title: Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.
abstract: An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.
authors.affiliation: Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri 63110.
authors.forename: S
authors.initials: S
authors.lastname: Kulakauskas
authors.forename: P M
authors.initials: PM
authors.lastname: Wikström
authors.forename: D E
authors.initials: DE
authors.lastname: Berg
mesh_terms: D000483:Alleles; D010582:Bacteriophage lambda; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D016254:Mutagenesis, Insertional; D010957:Plasmids; D014161:Transduction, Genetic