oriT-Directed Cloning of Defined Large Regions from Bacterial Genomes: Identification of theSinorhizobium meliloti pExo Megaplasmid Replicator Region Academic Article uri icon

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abstract

  • ABSTRACTWe have developed a procedure to directly clone large fragments from the genome of the soil bacteriumSinorhizobium meliloti. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (oriT) together with a plasmid replication origin capable of replicating large clones inEscherichia colibut not in the target organism. Supplying transfer genes in trans specifically transfers theoriT-flanked region, and in this process, site-specific recombination at theoriTsites results in a plasmid carrying the flanked region of interest that can replicate inE. colifrom the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with theoriTof the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from theS. melilotipExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacteriumAgrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of therepA,repB, andrepCgenes from otherRhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.

publication date

  • October 1, 2000