Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site.
We describe a simple method to select for transfer of mutant alleles from the Escherichia coli chromosome to a plasmid which formerly carried the wild-type (wt) allele. The wt allele on the plasmid is modified by introduction of a unique restriction site (e.g., XhoI) and transformed into a rec+ strain carrying the mutant allele on the chromosome. Upon homogenotization, the efficiency of which was increased by UV irradiation of the transforming plasmid [Chattoraj et al., Gene 27 (1982) 213-222], plasmids carrying the mutant allele are formed which are resistant to XhoI. These plasmids are selected from the population by resistance to XhoI digestion coupled with the low transformation efficiency of linear DNA molecules in recA- strain. The method is efficient and rapid and has particular advantages in situations where the mutant allele is difficult to detect by its phenotype.
D000483:Alleles; D002876:Chromosomes, Bacterial / Q000502:physiology; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics; D014466:Ultraviolet Rays
D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes