Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

Abstract

A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands ofa 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 -* 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.Molecular cloning methods have been used to develop a type of genetic analysis in which a cloned genetic element is first altered at predetermined sites and then assayed for changes in phenotype in the appropriate biological environment. This "reversed genetics" (1) allows construction and study of silent mutations that are often essential to correlate structure and function of a genetic element. In this approach, deletions or insertions can be introduced rather easily. However, methods to generate point mutations at predetermined sites in a DNA molecule by base-specific chemical treatment (2)(3)(4)(5) or by incorporation of nucleotide analogs (1) give rise to only a few mutants among a large background ofnonmutants. Because this yield is too low to permit saturation of a genetic element with point mutations, we developed an improved method for sodium bisulfite mutagenesis in vitro. This reagent has been used before for site-specific mutagenesis of sequences next to a unique restriction site in which a DNA sequence could be rendered single stranded to become a selective target for the mutagen (2). We have overcome these limitations by using a singlestranded phage vector system to clone either strand of the desired target DNA separately. Mutagenesis of the single-stranded recombinant DNA enabled us to introduce any number of C -* T base changes into one strand of a defined restriction fragment. The present study exemplifies the scope of the method by an extensive variation of the nucleotide sequence of the lac promoter/operator, a regulatory signal that controls the expression of the lac operon in Escherichia coli.MATERIALS AND METHODS E. coli C600 rm+ and KB35 and phages fdlO9 and fdlO9-2 were obtained from R. Herrmann (6). E. coli C600 galK (gal E'T+K-) and pKOl were obtained from K. McKenney (7). Construction of plasmids and recombinant phages, including transformation and cultivation of bacteria, and isolation of recombinant DNA was done essentially as described by Herrmann et al. (6). DNA se...

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