Fluorescent in situ hybridisation was performed on metaphase bovine fibroblast cells and showed that five of the seven probes hybridised most centromeres (E1A, E4A, Ba, W18, W22), one labelled the arms of all chromosomes (W5) and the H1A probe was specific to three chromosomes (ch14, ch20, and ch25). Moreover, FISH with H1A resulted in interpretable signals on interphase nuclei in 88% of the cases, while the other probes yielded only dispersed overlapping signals

Non-coding RNAs: potential therapeutic use

Les ARNs non codants constituent une classe d’acides nucléiques de plus en plus étudiée. D’abord pensés comme étant une catégorie anecdotique de produits de la transcription, il apparait clairement, que sous la forme de petites molécules (micro ARNs -ou miARNs-, siARNs et piARNs) ou de molécules beaucoup plus longues (long-non coding RNA ou lncRNAs), ils jouent un rôle crucial dans la régulation épigénétique de l’expression des gènes, dans le contexte général de l’interférence à ARN. Des molécules synthétiques mimant leur action peuvent aisément être produites (siARNs ou mimes de miARNs) et vont être capable d’inhiber l’expression des gènes (voire de miARNs naturels). L’intérêt thérapeutique de leur utilisation est donc évident dans quasiment tous les domaines de la médecine. Cette utilisation nécessite tout de même de détruire plusieurs verrous techniques ou technologiques, ce qui est clairement en passe de se produire.Non-coding RNAs are a class of nucleic acids increasingly studied. First conceived as an anecdotal product category of the genome transcription, it appears clear that under the form of small molecules (microRNAs -or miRNAs-, siRNAs, piRNAs) or much longer molecules (long non coding RNA or lncRNAs), they play a crucial role in the epigenetic regulation of gene expression, in the context of RNA interference. Synthetic molecules mimicking their action can be easily produced (siRNAs or miRNA mimics) and will be capable of inhibiting the expression of genes (or natural miRNAs). The therapeutic value of their use is evident in virtually all areas of medicine. This use still requires destroying several technical or technological obstacles, which is clearly going to happen

Mammals, sex, genes and genome : lessons from the intersex goat model

In goats, the PIS (polled intersex syndrome) mutation is responsible for the absence of horns in both males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation.Chez la chèvre domestique, la mutation PIS (polled intersex syndrome) entraîne à la fois une absence de cornes dans les deux sexes, et une inversion sexuelle touchant exclusivement les individus XX. Le mode de transmission est dominant pour les cornes et récessif pour l'inversion sexuelle. Chez les mutants XX PIS-/-, l'expression de gènes, spécifiques du testicule est observée de façon très précoce durant le développement gonadique. Par clonage positionnel, nous avons démontré que la mutation PIS consiste en la délétion d'un élément d'ADN de 11,7 kilobases, élément régulateur affectant l'expression de deux gènes, PISRT1 et FOXL2, qui agiraient de façon synergique pour déterminer le sexe femelle et promouvoir la différenciation ovarienne. L'extinction transcriptionnelle de ces deux gènes conduit à la formation très précoce de testicules chez les femelles génétiques mutantes (XX PIS-/-). En accord avec le profil d'expression et les données de la littérature, nous proposons l'hypothèse selon laquelle FOXL2 serait un gène-clef de la différenciation ovarienne et que l'ARN non codant PISRT1 pourrait agir comme un facteur répresseur de SOX9, gène-clef de la cascade de détermination mâle. Dans cette hypothèse, SRY, le facteur de détermination testiculaire, inhiberait ces deux gènes dans les gonades XY, pour permettre la différenciation testiculaire

Trophoblasts, invasion, and microRNA.

