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Ti
Plasmid
without
Tumorgenic
Genes - The disadvantage of Ti plasmid as a vector is its tumorigenic property. This problem is overcome by constructing a T-DNA that does not contain the tumorigenic genes.
Such a T-DNA has been constructed and cloned by at least three research groups.
One small plasmid created by Horsch and colleagues (1984) at the Monsanto Company (Murphy and Thompson, 1988), contains a T-DNA with a nopaline synthetase gene and the right hand border sequence of T-DNA that is essential for integration.
It also has a segment of DNA that is the same in nopaline type and octopine-type Ti plasmids; this segment allows insertion of the new T -DNA into an octopine type Ti plasmid by recombination.
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It contains a gene for resistance to streptomycin that aids in selecting the hybrid plasmids that contain the new T-DNA. Finally, it has a gene for neomycin phosphotransferase II. This gene makes the plant cells resistant to the antibiotic kanamycin. The kanamycin resistance trait can be used for selection of transformed plant cells.
In their 1984 study Horsch and his colleagues infected an A. tumefaciens strain with an octopine type Ti plasmid with the small plasmid containing the new T-DNA. This plasmid was not stable unless it recombined with the Ti plasmid; so a selection for streptomycin resistance picked out recombinants.
The recombinants had Ti plasmids with one left hand and two right hand border sequences.
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When these plasmids infected Nicotiana plumbaginifolia cells, at least some of the infections resulted in the transfer of only the new T-DNA genes to the plant genomes. Colonies of such cells could be identified by their resistance to kanamycin and their ability to regenerate plants.
Non transformed cells were not resistant to kanamycin and cells that received a larger T-DNA sequence, including the tumorigenic genes, could not regenerate plants. Of all the cells exposed to the A. tumefaciens, about 1 % were transformed; of all the transformants, about 10% were able to regenerate.
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A special feature of Horsch’s study was the demonstration of sexual transmission of the kanamycin resistant gene. Flowers from regenerated, kanamycin resistant plants formed seed following self-fertilization. The seeds were planted, and the new seedlings tested for resistance.
About three quarters were resistant to kanamycin, as would be expected if a single, dominant gene had been added to one chromosome of the parent plant's cells. The experiment shows the stability of the genes introduced by Ti plasmid transformation.
In an experiment reported by Murai of the University of Wisconsin and his collaborators from Agrigenetics, Inc. (Murphy and Thompson, 1988), a cloned gene for phaseolin, the storage protein in bean seeds, was inserted into cultured cells of sunflower.
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Three different hybrid Ti plasmids were used to infect the sunflower cells. In the first plasmid the phaseolin gene minus its promoter and its first 11 codons was fused to the promoter and first 88 Condons of the Ti plasmid octopine synthetase gene.
The octopine synthetase gene is normally expressed in all crown gall tissue. In the second plasmid the entire phaseolin gene with its own promoter was placed in the middle of the tml gene with the 5 -terminal end of the phaseolin gene toward the 5-end of the tml gene.
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The third plasmid was contructed like the second except that the 5 -end of the phaseolin gene pointed toward, the 3 -end of the tml gene. The tml gene is also normally expressed in crown gall tissue but we might expect that the expression of the phaseolin gene in the second and third plasmids would be controlled by the phaseolin promoter, which is normally turned on only in developing seeds and not in crown gall tissue.
Crown gall tissue infected with the first plasmid (octopine synthetase promoter) had sequences among its poly (A)+ RNAs that reacted with phaseolin cDNA and directed the synthesis in vitro of protein that reacted with the antibody to authentic phaseolin.
In this situation the octopine synthetase promoter (and not the phaseolin gene, its introns, or its 3' adjacent sequences) apparently controlled transcription. Crown gall tissue infected with the other two plasmids (phaseolin promoter) also produced phaseolin poly (A) + RNA but in much lower quantities, about one twentieth that of the first plasmid.This observation is consistent with the idea that in these cases also transcription was controlled by the promoter.
However, the amount of RNA, while low, was much more than would have been found in bean leaves and the amount of RNA in galls induced by the second and third plasmids was not always equal. Thus it is possible that sequences outside the gene, as well as species or tissue related factors, also influence the amount of transcription.
Although antiphaseolin reactive protein was detected in the crown gall tissue, the amount in each case was very low. It was found that proteases quickly degraded the phaseolin produced by translation. Breakdown of this gene product is another level of regulation of gene expression, though perhaps not a natural one in bean plants.
The experiments with phaseolin described above were performed with a Ti plasmid containing tumorigenic genes. Therefore it was not possible to regenerate sunflower plants and study the expression of the inserted genes in different tissues. Subsequently, tobacco cells were transformed with "disarmed" Ti plasmid containing a phaseolin gene.
Plants were regenerated from these cells and induced to flower. Phaseolin appeared primarily in the protein bodies of the seeds of these plants and not in other tissues. Apparently the transferred phaseolin gene contained regulatory elements that assured the correct localization of the gene product at both the tissue and sub cellular levels of organization, even in the genetic background of a plant from an entirely different family.
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