The objective of this proposal is an understanding of the mechanisms which control expression of the human thymidine kinase (tk) gene during the cell cycle and the onset of cell proliferation. The ultimate goal is a better understanding of those factors that are involved in the commitment of a cell to replicate. Our studies will involve measurement of transcription rates with nuclear run off assays, measures of steady state tk mRNAs levels, TK enzyme activities and TK protein levels with different cell stimuli and synchronization methods. We will rely heavily on serum stimulations of quiescent cells as well as separation of specific phases of the cell cycle by centrifugal elutriation. We will also make recombinant DNA constructs with different portions of the gene and the promoter to determine those sequences which are most important to the different modes of regulation and determine whether they work at transcriptional or post-transcriptional levels. Specific experiments using serum depletion or butyrate G1 blocks are designed to test for changes in mRNA stability at different stages of the cell cycle. Further experiments will be carried out to characterize the promoter region, both with deletion and mutation experiments as well as protein binding experiments to identify cruical promoter and/or regulatory regions of the gene. We will also follow up methylation studies to determine whether methylation changes observed upstream of the gene in germ line cell also correlate with possible methylation changes associated with the transformation process.
