Efficient circularization in Escherichia coli of linear plasmid multimers from Dictyostelium discoideum genomic DNA.
Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid 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 product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D. discoideum.
Barth
C
C
School of Microbiology, La Trobe University, Melbourne, Australia.
Wilczynska
Z
Z
Pontes
L
L
Fraser
D J
DJ
Fisher
P R
PR
D000818:Animals; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004270:DNA, Circular / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D016681:Genome, Fungal; D018503:Genome, Protozoan; D010957:Plasmids / Q000235:genetics*; D012093:Replicon; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004270:DNA, Circular; D004271:DNA, Fungal; D016054:DNA, Protozoan; D004262:DNA Restriction Enzymes