We propose to isolate and characterize the human gene coding for thymidine kinase (tk). Thymidine kinase gene is important because it plays an important role in the pyrimidine salvage pathway. It is a single copy gene expressed by all growing cells and it is regulated in a cell cycle dependent fashion. The tk gene is also unique in that it is inducible by several DNA viruses. It responds to adenovirus 12(Ad12) infection by high inducibility, chromosome "uncoiling" and chromatid and chromosome breaks. Human tk gene will be transferred to mouse L-cells by DNA mediated gene transfer. After 2-3 rounds of successive transfers, the DNA from the mouse cells containing the gene for human tk will be used to construct a genomic library using bacteriophage or cosmid vectors. The library will be screened with a human repeated DNA (Alu family) clone. DNA from positive clones will be tested for tk activity. The cloned DNA will be used to isolate the corresponding mRNA and to construct a cDNA clone. The cDNA clone will be subjected to complete nucleic acid sequencing. Deletions in the 5' and 3' regions of the gene will be induced in vitro and each of the deletion mutants tested for its ability to direct tk synthesis and inducibility in an in vivo system. The DNA will also be used to examine the molecular basis of inducibility. The purified DNA will be used to understand the basis for the Ad12-human cell interactions by studying the chromatin structure of the tk gene and surrounding regions when the gene is inactive, active, induced and hyperinduced by Ad12. Sensitivity to micrococcal nuclease and DNAase I will be used as the assays. All these studies are expected to provide information about the structure and function of an important, ubiquitously expressed, inducible single-copy gene.