Characterization of the pUC19– lacZC 141 reversion system for assaying chemical mutagenesis

Abstract

pUC19– lacZC 141 DNA contains a proline codon at positions 141 to 143, where an alanine codon normally appears in the original lacZ gene. pUC19– lacZC 141 DNA was produced using site-directed mutagenesis. After transfection of pUC19– lacZC 141 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 β-galactosidase activity. N-Methyl- N′-nitro- N-nitrosoguanidine (MNNG) and methyl-methanesulfonate (MMS) were used to characterize the pUC19– lacZC 141 DNA reversion assay. Mutagenesis resulting from methylated-DNA was examined in Escherichia coli strains JM109, BMH71–18 mutS, and SURE ®, which differ in their repair systems for DNA damage. In JM109 and BMH71–18 mutS, 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 (∼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– lacZC 141 assay was sufficiently sensitive to allow the detection of mutations in lacZ − hosts with different repair mechanisms. The pUC19– lacZC 141 DNA reversion system will permit the assaying of other chemicals not otherwise amenable to mutagenesis studies.