Ti Plasmid as Gene Vector,Foreign Genes,Natural Gene Vectors,Oncogenesis

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	Ti Plasmid as Gene Vector - Because of their natural ability to insert DNA into plant cells, the Ti plasmids have become the subject of extensive study for use in genetic engineering of plants. Ti plasmids are natural gene vectors which contain all the functions necessary for the transfer, stable incorporation and expression of genetic information in plants. Studies on the signals involved and required for the transfer and stabilization of the T -DNA have revealed how the Ti plasmid can be used as a vector for insertion of foreign genes into the plant genome. First, the transposon Tn7 inserted in the T-region is co transferred to and maintained in the plant cell without major rearrangements.
The co transfer of insertions at different positions in both the octopine and the nopaline T-region demonstrated involvement of "ends" for stabilization of the T-region. Therefore, one may expect that any segment between these "ends" would be co transferred and stabilized in the plant nucleus. Moreover, studies of the func tional organization of T-DNA have revealed that none of the genes involved in oncogenesis or suppression of differentiation are required for T -DNA transfer. However, the "ends", presumably including the DNA sequences around the 25 bp repeat box, have to occur in cis of the Ti plasmid.
A plasmid containing the T-region in trans of the Ti plasmid cannot induce tumors. Therefore, genes have to be engineered into the T-region of the Ti plasmids. Due to the large size of the Ti plasmids and the requirement of many genes for virulence, direct cloning into the Ti plasmid is impossible. So a technique for shuttling genes into the Ti plasmid via an intermediate vector. was worked out. The intermediate vector contains a region of homology with the T-region of the Ti plasmid into which genes can be inserted. The engineered segments of the intermediate vector have been worked out.
One is derived from a broad host range vector (W- and P-type plasmids), capable of replicating stably in Agrobacterium. Recombinants between vector and Ti plasmid can be isolated by selecting for cotransfer of the cointegrate plasmid during conjugation. This method requires conjugation in order to select for the first recombination, and subsequent screening for the second recombination or immediate selection by incompatibility for the substituted recombinants. The second type of intermediate vector is a pBR322 derivative, which can be mobilized efficiently by F- or I-type plasmids when the mob protein is complemented in trans (by a compatible ColE plasmid).
	Since the pBR322-derivatives cannot replicate in Agrobacterium, they are maintained only through homologous cointegration with the Ti plasmid. The second recombinant (or substituted T-region) is obtained after propagation of the cointegrates by screening for loss of the pBR322 vector markers. The three possibilities are outlined. As the final goal in the breeding of normally engineered plants, gene transfer by the I-DNA has to be separated from its tumorigenic activities. This has been shown to be possible since none of the T-DNA genes are essential for T-region transfer. Regenerates are fertile and transmit T-DNA linked genes in a Mendelian way to the progeny.

T-DNA can also be a source of expression signals for plant genes. Currently, T-DNA expression signals are being adapted for the insertion of foreign genes in an intermediate expression vector. Our knowledge about the Ti plasmids allows us to transfer any DNA sequence into plant cells, from which normal plants can be regenerated.

Recent work has shown that all that is needed for agrobacteria to infect and transform plant cells is an intact T-DNA region and another region of the Ti plasmid called 'vir'. More important from a practical standpoint is that these two regions do not have to be on the same plasmid.

Of an Agrobacterium harbors a Ti plasmid containing the vir region and another plasmid containing the T-DNA, the bacteria can transform plant cells and the T-DNA (and whatever other genes have been inserted into it) will be incorporated into the plant genome.