The three proposed specific aims described by Dr. Gorodetsky are as follows: 1) Determination of the level of expression of c/EBP family members during mammary gland development. Comparative analysis of the effect of c/EBP overexpression on the activity of the promoter region of rat and bovine beta- and kappa-casein genes in the mammary epithelial cell line HC11. 2) Identification of sequences responsible for tissue-specific expression of bovine kappa-casein genes using transgenic mice and transgenic embryo models. 3) Isolation of overlapping clones presenting a full sequence of the cluster of bovine casein genes and restriction mapping. These studies complement the ongoing research in Dr. Rosen's laboratory as follows: 1) Dr. Rosen's laboratory is focusing primarily on the characterization of other transcription factors, specifically the MGF and R/IRE factors, interacting with the beta-casein promoter involved in the hormonal and tissue-specific expression of the casein genes. Information about the c/EBP family members and their role in mammary gland differentiation will be required in order to understand the complex interactions of multiple factors interacting in the beta-casein gene promoter. 2) The lack of conservation of the kappa-casein gene promoter and its different evolutionary history from the other casein genes suggests that either different mechanisms may be regulating the expression of this gene or that the entire casein gene locus may be controlled by a locus control region. Once the factors regulating the beta-casein promoter are characterized it will be of interest to determine their effects of kappa-casein promoter activity. 3) The physical linkage and detailed map of the casein gene locus has not yet been established. The characterization of overlapping bovine cosmid clones containing the individual casein genes will complement similar rat casein gene locus mapping experiments underway in Dr. Rosen's laboratory. These studies are a necessary prerequisite to the identification of putative LCRs that may be critical determinants of gene activity within the casein gene locus. Cross-species hybridization experiments may help identify putative LCRs as well as facilitate gene walking experiments by eliminating the problems of species specific repetitive sequences. Identification of casein LCRs and the proteins interacting with these sequences will provide insights into the molecular mechanisms of mammary gland differentiation.
