The aim of the present study was to understand how the extracellular matrix (ECM) regulates at the gene level the prolactin (Prl)-induced signal transducer and activator of transcription 5 (STAT5)-dependent expression of the alpha s1-casein gene in mammary epithelial cells. CCAAT enhancer binding proteins (C/EBPs) are assumed regulators of beta-casein gene expression. Rabbit primary mammary cells express alpha s1-casein gene when cultured on collagen and not on plastic. Similar C/EBPbeta, C/EBPdelta, STAT5, and Prl-activated STAT5 were found under all culture conditions. Thus the ECM does not act through C/EBPs or STAT5. This was confirmed by transfections of rabbit primary mammary cells by a construct sensitive to ovine prolactin (oPrl) and ECM (6i TK luc) encompassing STAT5 and C/EBP binding sites. The mutation of C/EBPs binding sites showed that these sites were not mandatory for Prl-induced expression of the construct. Interestingly, chromatin immunoprecipitation by the anti-acetylhistone H4 antibody (ChIP) showed that the ECM (and not Prl) maintained a high amount of histone H4 acetylation upstream of the alpha s1-casein gene especially at the level of a distal Prl- and ECM-sensitive enhancer. Alpha6 integrin (a membrane receptor of laminin, the principal active component of the mammary ECM) was found at the surface of cells cultured on collagen but not on plastic. In cells cultured on collagen in the presence of anti-alpha6 integrin antibody, Prl-induced transcription of the endogenous alpha s1-casein gene was significantly reduced, without modifying C/EBPs and STAT5. Besides, histone H4 acetylation was reduced. Thus, we propose that the ECM regulates rabbit alpha s1-casein protein expression by local modification of chromatin structure, independently of STAT5 and C/EBPs.

No abstract

-Effective selection on the PrP gene has been implemented since October 2001 in all French sheep breeds. After four years, the ARR ''resistant'' allele frequency increased by about 35% in young males. The aim of this study was to evaluate the impact of this strong selection on genetic variability. It is focussed on four French sheep breeds and based on the comparison of two groups of 94 animals within each breed: the first group of animals was born before the selection began, and the second, 3-4 years later. Genetic variability was assessed using genealogical and molecular data (29 microsatellite markers). The expected loss of genetic variability on the PrP gene was confirmed. Moreover, among the five markers located in the PrP region, only the three closest ones were affected. The evolution of the number of alleles, heterozygote deficiency within population, expected heterozygosity and the Reynolds distances agreed with the criteria from pedigree and pointed out that neutral genetic variability was not much affected. This trend depended on breed, i.e. on their initial states (population size, PrP frequencies) and on the selection strategies for improving scrapie resistance while carrying out selection for production traits.genetic variability / scrapie resistance / molecular marker / pedigree / sheep *

-Effective selection on the PrP gene has been implemented since October 2001 in all French sheep breeds. After four years, the ARR ''resistant'' allele frequency increased by about 35% in young males. The aim of this study was to evaluate the impact of this strong selection on genetic variability. It is focussed on four French sheep breeds and based on the comparison of two groups of 94 animals within each breed: the first group of animals was born before the selection began, and the second, 3-4 years later. Genetic variability was assessed using genealogical and molecular data (29 microsatellite markers). The expected loss of genetic variability on the PrP gene was confirmed. Moreover, among the five markers located in the PrP region, only the three closest ones were affected. The evolution of the number of alleles, heterozygote deficiency within population, expected heterozygosity and the Reynolds distances agreed with the criteria from pedigree and pointed out that neutral genetic variability was not much affected. This trend depended on breed, i.e. on their initial states (population size, PrP frequencies) and on the selection strategies for improving scrapie resistance while carrying out selection for production traits.genetic variability / scrapie resistance / molecular marker / pedigree / sheep *

Previous studies have shown that the 5'HS4 DNaseI hypersensitive site of the chicken beta-globin locus is endowed with classic insulator activities: (i) it blocks the interaction between promoter and enhancers when it is inserted between them (ii) it confers expression of integrated foreign genes independent of their position in the chromatin. The aim of this present work was to determine whether the 5'HS4 element was able to stimulate the expression level and/or to increase the expression frequency of a luc+ reporter gene controlled by the rabbit WAP gene promoter. Two constructs with 5'HS4 insulator (p5'HS4-WAPluc) or without (pWAPluc) were introduced in mouse fertilised oocytes. All transgenic lines containing the 5'HS4 element (six lines) expressed the transgene whereas only two out of eight lines harbouring the pWAP-luc construct expressed the transgene to a significant level. Moreover, the mean level of expression was seven times higher in p5'HS4WAP-luc lines than in pWAP-luc lines. Even all these benefits on transgene expression, the 5'HS4 element did not confer a copy-dependent expression, did not decrease the ectopic expression of the reporter gene and did not decrease the variability of expression. Thus, the 5'HS4 element does not have all the properties of a perfect insulator on a construct containing the luc+ reporter gene controlled by the rabbit WAP promoter.