Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides.

Abstract

An efficient method for introducing 2 codons into a cloned gene was 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 (6-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 2 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.