DNA-SEQUENCE ANALYSIS OF HPRT MUTATIONS IN LYMPHOCYTES FROM SPRAGUE-DAWLEY ROTS TREATED WITH 7,12-DIMETHYLBENZ[A]ANTHRACENE

Treatment of female Sprague-Dawley rats with the potent mammary gland carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) results in the format ion of DNA adducts with dG and dA and in the induction of 6-thioguanin e-resistant (TG(r)) lymphocyte mutants. In this study, we have examine d the types of mutations induced in TG(r) lymphocytes from DMBA-treate d rats. DNA from 263 TG(r) lymphocyte clones was screened for mutation s in exons 2, 3, and 8 of the hprt gene by polymerase chain reaction ( PCR) amplification of the exons followed by heteroduplex analysis usin g denaturing gradient-gel electrophoresis. Twenty-five of the clones p roduced heteroduplexes in exon 2, 35 produced heteroduplexes in exon 3 , and 36 produced heteroduplexes in exon 8. Direct sequence analysis o f the heteroduplexes revealed 96 mutations, and at least 74 of these m utations were produced independently. Eighty-five of the total mutatio ns were simple bose pair (bp) substitutions, with A --> T and G --> T transversions being the predominant types. Seven mutations were deleti ons, three were complex bp substitutions, and one was an insertion. Th e results suggest that the types of mutations produced by DMBA in rat lymphocytes are specific to the DNA adducts produced by this compound. (C) 1996 Wiley-Liss, Inc.