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Sequence determination and characterization of the replicator region in the tumor-inducing plasmid pTiB6S3
S Tabata1, P J Hooykaas, A Oka
1Department of Biology, Faculty of Science, Nagoya University, Aichi, Japan.
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.
Insights
Researchers identified a 4.2-kb region sufficient for replication of the tumor-inducing plasmid pTiB6S3 in Agrobacterium tumefaciens. This region contains three replication genes (repA, repB, repC) crucial for plasmid stability.
Area of Science:
- Molecular Biology
- Microbiology
- Plant Pathology
Background:
- The tumor-inducing plasmid (pTiB6S3) is central to Agrobacterium tumefaciens-mediated plant transformation.
- Previous studies isolated a 6.8-kb miniplasmid containing the replicator region of pTiB6S3.
Purpose of the Study:
- To delineate the minimal DNA region required for pTiB6S3 replication.
- To identify and characterize the genes responsible for replication within this region.
- To compare the replication mechanisms with those of the hairy root-inducing plasmid (pRiA4b).
Main Methods:
- Subcloning of the pTiB6S3 replicator region into ColE1-based vectors.
- Site-directed mutagenesis of the identified replication genes.
- Assessing autonomous replication of mutant plasmids in Agrobacterium tumefaciens.
- Nucleotide sequence analysis and in vitro protein synthesis.
Main Results:
- A 4.2-kb DNA fragment was identified as sufficient for autonomous replication.
- Three open reading frames, designated repA, repB, and repC, were identified within the 4.2-kb region.
- Mutations in repA and repB attenuated replication, while repC mutations abolished it.
- The identified rep genes share similarities with those of pRiA4b, but functional complementation between the two plasmids was not observed.
Conclusions:
- The 4.2-kb region containing repA, repB, and repC is essential for pTiB6S3 replication.
- The rep genes exhibit functional conservation but also distinct characteristics compared to pRiA4b.
- Understanding these replication mechanisms is crucial for optimizing Agrobacterium-mediated genetic engineering in plants.