Identification and analysis of single nucleotide polymorphisms in the myosin VA (MYO5A) gene and its exclusion as the causative gene of the dilute coat colour locus in rabbit

[EN] Classical genetic studies have identified different coat colour loci in rabbit and comparative analyses have established corresponding loci across species. In particular, the rabbit dilute locus is determined by a recessive coat colour mutation that modifies the basic colours influenced by the agouti and extension mutations. In mice, similar phenotypic effects are determined by a similarly named locus. This locus encodes the myosin VA (Myo5a) gene, whose protein product is an unconventional myosin that plays an essential role in melanosome transport in the melanocytes. We selected the same gene as a strong candidate for explaining the dilute coat colour in rabbit. To this end, 1399 bp were re-sequenced, spanning 4 exons out of 41 exons and a portion of intronic regions of the rabbit MYO5A gene to identify polymorphisms that could be useful to confirm or exclude this gene as causative of the rabbit dilute locus. Nine polymorphisms were identified, one of which was used to follow the segregation of the blue and black colours in a Checkered Giant F1 family. The single nucleotide polymorphism (SNP) analysed did not co-segregate with the two colours. These results excluded the MYO5A gene as determinant of the dilute locus in rabbit. The two alleles of this SNP were also present in several other breeds with different coat colours, further indicating that this marker is not associated with the dilute mutation in rabbits. Other candidates should be investigated to identify the causative gene of this locus in rabbit.We thank several rabbit breeders and Associazione Nazionale Coniglicoltori Italiani (ANCI) for their collaboration in the sampling of biological materials. This work was supported by RFO and FAGenomicH project funds from the University of Bologna.Fontanesi, L.; Scotti, E.; Dall'olio, S.; Oulmouden, A.; Russo, V. (2012). Identification and analysis of single nucleotide polymorphisms in the myosin VA (MYO5A) gene and its exclusion as the causative gene of the dilute coat colour locus in rabbit. World Rabbit Science. 20(1):35-41. https://doi.org/10.4995/wrs.2012.1033SWORD354120

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

A first genotyping assay of French cattle breeds based on a new allele of the extension gene encoding the melanocortin-1 receptor (Mc1r)

The seven transmembrane domain melanocortin-1 receptor (Mc1r) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. Upon the binding of agonist (melanocortin hormone, α-MSH) or antagonist (Agouti protein) ligands, the melanosomal synthesis of eumelanin and/or phaeomelanin pigments is stimulated or inhibited, respectively. Different alleles of the extension gene were cloned from unrelated animals belonging to French cattle breeds and sequenced. The wild type E allele was mainly present in Normande cattle, the dominant ED allele in animals with black color (i.e. Holstein), whereas the recessive e allele was identified in homozygous animals exhibiting a more or less strong red coat color (Blonde d'Aquitaine, Charolaise, Limousine and Salers). A new allele, named E1, was found in either homozygous (E1/E1) or heterozygous (E1/E) individuals in Aubrac and Gasconne breeds. This allele displayed a 4 amino acid duplication (12 nucleotides) located within the third cytoplasmic loop of the receptor, a region known to interact with G proteins. A first genotyping assay of the main French cattle breeds is described based on these four extension alleles

Coloration de la robe chez le bovin (études fonctionnelles des allèles du gène Silver/Pmel17 bovin et, contribution à l identification de gènes impliqués dans le changement post-natal de la coloration chez la race Gasconne)

Le facteur de dilution bien connu des éleveurs et qui caractérise la race bovine charolaise et dans une certaine mesure la race Simmental a été récemment identifié à l échelle moléculaire par l UMR1061. Il s agit de deux allèles Dc (dilution Charolaise) et Ds (dilution Simmental) qui affectent le peptide signal de la protéine Silver/Pmel17. Cette dernière joue un rôle crucial dans la biogenèse du mélanosome. Dans l objectif de comprendre le mécanisme de dilution associé à ces deux allèles nous avons entrepris une approche souris et une étude in vitro. La richesse en séquences répétées du gène Silver murin ne nous a pas permis d obtenir les souris transgéniques porteuses des mutations identifiées chez le bovin. En revanche, l approche cellulaire nous a permis de montrer un effet inhibiteur de la mutation Dc sur le clivage du peptide signal en N-terminale de la protéine Silver Charolaise. Par ailleurs, l approche transcriptomique que nous avons mené afin de comprendre le changement post-natal de la couleur de la robe de la race Gasconne révèle que ce dernier s accompagne d une surexpression de tous les gènes de coloration étudiés à l exception du gène Agouti. En effet l expression de ce dernier reste inchangée quelque soit la coloration de la robe.Recently, our team identified at the molecular level the dilution factor, well known to breeders, which characterized the Charolais cattle breed and to some extent the Simmental breed. In fact, the two alleles Dc (Charolais dilution) and Ds (Simmental dilution) alleles, are associated to coat colour dilution, and both affect the signal peptide of Silver/Pmel17 protein. This latter is crucial for melanosome biogenesis. With the aim to understand the mecanism of dilution due to these two alleles, we have undertaken a Gene-targeting in mouse approach and an in vitro study. Given the abundance of repeated sequences in the murine Silver gene, we couldn t obtain transgenic mice that harbour the same mutations as cattles. On the other hand, the cellular approach revealed an inhibitor effect of the Dc mutation on the cleavage of signal peptide sequence, located to the N-terminus of Silver protein. Moreover, the transcriptomic approach that we conducted to understand the post-natal change of coat colour in Gasconne breed, shows an overexpression of all the analysed genes, except for Agouti gene. Indeed, Agouti expression remains unchanged whatever the coat colour is.LIMOGES-BU Sciences (870852109) / SudocSudocFranceF

Génétique moléculaire de la coloration (découverte d'un allèle A(br) du gène Agouti responsable de la bringeure de la race bovine Normande)

L'objectif de ce projet de recherche est l'identification de marqueurs moléculaires dans les gènes de coloration qui pourraient être utilisés pour la traçabilité des produits d'origine bovine. Parmi les gènes de coloration, Agouti code pour une protéine antagoniste de la fixation de l'hormone a-MSH sur le récepteur aux mélanocortines 1 (Mc1r), codé par le gène Extension, et permet le " switch " de la synthèse d'eumélanine (noir/marron) vers la synthèse de pheomélanine (jaune/rouge), dans les mélanocytes de la peau. Nous démontrons dans ce travail que trois promoteurs alternatifs dirigent l'expression de transcrits, porteurs de régions 5'UTRs différentes, dans de nombreux tissus, ce qui suggère un rôle probable d'Agouti dans différentes fonctions physiologiques, en plus de la coloration chez le bovin. La race Normande, de génotype Extension sauvage (E+/E+), a un phénotype bringé. La bringeure est une présence variable de poils noirs sur un fond coloré rouge/brun. Le gène Agouti est surexprimé chez la Normande. Un élément de type LINE, inséré dans la région 5' génomique, provoque la surexpression de transcrits alternatifs. Le niveau de méthylation de l'élément LINE est responsable de la répression aléatoire du gène Agouti dans la peau Normande. Ce nouvel allèle Agouti (Abr) est présent à l'état homozygote dans la race Normande. Enfin, l'immunodétection dans la peau bovine a révélé une forme libre tétramérique de la protéine Agouti et une forme liée à une protéine de grande tailleLIMOGES-BU Sciences (870852109) / SudocSudocFranceF

Génétique de la coloration de la robe chez les bovins (contribution à l'étude du rôle des gènes de la famille tyrosinase)

These works belong to the research thematic related to the identification of specific molecular markers for each French cattle breeds. They consist in the study of the tyrosinase family genes and of the RAB38 gene.The cloning and the polymorphism study of the coding region of TYR, TYRP1, DCT, RAB38 genes show a very good conservation at the genomic and proteic levels with other species. No racial polymorphism correlated to a phenotype has been detected. However, the transcriptional study shows that highly variable expression levels could explain the coat color differences.Notably, a lack in TYRP1 expression seems to be characteristic of the e/e animals at the EXTENSION locus. These works also confirm the epistatic property of the SILVER allele specific to the Charolaise breedLIMOGES-BU Sciences (870852109) / SudocSudocFranceF

Etude de la régulation épigénétique de l'allèle Abr du gène Agouti chez la race bovine normande

Le travail décrit dans ce mémoire est une contribution apportée aux recherches axées sur les mécanismes épigénétiques. Plus particulièrement, ont été étudiées les mécanismes épigénétiques qui régissent l'expression du promoteur LINE porté par l'allèle Abr. La couleur de robe de la race bovine Normande se caractérise par un mélande de poils noirs et de poils rouges (bringeure) dont la proportion est variable entre les animaux et au niveau des différentes régions de la peau du même animal. Notre équipe a identifié l'allèle Abr du gène Agouti qui est responsable de ce paron de coloration complexe. Dans la première partie de ce travail nous démontrons que le promoteur du LINE possède une activité transcriptionnelle en utilisant différents gènes rapporteurs. Par ailleurs nous avons démontré que les sites d'initiation de la transcription sont influencés par les séquences adjacentes au promoteur. Dans la deuxième partie nous montrons que la méthylation du promoteur du LINE est l'une des facettes du processus épigénétique qui régule son expression variable entre les animaux et au sein du même animal. En effet, nous avons montré que l'expression variable du gène Agouti est proportionnelle au degré de méthylation du promoteur du LINE. Par ailleurs, nous montrons que cette régulation épigénétique dépend du fond génétique des animaux à l'instar des données acquises pour un allèle similaire chez la souris.The work describeb in this manuscript is a contribution to the comprehension of the epigenetic mechanisms that control the variable expression ot the LINE promoter of the Abr allele. The coat color of the Normande cattle breed is characterized by a mixture of black and red hairs (brindle) whose proportion varies between animals and a different regions of the skin of the same animal. Our team has identified the Abr allele Agouti gene which is responsible for this coat color pattern. In the first part of this work we demonstrate, using different reporter genes, that the LINE promoter has a transcriptional activity. By RACE experiments wa have been able to determine the initiation sites of transcription which are influenced by adjacent sequences of the promoter. In the second part we show the methylation of the LINE promoter is one facet of epigenetic process that regulates its variable expression between animals and within the same animal. Indeed we have shown the the variable expression of Agouti gene is proportionnal to the degree of promoter methylation of the LINE. Furthermore, we show that this epigenic regulation depends on the genetic background of animals as reported for the allele of Agouti homologous in mice.LIMOGES-BU Sciences (870852109) / SudocSudocFranceF

The untranslated side of hair and skin mammalian pigmentation : beyond coding sequences

For several decades, tremendous advances in studying skin and hair pigmentation of mammals have been made using Mendelian genetics principles. A number of loci and their associated traits have been extensively examined, crossings performed, and phenotypes well documented. Continuously improving PCR techniques allowed the molecular cloning and sequencing of the first pigmentation genes at the end of the 20th century, a period followed by an intense effort to detect and describe polymorphisms in the coding regions and correlate allelic combinations with the observed melanogenic phenotypes. However, a number of phenotypes and biological events could not be elucidated solely by analysis of the coding regions of genes. Messenger RNA isolation, characterization and quantification techniques allowed groups to move ahead and investigate molecular mechanisms whose secrets lay within the noncoding regions of pigmentation genes transcripts such as MC1R, ASIP, or W. The untranslated elements contain specific nucleotidic sequences and structures that dramatically influence the mRNA half-life and processing thus impacting protein translation and melanin production. As we are progressively uncovering the complex processes regulating melanocyte biology, unraveling complete mRNA structures and understanding mechanisms beyond coding regions has become critical