International audienceMicroRNAs (miRNAs) have recently become essential actors in various fields of physiology and medicine, especially as easily accessible circulating biomarkers, or as modulators of cell differentiation. To this respect, terminal differentiation of trophoblasts (the characteristic cells of the placenta in Therian mammals) into syncytiotrophoblast, villous trophoblast, or extravillous trophoblast constitutes a good example of such a choice, where miRNAs have recently been shown to play an important role. The aim of this review is to provide a snapshot of what is known today in placentation mechanisms that are mediated by miRNA, under the angles of materno-fetal immune dialog regulation, trophoblast differentiation, and angiogenesis at the materno-fetal interface. Also, two aspects of regulation of these issues will be highlighted: the part played by oxygen concentration and the specific function of imprinted genes in the developing placenta

A genetic basis for a postmeiotic X versus Y chromosome intragenomic conflict in the mouse.

Intragenomic conflicts arise when a genetic element favours its own transmission to the detriment of others. Conflicts over sex chromosome transmission are expected to have influenced genome structure, gene regulation, and speciation. In the mouse, the existence of an intragenomic conflict between X- and Y-linked multicopy genes has long been suggested but never demonstrated. The Y-encoded multicopy gene Sly has been shown to have a predominant role in the epigenetic repression of post meiotic sex chromatin (PMSC) and, as such, represses X and Y genes, among which are its X-linked homologs Slx and Slxl1. Here, we produced mice that are deficient for both Sly and Slx/Slxl1 and observed that Slx/Slxl1 has an opposite role to that of Sly, in that it stimulates XY gene expression in spermatids. Slx/Slxl1 deficiency rescues the sperm differentiation defects and near sterility caused by Sly deficiency and vice versa. Slx/Slxl1 deficiency also causes a sex ratio distortion towards the production of male offspring that is corrected by Sly deficiency. All in all, our data show that Slx/Slxl1 and Sly have antagonistic effects during sperm differentiation and are involved in a postmeiotic intragenomic conflict that causes segregation distortion and male sterility. This is undoubtedly what drove the massive gene amplification on the mouse X and Y chromosomes. It may also be at the basis of cases of F1 male hybrid sterility where the balance between Slx/Slxl1 and Sly copy number, and therefore expression, is disrupted. To the best of our knowledge, our work is the first demonstration of a competition occurring between X and Y related genes in mammals. It also provides a biological basis for the concept that intragenomic conflict is an important evolutionary force which impacts on gene expression, genome structure, and speciation

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation

In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4–5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation

Gene expression regulation in the context of mouse interspecific mosaic genomes

The testis transcriptome of mouse strains containing homozygous segments of Mus spretus origin in a Mus musculus background was analyzed

Cytogenetical anchoring of sheep linkage map and syntenic groups using a sheep BAC library

In order to simultaneously integrate linkage and syntenic groups to the ovine chromosomal map, a sheep bacterial artificial chromosome (BAC) library was screened with previously assigned microsatellites using a sheep-hamster hybrid panel and genetic linkage. Thirty-three BACs were obtained, fluorescently labelled and hybridised on sheep-goat hybrid metaphases (2n = 57). This study allowed us, (i), to anchor all linkage groups on sheep chromosomes, (ii), to give information on the probable position of the centromere on the linkage map for the centromeric chromosomes, (iii), to contradict the previous orientation of the ovine × linkage group by the mapping of BMS1008 on OARXq38. Concerning our somatic cell hybrid panel, this study resulted in the assignment of all the previously unassigned groups to ovine chromosomes and a complete characterisation of the hybrid panel. In addition, since hybridisations were performed on a sheep-goat hybrid, new marker/anchoring points were added to the caprine cytogenetic map

Isolation of subtelomeric DNA sequences labelling sheep and goat chromosome ends

Two techniques that make it possible to isolate telomere DNA are presented, using sheep as an example. The first technique is based upon the screening of a sheep BAC library with PCR amplified DNA segments preserved from high-power laser beam irradiation. Twenty-three BACs hybridising to 13 subtelomeric regions in sheep and goats were obtained (out of 27 in the sheep complement), of which 13 recognised more than one region, telomeric or not. Twenty-three microsatellites were isolated from these BACs and 22 were genetically mapped on the sheep international genetic map, always consistently with the cytogenetical localisation in 17 cases out of 22. These results are discussed. The second technique is based upon the selective cloning of subtelomeric enriched DNA. Preliminary results were obtained by this approach