大腸菌における突然変異スペクトルの簡易解析法の開発に関する研究

抄録

The mutational specificities of mutagens provide important information about the mechanisms of mutagenesis and DNA repair. For the purpose to investigate the mutational spectrum, the Lac⁺ reverse mutation system with a set of six specific tester strains have been employed. The mutational target is an F’ plasmid-encoded mutant lacZ461 allele (CC101 to CC106), by which each of the six possible base-pair substitution mutations can be independently detected by measuring Lac⁺ revertant colonies. Using this system, we investigated the mutational specificities of several carcinogenic heterocyclic amines and revealed that G:C→T:A transversions generated by heterocyclic amines such as Methyl-IQ and Trp-P-2 could be easily detected with clear dose-response relationships. We also demonstrated that a recA730 lexA51 (Def) strain showed mutator activity generating A:T→T:A transversions most favorably. Compared with colony probe hybridization analysis or DNA sequencing analysis, one of the advantages of the Lac⁺ reverse mutation system is that very few transitions and transversions were quantitatively determined by this system.
We introduced uvrA mutations to increase the sensitivity of strains CC101-CC106 to various mutagens. However, the sensitivity did not improved as we expected. We also found that cells carrying both F’ and pKM101 plasmids showed impaired growth in minimal medium. We, therefore, transferred the F’ plasmids from E. coli K-12 (CC101 to CC106) to a derivative of E. coli B/r WP2uvrA. To further increase the sensitivity to mutagens, we isolated their rfa derivatives. Since no obvious growth delay was observed in E. coli B cells containing F’ and pKM101, we could construct two sets of tester strains, WP3101P to WP3106P (uvrA, pKM101), and WP4101P to WP4106P (uvrA, rfa, pKM101). With these tester strains, more than 40 kinds of mutagens including photomutagenic compounds were analyzed for their mutational spectra. The strains we developed were highly sensitive to detect not only mutational specificity with a Lac⁺ reversion assay, but also mutagenicity with an ordinary Trp⁺ reversion assay. It is expected that the mutational spectra of chemical mutagens in bacterial cells with different genetic background for DNA repair would provide novel findings on mutagenesis and DNA repair.