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• W2051796733 abstract "We have developed an approach for determining mutational spectra in exon 3 of the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene in splenic T-lymphocytes of B6C3F1 mice. Hprt- mutants from treated animals were isolated by culturing splenic T-cells in microtiter dishes containing medium supplemented with IL-2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking the exon 3 region of hprt. Identification of samples containing mutant exon 3 sequences and purification of mutant DNA from contaminating wild-type hprt DNA was accomplished using denaturing gradient gel electrophoresis. Purified mutant sequences were then sequenced. This approach is being used to study the sequence specificity of ethylene oxide (ETO). 12-day-old mice were given single i.p. injections of 100 mg ETO/kg every other day or 30, 60, 90 or 120 mg ETO/kg daily for 5 days to achieve different cumulative doses of this compound. In mice exposed every other day, cumulative doses of 200, 600 and 900 mg ETO/kg produced average mutant frequencies of 15 +/- 12.8, 45 +/- 13.2, and 73 (70, 75) x 10(-6), respectively, 8 weeks after the first treatment. In mice exposed daily, cumulative doses of 150, 300, 450 and 600 mg ETO/kg produced average mutation frequencies of 4.2 +/- 10.4, 8.2 +/- 10.4, 11.1 +/- 1.0 and 15.5 +/- 10.7 x 10(-6), respectively, 20 weeks after the first treatment. The mutant fraction in control mice was less than 3 x 10(-6). 123 hprt- mutants from mice exposed to 600 or 900 mg ETO/kg were isolated and analyzed for mutations in exon 3. 18 were located in exon 3 (14.6%). DNA sequencing revealed that 11/18 mutations were base-pair substitutions at 8 different sites in exon 3. Four AT transversions, three AT transitions, two GC transversions, and two GC transitions were observed. Three of the substitutions (2 AT-->CG, 1 AT-->GC) occurred at one base (203) in a single animal. The remaining 7 mutations, isolated from 4 different animals, were the same +1 frameshift mutation in a run of 6 consecutive guanine bases (207-212) in exon 3. These results suggest the involvement of both modified guanine and adenine bases in ETO mutagenesis. The mouse T-cell cloning/sequencing assay for hprt described here represents a useful system for studying the molecular mechanism of chemically-induced mutation occurring in vivo at an endogenous gene." @default.
• W2051796733 created "2016-06-24" @default.
• W2051796733 creator A5039613182 @default.
• W2051796733 creator A5087455689 @default.
• W2051796733 date "1993-07-01" @default.
• W2051796733 modified "2023-10-18" @default.
• W2051796733 title "A mouse model for the study of in vivo mutational spectra: Sequence specificity of ethylene oxide at the hprt locus" @default.
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• W2051796733 doi "https://doi.org/10.1016/0027-5107(93)90216-3" @default.
• W2051796733 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7686258" @default.
• W2051796733 hasPublicationYear "1993" @default.
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