IN-VIVO INTERMOLECULAR RECOMBINATION IN ESCHERICHIA-COLI - APPLICATION TO PLASMID CONSTRUCTIONS

Repair of a double-strand break (DSB) was investigated by intermolecul ar recombination in Escherichia coli (Ec) recBC sbcBC cells with restr iction enzyme-cleaved model plasmids. Circular plasmids were generated when a linearized plasmid (vector) containing an origin of replicatio n was co-transformed with a DNA fragment (template) containing a homol ogous sequence. The influence of the position of the DSB in the vector was analyzed using templates which contain various genetic markers, n on-homologous sequences and/or deletions relative to the vector. In al l cases, when a DSB occurs within a marker, this marker is lost in the resulting plasmid, whereas markers flanked by homologous regions loca ted in the vicinity of a DSB are transmitted. Insertions (deletions), substitutions and shuffling of genetic markers are possible by in vivo recombination using Ec and can be applied to plasmid constructions. I t is shown that recombination can occur from both template ends or fro m one vector and one template end. A D-loop nuclease is suggested to p articipate in the resolution of the recombination intermediates.