Site-Specific Recombination Directed by Single-Stranded Crossover Linkers: Specific Deletion of the Amino-Terminal Region of the β-Galactosidase Gene in pUC Plasmids

Abstract

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.

Mechanism of Streptococcus mutans glucosyltransferases: hybrid-enzyme analysis

“…6). This finding is consistent with a previous suggestion that a 5-bp homologous sequence is sufficient to generate recombination between single-stranded plasmid DNA fragments (29). Hybrid proteins BD701, BD725, and BD742 were apparently generated following homologous recombination of linearized plasmid molecules which were not ligated prior to transformation.…”

Synthesis and characterization of extended and deleted recombinant analogs of parathyroid hormone-(1-84): correlation of peptide structure with function

“…An in vivo DNA polymerization would subsequently repair the gap at the single-stranded region to yield a circular plasmid with nicks. However, this ability to direct in vivo DNA polymerization appeared to be limited, as it failed to generate mutants if the gap to be filled in was wider than 22 bases, as shown in other studies (Sung et al, 1989;Sung & Zahab, 1987). This limited capability indicated that the initial exonucleolytic reaction may be controlled or directed (Radding, 1973;Cassuto & Radding, 1971) such that the degradation would not be so extensive as to generate a gap which was too wide to be repaired.…”

“…The resultant plasmid pProPTH-1 still maintained a residual /3-Gal sequence fused with the ProPTH gene. The second cross-over linker, COL-3 (Sung & Zahab, 1987), with a 22-base homology-searching sequence targeting the ribosome-binding site of the lacZ gene, efficiently deleted the /3-Gal gene residue to yield plasmid pProPTH-1 B. The restored £coRI site of pProPTH-1 B was eliminated by linearization.…”

“…In the case of the expression plasmid encoding hProPTH-(-6-*-+84) a "single-stranded cross-over linker" technique (Sung et al, 1989;Sung & Zahab, 1987) was employed. The mechanism for recombination directed by a single-stranded cross-over linker, such as PRO-1 used here, may be a variation of pathways originally proposed for related recombination in other cell lines (Lin et al, 1984).…”