EFFICIENT CIRCULARIZATION IN ESCHERICHIA-COLI OF LINEAR PLASMID MULTIMERS FROM DICTYOSTELIUM-DISCOIDEUM GENOMIC DNA

Transformation of Escherichia coli with Dictyostelium discoideum genom ic DNA containing integrated shuttle vectors in multicopy, tandemly du plicated format resulted in the establishment of the linear plasmid mo lecules as circular monomeric replicons. The transformation efficienci es were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digesti on of the genomic DNA prior to the transformation using restriction en zymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmi ds carried deletions. Our results provide evidence that the linear pla smid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene pro duct but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformatio n with plasmid multimers of a recB recC sbcB mutant indicated the pres ence of an additional pathway for recombinational recircularization in dependent of these gene products. Unlike previous studies using as a D NA source Linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. disc oideum genome ensured that none of the E. coli transformants we obtain ed could be attributed to low levels of uncut circular plasmid molecul es. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful teal in the characterization of such plasmid insertions in the genome of D. disco ideum. (C) 1996 Academic Press, Inc.