The goal of the proposed research is to understand the molecular mechanisms responsible for the regulation of thymidine kinase during the cell cycle of actively proliferating cells. Our previous work has shown that the level of the 24 kd thymidine kinase polypeptide is controlled at two levels. (1) The efficiency of translation of thymidine kinase mRNA increases significantly during S phase resulting in a large accumulation of the enzyme, and (2) the accumulated thymidine kinase protein is rapidly and specifically degraded at cytokinesis. We propose a series of genetic and biochemical experiments aimed at defining the molecular details of both of these regulatory mechanisms. By constructing a series of mutants with lesions in the thymidine kinase cDNA, we will identify sequence elements that are required in cis for the modulations of translational efficiency and protein stability during the cell cycle. We will identify the trans- acting factors involved in the two regulatory pathways by analyzing in vitro systems derived from cells at specific states of the cell cycle.
