Characterization of c-Ki-ras oncogene alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced tumors

Abstract

Activated c-Ki-ras genes in liver tumors from rats exposed to the potent hepatocarcinogen aflatoxin B1 were analyzed to determine the nature of their activation by characterization of two c-Ki-ras alleles present in tumor-derived NIH 3T3 mouse transformants. Using selective hybridization of synthetic oligonucleotides to transformant DNA, we have determined that a single G X C to A X T base transition in either the first or second position of the 12th codon is associated with activation of the gene. Such mutations would lead to amino acid substitutions of aspartate or serine for glycine in the mutant proteins. To confirm these findings, we applied a technique for direct sequence analysis of a 90-base-pair region of the rat c-Ki-ras gene produced by primer-directed enzymatic amplification. Findings produced by this approach, which provides a convenient method to characterize mutations in multiple alleles without the necessity to clone individual genes, confirmed the presence and identity of the 12th codon mutations in the activated oncogene, as initially determined by the oligonucleotide hybridization technique.