The proposal describes approaches to define the spectrum of mutations induced two model chemical carcinogens in an endogenous mammalian gene at the DNA sequence level. The carcinogens we used were N-acetoxy-N-2-acetylaminofluorene (AAAF) and (plus/minus)-r- 7,t-8-dihydroxy-t-9, lO-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE). The target for mutation was the dominant autosomal dihydrofolate reductase (dhfr) gene of Chinese hamster ovary cells. DHFR- mutants are triple auxotrophs for glycine, thymidine, and hypoxanthine and were selected starting with a hemizygote cell line (dhfr+/delta). Characterization of the induced mutants by Southern blot analysis showed that in the case of AAAF, a significant number (28%) carried gene rearrangements (deletion, translocation, large insertion, or inversion). In the case of BPDE, the dhfr- mutants virtually all carried small lesions (base substitution, + or -1 frameshift mutation). We also examined the expression phenotype of the induced mutants by Northern analysis and RNA heteroduplex mapping. About 50% of the putative point mutants showed markedly reduced steady-state levels of poly(A)+ dhfr mRNA. By cloning and sequencing, we determined that two mutants displaying a low mRNA phenotype carried chain termination codons at different positions in the dhfr coding sequence. Our proposal describes plans for the further study of these mutants. We describe methods to facilitate the DNA sequence analysis of the putative point mutants. We intend to isolate dhfr- mutants induced with a related aromatic amine, N- hydroxy-AF in order to determine if the mutational spectra of AAAF is associated with the conformational changes in DNA structure induced by the dG-adduct. In collaboration, we will determine if chromosomal location influences the frequency and spectra of induced mutations. Finally, we will attempt to suppress the nonsense mutations by transfection of Su+ tRNA and selection for a DHFR+ phenotype. We will then evaluate the dhfr mRNA levels in these transfected cells.
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