Characterization of the pUC19-lacZC141 reversion system for assaying chemical mutagenesis.
pUC19-lacZC141 DNA contains a proline codon at positions 141 to 143, where an alanine codon normally appears in the original lacZ gene. pUC19-lacZC141 DNA was produced using site-directed mutagenesis. After transfection of pUC19-lacZC141 DNA into lacZ hosts, the transformants produce white colonies on an agar plate containing X-gal and IPTG. lacZ+ revertants can be identified by their dark- and light-blue colony color against a background of non-mutant white colonies, indicating restoration of beta-galactosidase activity. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylmethanesulfonate (MMS) were used to characterize the pUC19-lacZC141 DNA reversion assay. Mutagenesis resulting from methylated DNA was examined in Escherichia coli strains JM109, BMH71-18mutS, and SURE, which differ in their repair systems for DNA damage. In JM109 and BMH71-18mutS, mostly G:C-->A:T transitions and some G:C-->C:G or G:C-->T:A transversions were observed. E. coli SURE produced, in addition, frameshift mutations (approximately 10%). The DNA sequence analysis of 174 induced mutants indicated that the major effect of methylation is on single base-pair substitutions with a slight effect on deletion frameshifts. All mutations are consistent with miscoding of guanine or cytosine adducts or lesions. Transitions account for 158 of 165 (96%) induced base substitutions. Approximately 93% of the base-substitution mutations occurred at the expected positions 141 to 143 in the lacZ gene. The pUC19-lacZC141 assay was sufficiently sensitive to allow the detection of mutations in lacZ- hosts with different repair mechanisms. The pUC19-lacZC141 DNA reversion system will permit the assaying of other chemicals not otherwise amenable to mutagenesis studies.
Ogawa
H
H
Hokkaido Institute of Public Health, Hokkaido, Japan.
Ohyama
T
T
Katsura
E
E
Katoh
Y
Y
D019175:DNA Methylation; D004274:DNA, Recombinant / Q000187:drug effects* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D010957:Plasmids
D004274:DNA, Recombinant