PMID:7041119

Citation	Weiher, H and Schaller, H (1982) Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element. Proc. Natl. Acad. Sci. U.S.A. 79:1408-12
Abstract	A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.
Links	PubMed PMC345982
Keywords	Base Sequence; DNA, Recombinant; Escherichia coli/genetics; Lac Operon; Mutation; Operon; Plasmids; Sulfites
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