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Journal of Bacteriology, October 2000, p. 5486-5494, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

oriT-Directed Cloning of Defined Large Regions from Bacterial Genomes: Identification of the Sinorhizobium meliloti pExo Megaplasmid Replicator Region

Patrick S. G. Chain, Ismael Hernandez-Lucas, Brian Golding, and Turlough M. Finan^*

Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1

Received 1 March 2000/Accepted 29 June 2000

We have developed a procedure to directly clone large fragments from the genome of the soil bacterium Sinorhizobium 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 in Escherichia coli but not in the target organism. Supplying transfer genes in trans specifically transfers the oriT-flanked region, and in this process, site-specific recombination at the oriT sites results in a plasmid carrying the flanked region of interest that can replicate in E. coli from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the oriT of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S. meliloti pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, and repC genes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.

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^* Corresponding author. Mailing address: Department of Biology, McMaster University, Hamilton, Ontario, Canada L85 4K1. Phone: (905) 525-9140, ext. 24400. Fax: (905) 522-6066. E-mail: finan{at}mcmaster.ca.

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Journal of Bacteriology, October 2000, p. 5486-5494, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

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