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Mapping and characterization of mutations induced by benzo[a]pyrene diol epoxide at dihydrofolate reductase locus in CHO cells

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Somatic Cell and Molecular Genetics

Abstract

Chinese hamster ovary cells were mutagenized with benzo[a]pyrene diol epoxide (BPDE), an aromatic hydrocarbon carcinogen, and mutants at the dihydrofolate reductase (dhfr) locus were isolated. Of 15 mutants analyzed by Southern blotting, one contained a large deletion that spanned all six exons of the 25-kbdhfr gene; the remaining mutants exhibited no detectable changes. Three of these putative point mutations were localized by the loss of a restriction site: a SacI site in exon III, an MspI site in exon III, and a KpnI site in exon VI. The affected regions in two of these mutants were cloned and sequenced. The SacI− mutant was caused by a G:C → T:A transversion resulting in an amber termination codon. In the MspI− mutant, the deletion of a single C:G resulted in a frameshift and a downstream ochre termination codon. On the basis of overlapping restriction site sequences, the KpnI− mutant was deduced to be a splicing mutant involving the most 3′ G in intron V. The location of these and the remaining 11 putative point mutations was sought using RNA heteroduplex mapping. Mismatched bases between riboprobes complementary to wild-typedhfr mRNA and mutant mRNA molecules were detected in 10 of the 14 mutants analyzed. These mutations mapped to four of the six exons or exon splice sites. Surprisingly, over half of these mutants exhibited greatly reduced (approximately 10-fold) steady-state levels ofdhfr mRNA.

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Authors and Affiliations

  1. Institute of Cancer Research, Columbia University, 10032, New York, New York

    Adelaide M. Carothers & Dezider Grunberger

  2. Department of Biological Sciences, Columbia University, 10027, New York, New York

    Gail Urlaub & Lawrence A. Chasin

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  1. Adelaide M. Carothers
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Carothers, A.M., Urlaub, G., Grunberger, D. et al. Mapping and characterization of mutations induced by benzo[a]pyrene diol epoxide at dihydrofolate reductase locus in CHO cells. Somat Cell Mol Genet 14, 169–183 (1988). https://doi.org/10.1007/BF01534402

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  • DOI: https://doi.org/10.1007/BF01534402

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