I describe here an anticipated mutation assay which estimates the frequency and spectrum of somatic point mutations in any genes of any samples by using SMRTTM (Single Molecule Real Time) DNA sequencing technology, which will be released in 2010. The basic concept of the mutation assay is very simple: just prepare the target template and sequence it accurately. In this paper, I propose ideas on how to make the template and how to eliminate artifactual mutations caused by damage to the DNA template.
Sidestream smoke from smoldering cigarette is the major source of environmental cigarette smoke. Cigarette smoke condensate (CSC) from sidestream smoke has been known to be mutagenic in Ames' Salmonella strains in the presence of an S9 metabolic activation system. We investigated a novel photomutagenic property of CSC with UVA-irradiation in the absence of metabolic activation. CSCs were collected from sidestream smoke of three Japanese brands of cigarettes using a smoking machine according to the ISO Standard. Bacterial cells with or without CSC were irradiated using a UVA light (320-400 nm) for 10-30 min. Photomutagenicity of CSC was more evident in E. coli WP2uvrA/pKM101 than in S. typhimurium TA100 and TA98. Photomutagenic potency (number of induced revertants/μg CSC) of sidestream CSC from Mild Seven One, an ultra-light tar cigarette, showed equal mutagenic potency to that from Seven Stars, a high tar cigarette. Photomutagenic potency of Pianissimo One, an ultra-light tar cigarette, was significantly higher than that of Seven Stars or Mild Seven One. We could not detect any increase of 8-OHdG formation in DNA isolated from cells irradiated with UVA in the presence of CSC. CSC with UVA irradiation caused A:T→T:A transversions most frequently, followed by A:T→G:C transitions. CSC was not photomutagenic in WP2/pKM101, indicating that the DNA damages generated by UVA-irradiated CSC were efficiently repaired by a nucleotide excision repair system. In addition, an error-prone translesion DNA synthesis by DNA polymerase RI encoded by mucAB genes on pKM101 is considered to be involved in photomutagenesis of CSC. We speculate a possible mechanism of production of radicals followed by DNA bulky adduct formation.
When 8-oxo-7,8-dihydro-2'-deoxyguanosine in potassium phosphate buffer of pH 7.4 was irradiated by UV light from a mercury lamp, two major products were formed. They were identified as the diastereomers of spiroiminodihydantoin deoxyribonucleoside on the basis of their identical ESI-MS and UV spectra and HPLC retention times with those of the authentic samples. Degradation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by UV light was faster than those of 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxyadenosine, and thymidine. Sunlight also generated the diastereomers of spiroiminodihydantoin from 8-oxo-7,8-dihydro-2'-deoxyguanosine. The results suggest that sunlight may induce 8-oxo-7,8-dihydroguanine oxidation in DNA, generating spiroiminodihydantoin in humans.
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