Mutational spectrometry: a general approach for hot-spot point mutations in selectable genes.
To observe point mutational spectra with a high degree of precision, independent large cultures of human lymphoblastoid cells were treated with a mutagen, benzo[a]pyrene diol epoxide, and mutants at the HPRT gene were selected en masse by 6-thioguanine resistance. An average of 1.6 x 10(4) 6-thioguanine-resistant mutants were created per experiment and the kinds, positions, and numbers of the most frequent mutations were examined in exon 3 of the HPRT gene by using a high-fidelity polymerase chain reaction and denaturing gradient gel electrophoresis. Sixteen exon 3-specific mutations were found to be predominantly G----T transversions and corresponded to an average of 3500 induced mutants per experiment. Of these mutations, 6 occurred within a run of 6 guanines and 5 occurred in the sequence 5'-GAAGAG-3'. The variation among independent experiments is consistent with the numerical expectation that all 16 mutations fulfill reasonable statistical criteria for mutational hot spots. The agreement with data from various systems using clone-by-clone analysis shows that the protocol reported herein can be a useful tool to study hot-spot point mutational spectra for DNA sequences for which phenotypic selection systems exist.
Keohavong
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Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, PA 15261.
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D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D016368:Frameshift Mutation; D005784:Gene Amplification; D005821:Genetic Techniques; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013866:Thioguanine / Q000494:pharmacology*
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine