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Use
of
CaMV
as
Vector -
From our knowledge of GaMV's structure and its infection process, we can guess that this virus might help researchers to introduce genes into plants in a way that would encourage their spread and expression. GaMV has several features in complete contrast to those of Ti plasmid, some of which make it quite attractive as a vector.
One useful feature is that the naked DNA is, infective, being able to enter plant cells directly if rubbed onto a leaf with a mild abrasive. Once inside the cells, the DNA is replicated and encapsidated within virus particles, which then invade the rest of the plant.
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Although the CaMV DNA does not become integrated into the chromosomal DNA and is therefore not certain to be handed on to all cells during cell division, its spread throughout the plant means that transformed plants can be effectively cloned by vegetative propagation.
But there are several problems associated with the use of CaMV as a gene cloning vector for plants. Firstly, the genome is so tightly packed with coding regions that there is little room to insert foreign DNA. Most deletions of any significant size destroy virus infectivity except for small modifications in a specific region.
Inserts up to 0.4 kbp long are tolerated but those over 1.3 kbp destroy infectivity of the DNA. Attempts have been made to sidestep this size limitation problem by using a helper virus system, wherein a sub stantial proportion of the viral genome is deleted and replaced with foreign DNA.
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The loss of function could be complemented by coinfection with a normal viral DNA, or viral DNA deleted for a different function.
However, the rescue of viral functions in all experiments has occurred by recombination between the inactive viral genomes and only normal infectious virus recovered.
For a helper virus system to be of any use, the recombinational rescue of altered genomes must be suppressed, albeit the "retroviral like" mode of replication produces a high recombination frequency and alteration of this would affect viral replication.
Secondly, the infection, once established, becomes systemic, spreading throughout the whole plant. This lack of inheritance through the germ line might be advantageous in that the CaMV DNA, and any inserted gene sequence, would be highly amplified in the host plant cells, potentially permitting the expression of large quantities of the foreign gene product.
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How ever, it appears that to propagate CaMV and to allow its movement through out the vasculature of the plant, the DNA must be encapsidated; this would impose serious constraints on the size of foreign DNA to be inserted into the viral genome.
Thirdly, CaMV DNA has multiple cleavage sites for most of the commonly used restriction endonucleases. This would limit the usefulness of wild isolates of GaMV.
To date, the infectivity of the virus particle and its naked DNA, are the most useful assets in terms of GaMV utility and development as a gene cloning vector for plants.
Not only does CaMV have a unique mode of replication, it also displays a peculiar translational coupling of at least three genes that imposes some serious constraints on the use of the virus to express foreign genes in plants.
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Only small genes that do not exceed 300 base pairs in length and are devoid of introns are stably maintained and expressed by CaMV. The only gene of the virus that may be replaced by a foreign gene is the one encoding the aphid acquisition factor, a protein necessary for the spread of the virus in nature.
Genes that have been successfully expressed after introduction into plant cells by CaMV are a bacterial dihydrofolic reductase gene and a human interferon gene. In addition, the CaMV genome contains two strong promoters of gene expression in plant cells that are often spliced into other vectors, including the Ti plasmid, to drive the expression of foreign genes in plants.
One report of the insertion of a foreign gene into a plant describes the use of GaMV. In this experiment a gene from E. coli was inserted into the CaMV genome in place of a gene that was unnecessary for viral replication. GaMV infected turnip cells demonstrated the presence and expression of this gene in several ways.
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The gene chosen for introduction was the dihydrofolic reductase gene (DHFR) from E. coli strain 67. The enzyme coded by this gene is very resistant to the antibiotic methotrexate, whereas most other DHFR enzymes are inhibited by this compound.
The investigators, Brisson and his colleagues from the Frederich Miescher Institute in Switzerland(1984) removed most of open reading frame II with restriction enzymes and ligated the DHFRXN gene in the same place.
Brisson took particular care to reduce the number of excess intergenic bases between the DHFR gene and the adjacent CaMV genes (I and III), which proved to be important for reproduction of the virus.
Infected leaves, ones that developed several days after viral DNA was rubbed on older leaves, contained the E. coli DHFR. This was demon strated by a Western blot, showing that the cells had an enzyme protein that reacted with antibody to the E. coli enzyme. Uninfected leaves lacked the antibody reactive protein.
The plants infected with virus were also resistant to methotrexate. They could incorporate 32p into DNA in the presence of this drug and survive several spray treatments. Control plants were unable to synthesize DNA in the presence of the drug and died.
This experiment shows the feasibility of inserting and expressing a foreign gene, in fact a prokaryotic gene, in a plant cell. As we might expect, the gene was present only in infected cells and there was no evidence that it moved into a stable position in the plant genome.
In fact, when a larger piece of DNA containing the DHFR gene was inserted into the CaMV DNA, it was unstable even in the viral genome and tended to disappear, being undetectable in the progeny virus.
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