The experiments described in this proposal are intended to elucidate changes in the structure and expression of endogenous oncogenes during the process of transformation of cultured human epidermal keratinocytes by the oncogenic virus, SV40. The proposed studies are to be carried out in three parts: 1. In the initial phase we will continue ongoing experiments to screen for oncogenes which appear to be activated after SV40 infection by two methods: a) RNA and DNA blot hybridization analyses of oncogene sequences in viral transformants; cloned oncogenes will be used as probes, b) focus formation assays in NIH 3T3 cells transfected with genomic DNAs from viral transformants. Cells derived from isolated foci will be screened for the presence of transfected, presumably activated, oncogenes by DNA blot hybridization analyses. 2. We will test the effect of various exogenous oncogenes on phenotype expression in keratinocytes by transfection of a number of cloned retroviruses and oncogenes into cultured keratinocytes. We will utilize various phenotypic properties developed for SV40 including expression of keratin proteins as markers. 3. Using the lambda bacteriophage system, we will clone oncogene sequences showing structural alterations of interest in order to carry out more detailed analyses. We can begin this phase by cloning myc sequences for which we now know interesting structural alterations related to SV40 exist.
