Mutational Spectrometry: A General Approach for Hot-Spot Point Mutations in Selectable Genes

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 10; pp. 4623 - 4627
• Main Authors: Keohavong, Phouthone, Thilly, William G.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15.05.1992; National Acad Sciences; National Academy of Sciences
• Subjects: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide - pharmacology; Amino Acid Sequence; Base Sequence; benzo(a)pyrene diol epoxide; Biological and medical sciences; Cell culture techniques; Cell Line; Cell lines; Cellular biology; Cultured cells; Exons; Frameshift Mutation; Fundamental and applied biological sciences. Psychology; gel electrophoresis; Gels; Gene Amplification; genes; Genetic mutation; Genetic Techniques; Genetics; hot spots; HPRT gene; Humans; hypoxanthine phosphoribosyltransferase; Hypoxanthine Phosphoribosyltransferase - genetics; identification; man; Medical research; Melting; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis; Mutagenesis. Repair; Mutagens; Point mutation; Polymerase chain reaction; Thioguanine - pharmacology; TK6 cells; Human; Polymerase chain reaction; Cell culture; Exon; Gene; Point mutation; Tumor cell
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.10.4623

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 * 1046-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.