Analysis at the molecular level of carcinogen-induced mutagenesis is important for elucidating the mechanism of initiation of carcinogenesis. Two approaches will be developed for the molecular analysis of mutations induced in mammalian cells by chemical carcinogens. Both approaches are based on the use of a recombinant DNA shuttle vector composed of the SV40 early region, sequences derived from the bacterial plasmid PBR322, and the thymidine kinase gene of Herpes simplex virus (HSV-tk). In the first model system, mutations in the HSV-tk gene will be fixed by replication of the vector as a plasmid in monkey or human cells. In the second model system, the vector will be used to transform human cells to yield multiple tandemly duplicated copies of the vector integrated in host chromosomes. The transformed cells will be treated with a mutagen and vector DNA will be isolated by restriction enzyme digestion of cellular DNA. For both approaches, the isolated vector DNA will be used to transform E. Coli for selection of DNA melecules bearing mutant HSV-tk genes. The induced mutations will be mapped by recombinational analysis and characterized by DNA sequence analysis. The long-term goals of this project are to determine the types of DNA sequence changes induced in mammalian cells by chemical carcinogens, to characterize host processes that determine the frequency or types of mutations induced by chemical carcinogens, and to identify the primary determinants of sequence specificity in mammalina mutagenesis. Three specific questions are posed in this grant application: (1) Is mutagenesis modified by expression of the target gene? (2) Are the types of mutations induced in cells from certain genetically cancer-prone individuals different from those induced in wild-type human cells? (3) How is mutagenesis influenced by chromatin structure? In addressing these questions, the types of mutations induced by several carcinogens will be studied. These include ethylnitrosourea, N-acetoxy-2-acetylaminofluorene, and (plus and minus)7Beta,8Alpha-dihydroxy-9Alpha, 10Alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.
