The developmental regulation of two recently described cytoskeletal proteins (endo A and endo B), which are found in parietal endodermal cell lines but not in a variety of mesodermal cell lines or keratinocytes, will be studied in preimplantation mouse embryos, parietal endodermal cell lines, and cell lines derived from murine teratocarcinomas. The cytoskeletal proteins of blastocyst stage mouse embryos will be compared to the endo A and B cytoskeletal proteins by immunofluoresence, immunoprecipitation, and two-dimensional gel electrophoresis in order to determine if these proteins are associated with trophectoderm, the first differentiated cell type to appear during development. Amino acid sequences of the N-terminal regions of cyanogen bromide fragments generated from purified endo B protein will be determined. Cell-free translation of the poly(A)+ RNAs of embryonal carcinoma (ec) cells, parietal endodermal cells, and of embryonal carcinoma cells induced to differentiate by exposure to retinoic acid will be used to define differences in the mRNA populations of ec and parietal endodermal cells. The time of appearance of translatable endo B mRNA will be compared to the time of appearance of the proteins in ec cells exposed to retinoic acid. The endo B mRNA will be purified from polyribosome preparations by immunochemical means, and the endo B cDNA will be cloned in the pBR322 recombinant plasmid and subcloned in M13 phage. The DNA sequence of the cloned gene will be determined. The cloned endo B cDNA will be used as a probe to determine the methylation state of certain endo B coding sequences in ec cells, retinoic acid-induced ec cells, parietal endodermal cells, fibroblasts, and myoblasts. The state of endo B sequences within nuclei of the same cell types will be compared by brief digestion of isolated nucleic with DNase I followed by restriction enzyme digestion of the DNA and Southern blot hybridization analysis. (M)
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