The central aims of the proposed research are to understand the role in the process of mammalian embryonic development and to characterize the regulation of expression of two placental proteins in the prolactin - growth hormone family. The expression and action of these two proteins, proliferin and proliferin-related protein, will be studied in the mouse and in mouse cell cultures. The effect of proliferation (which was originally identified as a serum-inducible mRNA and protein in cultured mouse fibroblasts) on the differentiation of myogenic and other mouse cell lines, and on the growth of primary embryo cultures, will be investigated using transient DNA transfections and stable DNA transformations, and by administration of purified protein. Mutant proliferin proteins and chimaeric proteins combining proliferin and prolactin or placental lactogen (another member of the prolactin family) domains will be generated and tested for activity in differentiation growth, and for the ability to interact with specific cell surface receptors. Placental cultures will be assayed for the synthesis and secretion of proliferin-related protein, and this protein will be purified. The structures of the carbohydrate moieties on proliferin and proliferin-related protein will be compared by digestion with various glycosidases, and the relative activities of the different modified forms will be examined. Levels of proliferin-related protein during pregnancy will be determined by Western blotting and radioimmunoassays, and receptor binding assays will be performed with purified proliferin-related protein and membranes from various tissues isolated during pregnancy. A genomic mouse DNA library will be constructed and screened for the proliferin-related protein gene. Expression of proliferin and proliferin-related protein genes will be followed in transfected mouse cells and using an in vitro transcription system. The activity of these genes in transcription extracts generated from placenta and other tissues will be compared, and he regions of the promoters essential for tissue- specific expression will be mapped by mutagenesis. These studies may contribute to a more complete description of hormonal effects on embryonic development, and in particular on the regulation of cell growth and differentiation. Characterization of the placental-specific expression of proliferin and proliferin- related protein, and the serum-inducible expression of proliferin in cultured mouse cells, may also reveal novel aspects in the regulation of gene expression.
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