Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides.
An efficient method for introducing two codons into a cloned gene has been applied to studying functional regions of the pBR322-encoded tetracycline-resistance gene and beta-lactamase (ampicillin-resistance) gene. Single-stranded hexameric linkers are inserted into a preexisting cohesive end restriction site to create a new (six-base recognition) restriction site. Insertion mutations are enriched by using biochemical selection or are selected by using a kanamycin-resistance cassette (biological selection). Phenotypes of insertion mutations isolated in the tetracycline-resistance gene support the hypothesis that it is comprised of two domains connected by a central hinge. Mutations in the beta-lactamase gene are temperature sensitive and demonstrate altered sensitivity to various beta-lactams and inhibitors.
D000595:Amino Acid Sequence; D003062:Codon; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000235:genetics; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D010957:Plasmids; D013752:Tetracycline / Q000494:pharmacology; D001618:beta-Lactamases / Q000235:genetics
D003062:Codon; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D001618:beta-Lactamases; D013752:Tetracycline