Steroid hormones function via specific receptors to exert dramatic effects on gene expression in virtually all animal cells. We propose to study the molecular biology of glucocorticoid action in well-defined tissue culture systems derived from mouse mammary carcinoma and rat hepatoma cells. The production of mouse mammary tumor virus (MMTV) RNA is stimulated by glucocorticoid hormones in the mammary cells and in MMTV-infected rat hepatoma cells. Using a variety of molecular techniques (including nucleic acid hybridization and RNA sequencing) we will study the biochemical processes by which viral RNA synthesis is stimulated; in particular, we will attempt to identify the promotor site(s) at which viral RNA synthesis is initiated. Transcription of viral genes will be studied in isolated nuclei and chromatin in order to eventually reconstruct hormone dependent transcription systems in vitro. Using immunological selection techniques, genetic mutants of the rat hepatoma cells will be developed in which one or more of the components involved in the steroid response is defective. These mutants would be used to identify the molecular components involved in hormone action and to serve as genetic controls in correlating biochemical observations with the events which occur in vivo. These approaches may serve not only to elucidate the molecular mechanisms of steroid hormone action but also to provide some further insights into eukaryotic gene regulation in general and the events leading to virally induced tumorigenesis.
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