The proposal describes approaches to define the spectrum of lesions induced by mutagens in a mammalian gene at the DNA sequence level. Mutations occurring in an endogenous eukaryotic gene differ qualitatively. We will examine carcinogen-induced mutations in the Chinese hamster ovary locus encoding dihydrofolate reductase (DHFR). In this system, we can characterize point mutations, as well as chromosomal events (translocation, deletion, sequence duplication, and insertion). DHFR-deficient cells have been isolated that are either hemozygous (dhfr+/d) or deleted for the locus (dhfr(d/d)). DHFR- mutants are triple auxotrophs for glycine, thymidine, and hypoxanthine. Mutants will be selected starting with the hemizygote. The gene structure has been well characterized. Southern blot analysis of the induced dhfr-/d genomic DNAs will show changes arising from chromosomal events. Using dhfr-specific cosmid vectors, we will clone the locus from DHFR-cells carrying point mutations. We will map these mutations by marker rescue relying on in vivo or in vitro recombination of the dhfr- cosmid with dhfr+ fragments to correct the lesion. After transfection of the mutant and wild-type DNAs into dhfr(d/d) cells, prototrophic clones will be selected. Upon further mapping, we will sequence regions containing the mutations. Another approach for studying the nature of induced mutations in animal cells similar to the Ames assay is described. We will construct by site-directed mutagenesis 4 different minigenes (full length cDNA fused to the dhfr promoter). The changes to be introduced will create transition, transversion, +1, and -1 frameshift mutations. We will transfect the minigenes into dhfr(d/d) cells selecting for another marker (XGPT). These clones will then be mutagenized to induce reversion to DHFR+. If we can induce reversion, this system will be useful for screening the mutational potential of drugs and chemicals. Also, other parameters influencing the spontaneous and induced frequencies of reversion such as chromosomal location and DNA excision repair will be investigated.
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