| Co-Investigator(Kenkyū-buntansha) |
OGAWA Hiroaki Kyushu Inst.Tech., Fac.Engineering, Associate Professor, 工学部, 助教授 (50108685)
NUNOSHIBA Tatsuo Tohoku University, Graduate School of Science, Research Associate, 大学院・理学研究科, 助手 (10270802)
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| Research Abstract |
Escherichia coli K12 strain KS40 and plasmid pKY241 had been previously designed for easy screening of supF mutations in plasmid. KS40 has mutation of nalidixic acid (Nal) -resistant (gyrA) and streptomycin (Sm) -resistant (rpsL) mutations. Using in vitro mutagenesis, an amber mutation was introduced into the cloned rpsL structural gene, of E.coli, to give pOF105, a derivative of pKY241 which carries gyrAam mutation. When KS40 containing pOF105 (designated KS40/ pOF105) is transformed with plasmid having supF gene, KS40/ pOF105 give rise to Nal-sensitive and Sm-sensitive colonies. If the supF gene on the plasmid carries an inactivating mutation, then KS40/ pOF105 will form Nal-resistant and Sm-resistant colonies. We have further constructed a vector plasmid carrying the pBR322 replication origin, beta-lactamase gene, M13 replication origin which permits easy preparation of single-stranded plasmid DNA,and supF gene.
By using the system, oxygen species induced mutations were characterized. When the vector plasmid was treated with ferrous ion in the presence of EDTA,G : C to C : G transversion mutations were predominantly observed followed by G : C to T : A transversion. Ferrous ion resulted in increased formation of 8-hydroxydeoxyguanine (8-oxodG) in plasmid DNA.We can therefore indicate that the origin of Fe^<2+>-induced G : C to T : A transversion is 8-oxodG ; the origin of G : C to C : G is unidentified. When the plasmid was directly treated with cobalt (II), which is assumed to bind DNA,8 to 50 base deletions were predominantly observed followed by frameshift mutations. The sites of deletion junction have repeated sequences. Therefore, DNA-cobalt complex can interfere DNA replication, facilitating aberrant slip-mispairing DNA replication forming deletion mutations.
The design of E.coli host as well as vector plasmid will establish the screening system for environmental mutagens.
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