A major difference between normal and transformed cells in culture is the ability of normal cells to limit their growth when the cells become confluent, while transformed cells continue to divide under these circumstances. Understanding the basis of this difference is of obvious importance to the study of oncogenic transformation. In such contact inhibited normal cells, the levels of many of the enzymes involved in DNA metabolism are very low. In growing cells, the levels of these eyzymes are linked to the cell cycle, being very high in S phase, and low at other times in the cell cycle. The goal of the research proposed in this application is to understand the molecular basis of this regulation, and the effect that infection with the virus SV40 has upon it. The specific gene that will be studied is the human thymidine kinase (Tk) gene. Thymidine kinase is an enzyme in the pyrimidine salvage pathway, and catalyzes the phosphorylation of thymidine to dTMP. The molecular basis of Tk regulation will be investigated using a cloned Tk gene and various techniques including in vitro mutagenesis, DNA transfections, and mRNA and enzyme analyses, in order to determine the human DNA sequences required for normal regulation, and the stage at which regulation occurs. When confluent cells are infected with the virus SV40, Tk and other enzyme levels are induced 2-20 fold. This induction will also be investigated using similar molecular techniques.
