The molecular mechanisms that regulate the expression of the herpes simplex virus (HSV) thymidine kinase (tk) gene will be studied. Two general methods will be used to approach this problem. First, the DNA sequences that serve as transcriptional control signals will be identified by the construction and assay of mutated tk genes. Mutation-bearing genes will be generated by four different methods of in vitro mutagenesis: (i) oliogonucleotide-directed mutagenesis; (ii) sodium bisulfite mutagenesis; (iii) deletion and insertion mutagenesis; and (iv) linker scanning mutagenesis. Individual mutants will be characterized by direct DNA sequencing and then assayed for their expression phenotype in environments that allow only constituitive transcription, or that reconstitute regulated transcription. The two assays that are used to monitor constituitive transcription are the mouse L-cell transformation procedure and the amphibian oocyte microinjection procedure. The assay that will be used to monitor the regulated transcription of the HSV tk gene relies on homologous in vivo recombination of mutated tk genes into the intact HSV chromosome. The second approach to the problem of transcriptional control of the HSV tk gene will involve the identification and isolation of cellular and viral transcription factors that mediate the constituitive and regulated expression of the gene. Cellular transcription factors of the amphibian oocyte nucleus will be identified by a DNA footprinting assay. Viral transcription factors will be purified from virally-infected tissue culture cells and used to supplement both the amphibian oocyte transcription assay and an in vitro transcription assay in order to reconstitute proper transcriptional regulation. A detailed description of the mechanisms that underlie the expression of this model eukaryotic gene will promote studies of more complex cellular genes. A resolution to the problem of transcriptional control in eukaryotic cells will serve a key role in our understanding of the basis for human genetic disorders and cancer. Moreover, a detailed description of the mechanism of transcriptional regulation of the HSV tk gene will help to provide a better understanding of the life cycle of the virus. This, in turn, may lead to the discovery of new and better treatment schemes for HSV-infected patients, and to the establishment of effective measures for preventing the spread of herpes simplex virus.
