The long-term objective of this project is a complete understanding of the modes of regulation of the thymidine kinase gene. This includes cell-cycle regulation, induction of normally quiescent cells by factors such as by small DNA tumor viruses, and eventually its mode of repression in terminally differentiated tissues such as heart and brain. There are a number of different health-related problems relevant to this. One is that DNA replication enzymes, including thymidine kinase, must be restimulated for abnormal growth of cells such as in tumors. Also, thymidine kinase regulation is an important part of normal differentiation. Lastly, in the long term an understanding of how this gene is turned off in terminally differentiated tissues may help to understand how to turn it and similar genes back on, to regenerate damaged tissues in heart or nervous tissue. The methodology will primarily center on the use of the cloned human cDNA and gene for thymidine kinase. Mutational studies will be used to delineate the crucial transcriptional regulation sequences. The regulatory sequences in both the normal cell cycle and in papovavirus-induced thymidine kinase activity will be compared. Following the creation of a thymidine kinase antisera, a complete analysis of RNA, protein, and enzyme levels will be carried out. This will help to define possible secondary controlling influences in this system. The complete primary structure of this gene and its cDNA will be determined and studied in the process of this work. (N)
