Genes and Environment
Online ISSN : 1880-7062
Print ISSN : 1880-7046
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Volume 35 , Issue 4
Showing 1-4 articles out of 4 articles from the selected issue
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REVIEW
  • Takashi Yagi
    Volume 35 (2013) Issue 4 Pages 93-98
    Released: November 12, 2013
    [Advance publication] Released: July 01, 2013
    JOURNALS FREE ACCESS
    Shuttle vector plasmids have made major contributions to the advancement of mutation research for the last two decades. I have been involved in the development of shuttle vector plasmids and their experimental protocols, and herein provide a chronicle of shuttle vector studies. In the late 1960s, in vitro mammalian cell culture became a common technique in cell biology. Geneticists established a method to detect gene mutation as 8-azaguanine or 6-thioguanine resistance after cells had been exposed to radiation or chemicals. The resistance of the cells was found to be due to a mutation of the hypoxanthine-guanine phosphoribosyl transferase gene; however, which base was changed in the gene remained difficult to determine until DNA sequencing techniques were developed. After the Sanger method of DNA sequencing became available to geneticists, the next issue was how to identify numerous base changes in mutants easily and rapidly. A breakthrough on this issue was the use of the shuttle vector plasmid pZ189, which was developed for practical applications by Seidman and colleagues. Along with the development of new molecular biology techniques such as polymerase chain reaction and automatic DNA sequencing, pZ189 has been modified to several forms as adaptations to the new techniques. These shuttle vector plasmids remain useful tools to reveal what types of mutations are induced by newly identified mutagens at the DNA sequence level. This article describes some of the contents of my JEMS award lecture in 2011.
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REGULAR ARTICLE
  • Keiko Inami, Hiroki Yasui, Hirotaka Tsugumi, Satoko Ishikawa, Masataka ...
    Volume 35 (2013) Issue 4 Pages 99-104
    Released: November 12, 2013
    [Advance publication] Released: October 01, 2013
    JOURNALS FREE ACCESS
    N-Alkyl-N-(3-carboxypropyl)nitrosamines are known to selectively induce urinary bladder tumor in rats and mice. To detect DNA damage by N-alkyl-N-(3-carboxypropyl)-nitrosamines, we evaluated their mutagenicity using the Ames assay in S. typhimurium and E. coli under oxidative conditions of chemical model for cytochrome P450. The activation system consisted of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrinatoiron(III) pentachloride (4-MPy) plus an oxidant. The N-alkyl-N-(3-carboxypropyl)-nitrosamines; N-methyl-N-(3-carboxypropyl)nitrosamine (MCPN), N-ethyl-N-(3-carboxypropyl)nitrosamine (ECPN), N-propyl-N-(3-carboxypropyl)nitrosamine (PCPN), N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), were treated with 4-MPy and t-BuOOH in acetonitrile for 30 min at room temperature, the reaction mixture was extracted with dichloromethane, and the organic phase was assayed for their mutagenicity in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA. The dichloromethane extract derived from the reaction mixture of MCPN, ECPN, PCPN or BCPN with 4-MPy plus t-BuOOH was mutagenic in both of the strains, indicating that N-alkyl-N-(3-carboxypropyl)nitrosamines were oxidized to direct-acting mutagens by the 4-MPy plus t-BuOOH. The mutagenicity of oxidized BCPN extract in S. typhimurium YG7108 was higher than that in S. typhimurium TA1535, suggesting that the mutagenicity derived from BCPN was due to DNA alkylation. Furthermore, the DNA seemed to be butylated, not 3-carboxypropylated, exerting the mutagenicity of BCPN in the presence of 4-MPy and t-BuOOH.
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SHORT COMMUNICATION
  • Hiroyuki Kamiya, Masahiro Kurokawa
    Volume 35 (2013) Issue 4 Pages 105-109
    Released: November 12, 2013
    [Advance publication] Released: July 01, 2013
    JOURNALS FREE ACCESS
    DNA polymerase λ preferentially inserts dCTP opposite 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) in vitro, and this function is considered to be important after A base removal from GO:A pairs by the MUTYH DNA glycosylase. However, dCTP incorporation would promote A:T→C:G transversion mutations induced by 8-oxo-7,8-dihydro-dGTP (dGOTP), the mutation triggered by the incorporation of dGOTP opposite A in the template DNA. In this study, double-stranded plasmid DNA containing the GO:A pair, an intermediate in the dGOTP mutagenesis pathway, was transfected into human cells in which DNA polymerase λ was knocked down. The knockdown of DNA polymerase λ significantly reduced the frequency of A:T→C:G transversion mutations induced by the GO:A pair although the knockdown effect was small. These results suggested that DNA polymerase λ is involved in the mutagenesis processes of GO, generated by dGOTP, in the nucleotide pool.
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  • Kohta Nagira, Sayaka Tamura, Shinji Kawano, Shogo Ikeda
    Volume 35 (2013) Issue 4 Pages 110-114
    Released: November 12, 2013
    [Advance publication] Released: August 01, 2013
    JOURNALS FREE ACCESS
    Cu,Zn-superoxide dismutase (SOD1) is a critical enzyme in the cellular antioxidant system. The yeast Saccharomyces cerevisiae SOD1 mutant (SOD1Δ) exhibits a moderate mutator phenotype under aerobic conditions. The mutation frequency of a SOD1Δ strain determined by a CAN1 forward-mutation assay was about 12-fold higher than that of the parental strain. Base substitutions G·CT·A, G·CA·T, and A·TC·G were most commonly observed in CAN1 mutants, indicating that the mutations are caused mainly by oxidative DNA damage. The mutation frequency of SOD1Δ was reduced in a dose-dependent manner by cultivating it in the presence of ascorbic acid, implying that the SOD1Δ mutant can be used as a tester strain for small molecule antioxidants. Exogenous glutathione and N-acetylcystein also alleviated the mutator phenotype. The results indicate that ascorbic acid and thiol antioxidants are able to efficiently protect cells against oxidative damage-induced mutagenesis. In this assay, no apparent mutation suppression was seen for other categories of antioxidants including resveratrol, Trolox and melatonin.
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