A sectored colony assay for monitoring mutagenesis by specific carcinogen-DNA adducts in Escherichia coli.
To study the mutagenicity of various carcinogen-DNA adducts in Escherichia coli, a cassette plasmid was developed that permits positioning of specific carcinogen-modified bases within the ATG initiation codon of the lacZ' alpha-complementation gene. Adduct-induced mutations inactivate the gene and lead to formation of blue and white sectored colonies when transformants from an alpha-complementing version of E. coli strain AB1157 are grown on media containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside. In the absence of mutation, blue colonies are produced. This system has been used to measure the mutagenicity of O6-methyl-, O6-ethyl-, and O6-benzyl-2'-deoxyguanosine residues incorporated in place of the normal 2'-deoxyguanosine of the ATG initiation codon. Although a low percentage of sectored colonies was produced in this repair-proficient strain, pretreatment of the bacteria with N-methyl-N'-nitro-N-nitrosoguanidine to disable DNA repair led to a dose-dependent increase in the percentage of sectored colonies. This percentage increased as a function of modified guanine in the order O6-benzyl- less than O6-methyl- less than O6-ethyl-2'-deoxy-guanosine. The only mutations detected at the site of incorporation of these O6-substituted guanines were G-to-A transitions. This sectored colony assay system permits convenient screening of large numbers of colonies and simplifies quantification of modified-base-induced mutations whether they be single-base changes, frameshifts, insertions, or deletions.
Pauly
G T
GT
Chemistry of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702.
Hughes
S H
SH
Moschel
R C
RC
D001483:Base Sequence; D002273:Carcinogens / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000187:drug effects*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000633:toxicity; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D010766:Phosphorylation; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics
D002273:Carcinogens; D004269:DNA, Bacterial; D001616:beta-Galactosidase; D003849:Deoxyguanosine