Site-specific recombination directed by single-stranded crossover linkers: specific deletion of the amino-terminal region of the beta-galactosidase gene in pUC plasmids.
The "duplex crossover linker" technique was simplified and used to delete the beta-galactosidase (beta-Gal)-coding sequence upstream from the multiple restriction sites in pUC plasmids. A single-stranded crossover linker, with a homology-searching sequence as short as 5 bases, was initially ligated to a linearized plasmid. Inside Escherichia coli, the plasmid was circularized by intramolecular, homologous recombination between the (5'-or 3'-) protruding homology-searching sequence and a targeted region in the opposite terminus. As a consequence, sequences beyond the point of integration were deleted. Specific deletion of sequences up to 1472 bp was demonstrated. The single-stranded linkers apparently avoided generation of undesirable mutants associated with the usage of duplex linkers. A mechanism has been proposed for the intramolecular recombination directed by the crossover linkers. It principally involves either 3'- or 5'-exonucleolytic breakdown of the homologous terminus of the plasmid, circularization by spontaneous pairing of the exposed complementary strands, and subsequent degradation of any redundant sequence.
D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005822:Genetic Vectors; D010281:Parathyroid Hormone / Q000235:genetics; D010957:Plasmids; D011995:Recombination, Genetic; D013753:Tetracycline Resistance; D001616:beta-Galactosidase / Q000235:genetics*
D004274:DNA, Recombinant; D010281:Parathyroid Hormone; D005696:Galactosidases; D001616:beta-Galactosidase