Sequence determination and characterization of the replicator region in the tumor-inducing plasmid pTiB6S3

Abstract

The replicator region of the 195-kilobase-pair (kb) tumor-inducing plasmid pTiB6S3 was previously identified by isolation of a 6.8-kb miniplasmid (B.P. Koekman, P.J.J. Hooykaas, and R.A. Schilperoort, Plasmid 7:119-132, 1982). This miniplasmid was joined to ColE1-based vectors and subjected to mutagenesis. The resulting mutant plasmids were examined for their ability to replicate autonomously in Agrobacterium tumefaciens. It was found that a 4.2-kb region was sufficient for displaying replication characteristics similar to those of the parental pTiB6S3. Nucleotide sequence analysis of this 4.2-kb region revealed the presence of three possible reading frames in the same direction (repA, repB, and repC). Proteins coded for by these frames were identified by in vitro synthesis in a coupled transcription-translation system. The replicating ability became attenuated by repA and repB mutations but was completely abolished by repC mutations. The size, arrangement, and mutational effects of the three rep genes were quite similar to those of the rep genes that were previously identified in the hairy root-inducing plasmid pRiA4b. However, defects caused by rep mutations in one plasmid were unable to be complemented by corresponding functions in the other plasmid.