The function of intermediate filaments composed of Endo A and B proteins will be investigated in differentiating cultures of murine embryonal carcinoma cells and mouse embryos. Plasmid DNAs designed to expresss an RNA complementary to the 5' end of Endo B mRNA will be introduced into embryonal carcinoma cells. The effect of the expression of the antisense RNA upon intermediate filament formation and visceral endoderm epithelium maturation will be assessed by immunofluorescence, immunoprecipitation, and nucleic acid hybridization techniques. The gene coding for Endo B will be cloned and characterized. DNase-hypersensitive sites of the chromatin state of the gene will be mapped and compared in embryonal carcinoma cells and their differentiated derivatives. Proteins which bind to specific sequences of the Endo B gene will be identified by DNA binding assays and chromatin footprinting experiments. Finally, the regulatory sequences of the Endo B gene will be identified by deletion analyses of recombinants designed to express the chloramphenicol acetyl transferase indicator gene. Genomic sequences will be tested for tissue-specific transcriptional enhancer activity.