Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells.
- 1 September 1986
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 83 (17), 6519-6523
- https://doi.org/10.1073/pnas.83.17.6519
Abstract
To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes < 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (< 35 kb) with 5'' and 3'' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5''flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5'' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC .fwdarw. TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%)to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed.This publication has 34 references indexed in Scilit:
- DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coliJournal of Molecular Biology, 1985
- Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coliJournal of Molecular Biology, 1984
- On the nature of the mutations induced by the diolepoxide of benzo[a]pyrene in mammalian cellsCarcinogenesis: Integrative Cancer Research, 1984
- Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblastsMutation Research, 1984
- Structure of mutant alleles at the aprt locus of Chinese hamster ovary cellsJournal of Molecular Biology, 1983
- Quantitative forward-mutation specificity of mono-functional alkylating agents, ICR-191, and aflatoxin B1 in mouse lymphoma cellsMutation Research, 1982
- Metabolism of benzo[a]pyrene and DNA adduct formation in cultured human epidermal keratinocytesCarcinogenesis: Integrative Cancer Research, 1981
- Circular dichroism and proton magnetic resonance studies of dApdG modified with 2-aminofluorene and 2-acetyl-aminofluoreneCarcinogenesis: Integrative Cancer Research, 1980
- Detection of specific sequences among DNA fragments separated by gel electrophoresisJournal of Molecular Biology, 1975
- Physical studies on deoxyribonucleic acid after covalent binding of a carcinogenBiochemistry, 1972