To understand cellular events and biochemical mechanisms involved in cell transformation, we will focus on a single viral protein, the human adenovirus (Ad) ElA 243R oncoprotein. We will study (i) the collective functions of 243R in modulating (activating or repressing) expression of cellular genes that may be involved in oncogene cooperation and cell immortalization and (ii) the individual biochemical functions of 243R in transcriptional repression and the induction of cellular DNA synthesis. The hypothesis that ElA 243R modulates expression of critical cellular genes will be investigated. It will be determined whether expression of subsets of """"""""primary response"""""""" genes (rapidly activated when quiescent cells are induced to proliferate) and """"""""late response"""""""" genes are induced by introduction of EIA 243R into quiescent cells. To identify previously unrecognized cellular genes regulated by ElA 243R, hybridization enriched cDNA libraries from mRNA populations expressed early after ElA introduction into quiescent cells will be analyzed. To determine whether cellular genes modulated by ElA are """"""""primal genes"""""""" whose overexpression is essential and sufficient for ElA oncogene cooperation and/or immortalization, cDNA clones under control of a strong constitutive promoter will be assayed in baby rat kidney cells. To study ElA 243R transcriptional repression and cellular DNA induction functions, essential ElA domains will be clarified and attempts made to synthesize biologically active, minimal domain ElA peptides. It will be determined whether these minimal ElA peptides induce a subset of cellular genes modulated by native ElA 243R. To distinguish between models of ElA repression, a novel cell microinjection assay will be used to determine whether de novo protein synthesis is required for repression. To provide functional assays for cellular proteins that associate with ElA 243R functional domains and that may play a role in repression or DNA induction, attempts will be made to develop in vitro repression and cell complementation assays.
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