Mutations activating human c-Ha-ras1 protooncogene (HRAS1) induced by chemical carcinogens and depurination.
In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells.
D000099:Acetoxyacetylaminofluorene / Q000494:pharmacology; D001080:Apurinic Acid / Q000235:genetics*; D001483:Base Sequence; D001580:Benzopyrenes / Q000494:pharmacology; D002273:Carcinogens; D055598:Chemical Phenomena; D002621:Chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics; D011119:Polynucleotides / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*
D001580:Benzopyrenes; D002273:Carcinogens; D009841:Oligonucleotides; D011119:Polynucleotides; D011518:Proto-Oncogene Proteins; D001080:Apurinic Acid; D000099:Acetoxyacetylaminofluorene