Our long-term goal is to understand in detail the molecular mechanisms involved in cell transformation by the human adenoviruses. To accomplish this objective, We propose to identify and analyze cellular genes that regulate the transformed phenotype and to study mechanisms of interaction between the Ad T antigens and cellular and viral macromolecules.
Our specific aims are the following: 1. To identify cellular genes that may be positively or negatively regulated in Ad-transformed cells. We will construct and analyze extensive random and hybridization-subtracted cDNA libraries in AGT10 from parallel Ad-transformed and parental non-transformed cells and screen cDNA libraries with a sensitivity to detect expression of single-copy genes (1 mRNA in 100,000). 2. To prove that the modified expression of specific cellular genes is a function of E1A or E1B T antigen synthesis. We will generate and analyze stable cell lines containing individual Ad E1A and E1B genes regulated by inducible promoters. 3. To study structure and function of highly purified, in vitro phosphorylated, E. coli-produced Ad12 E1A 266R (13S mRNA) and 235R (12S mRNA) T antigens. We will determine whether phosphorylated T antigen binds to specific DNA sequences and regulates transcription in vitro. 4. To identify and isolate putative cellular (and viral) proteins and nucleic acids that associate with E1A T antigens within cells and that bind to purified phosphorylated Ad12 E1A antigens in vitro. 5. To determine the domains of the E1A T antigens that are responsible for the immortalization and partial transformation functions. We will study Ad E1A single base-pair mutants that saturate specific domains of E1A T antigens. 6. To identify and study known and theoretical adenovirus proteins. We will synthesize antipeptide antibodies targeted to putative URF 10 and URF 11 proteins encoded in Ad2 E1 1-strand, putative 6.1K protein encoded by the Ad2 E1A-9S mRNA, early Ad2 L1 52K/55K protein(s), putative Ad2 angnoprotein encoded at 21-22.5 mu and putative Ad2 protease at 60.0-61.7. We will use antipeptide antibodies to analyze the corresponding viral proteins and to study mutants in URF 10, URF 11, and L1 52K/55K.
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