Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome.
To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ- transposable element was constructed to insert the lacZ-(Trp570 --> Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop --> LacZ+; candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14000 colonies, and named plm-1 (preferential lacZ mutagenesis) and plm-2. The frequency of LacZ- --> LacZ+ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac+ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan(r) gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ+ --> LacZ- and Kan(r) --> Kan(s) mutant frequencies of these Lac+ revertants were in the range of 10(-3) to 10(-2), indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome.
Liu
S K
SK
Department of Biology, National Sun Yat-sen University Kaohsiung, Taiwan, Republic of China.
Tseng
J N
JN
Shiuan
D
D
Hanawalt
P C
PC
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D008040:Genetic Linkage; D007613:Kanamycin Resistance / Q000235:genetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial