Directed mutagenesis method for analysis of mutagen specificity: application to ultraviolet-induced mutagenesis.

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A directed mutagenesis method has been developed for the analysis of mutagen specificity. The method is based on the construction of a plasmid damaged by the mutagen at a specific segment within a given marker gene, followed by screening for mutant plasmids and nucleotide sequence analysis of the damaged segment. By using this method plasmid pXf3 has been specifically damaged by UV radiation at the BamHI-Sph I segment in the tetracycline resistance (tet) gene(s) and used to transform SOS-induced Escherichia coli. Fourteen ampicillin-resistant, tetracycline-sensitive mutants of pXf3 were isolated and subjected to sequence analysis. The data revealed the induction of transitions, a transversion, a frameshift mutation, and deletions. The single base changes all were located within runs of pyrimidines, and the deletions mapped between direct repeats of polypyrimidine tracts. In addition, mutant plasmids were found with no mutation within the damaged segment. In these cases it is likely that "untargeted mutagenesis" in other portions of the tet gene(s) is responsible for the mutant phenotype. The method can be applied to any mutagen that reacts with DNA, and it also can be used for genetic analysis as a means to mutate specific segments of DNA.

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