Murine embryonal carcinoma (ED) cells, the malignant stem cells of teratocarcinomas, can grow as undifferentiated cells or can differentiate to form various types of benign somatic cells. This proposal will use genetic and molecular approaches to examine regulation of gene expression and DNA replication during the growth and differentiation of EC cells. Undifferentiated ED cells are refractory to infection by wild-type polyoma virus, the infection process being blocked at a stage after virus adsorption and penetration but before expression of viral early gene products. Differentiated cells derived from EC cells are usually permissive for infection by wild-type polyoma. Several host range mutants of polyoma virus that productively infect undifferentiated EC cells have been isolated and characterized. Mutants isolated on different EC cell lines show differences in their host range properties, suggesting that different host range mutants may provide probes that are specific for different cell types with respect to gene expression and DNA replication. To determine viral DNA sequences relevant to expression in EC cells, host range mutants of polyoma virus will be isolated on several different EC cell lines. Using immunofluorescence and infectious centers assays, the host range properties of these mutants among different cell lines will be examined. The genomic sequence alterations leading to altered host range properties will be studied by restriction analysis, marker rescue, and DNA sequencing. Mixed infection experiments will be performed to characterize the ability of these host range mutants to rescue replication of wild-type polyoma DNA in different EC cell lines. As a means of identifying possible regulatory compartments and/or proteins in EC cells, the specificity of interaction of wild-type and mutant polyoma DNAs with the nuclear matrix and with solubilized nuclear proteins will be tested by dot-blot hybridization and by protein blotting techniques.
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| Fujimura, F K (1988) Point mutation in the polyomavirus enhancer alters local DNA conformation. Nucleic Acids Res 16:1987-97 |
| Fujimura, F K (1986) Nuclear activity from F9 embryonal carcinoma cells binding specifically to the enhancers of wild-type polyoma virus and PyEC mutant DNAs. Nucleic Acids Res 14:2845-61 |
