Detailed knowledge of the mechanisms by which chemical carcinogena induce mutations in mammalian cells is a prerequisite for understanding the initiating events of carcino-genesis. As an approach to elucidating the molecular basis for carcinogen-induced mutations, recombinant DNA shuttle vectors were developed for analysis of mutagenesis in human lymphoblastoid cells. These shuttle vectors, which contain the oriP element from Epstein-Barr virus, a bacterial plasmid replicon, and the herpes simplex virus type 1 thymidine kinase (HSV-tK) gene, replicate as plasmids and are stably maintained in human cells. Mutations in the target HSV-tK gene are fixed by replication of the vector in human cells and mutant plasmids are selected after transformation of E. coli. this approach allows the facile molecular characterization of carcinogen- induced and spontaneous mutations and the determination of the roles in mutagenesis of functional characteristics of the target gene and the host cell genetic background. These oriP-tk shuttle vectors will be used to address four specific questions related to mutagenesis in human cells. 1) Does the spectrum of mutations induced by aralkylated bases in DNA depend on the structure of the DNA adduct? The efficiencies and specificities for mutagenesis in human cells will be compared for N-hydroxy-2-amino-fluorene, N-hydroxy-4- aminobiphenyl, N-hydroxy-N'-acetylbenzidine and N-benzoyloxy-N-methyl-4- aminoazobenzene. 2) Does transcriptional expression of the target gene influence the frequency, distribution, or specificity of chemically- induced mutations? Mutagenesis by N-ethyl-N-nitrosourea and N-hydroxy-2- aminofluorene will be studied using plasmids which result in a high level of HSV-tk gene expression or no detectable transcription in human cells. The relative importance of DNA repair, differential DNA modification, and interaction between the replication and transcription complexes will be determined. 30 Are mutations differentially fixed by leading or lagging strand DNA synthesis during DNA replication? The strand bias for chemically-induced mutations will be compared for plasmid vectors in which the direction of replication fork migration is either 5'3' or 3'5' through the HSV-tk gene. 4) Do the frequencies of specificities of spontaneous mutations induced in cells derived from cancer-prone Bloom's syndrome or ataxia telangiectasia patients differ from those induced in normal cells? Clones of plasmid-bearing cells of these three genotypes will be isolated and propagated in culture for analysis of the rate and specificity of spontaneous mutagenesis.
