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Analysis of solvent control and 1‐Nitrosopyrene‐induced chinese hamster ovary cell mutants by southern and northern blots and the polymerase chain reaction
Author(s) -
Newton Ronald K.,
Mittelstaedt Roberta A.,
Heflich Robert H.
Publication year - 1992
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.2850190209
Subject(s) - chinese hamster ovary cell , mutant , microbiology and biotechnology , southern blot , biology , northern blot , complementary dna , mutagen , blot , gene , dna , ethyl methanesulfonate , biochemistry , genetics , cell culture
1‐Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1‐nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N ‐(deoxyguanosin‐8‐yl)‐1‐aminopyrene, is produced in CHO cells treated with 1‐nitrosopyrene, and this adduct is found in rats and mice exposed to 1‐nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1‐nitrosopyrene‐treated cultures). Pst l‐ and Bam Hl‐digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1‐nitrosopyrene‐induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein‐coding region could be amplified from 23 of the 1‐nitrosopyrene‐induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1‐nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1‐nitrosopyrene acts as a point mutagen. A large number of both control and 1‐nitrosopyrene‐induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequences. These alterations may contribute to the 6‐thioguanine‐resistant phenotype.