Biomedical Engineering Reference
In-Depth Information
gene transfer from prokaryote to eukaryote occurring with moderate frequency in
nature. The genes coding the information required for the insertion of this small
piece of DNA including the insertion sequence itself, are carried on a plasmid,
described in Chapter 2, called Ti plasmid whose structure is shown in Figure 9.4.
The process of infection is stimulated by exudate from a plant which has been
wounded by some means not necessarily the result of infection, for example by
frost damage. The vir genes are activated leading to nicks being introduced at the
borders of the T-DNA leading to the release of one of the strands of the double
stranded DNA. A copy is made of the remaining strand by the usual methods of
DNA synthesis and repair thus restoring the status quo of the Ti plasmid. The
single stranded piece of T-DNA is free to be transferred into the plant cell through
a wound site and on into the plant cell nucleus. Here the complementary strand
is synthesised in the normal way using the plant cell enzymes and the resulting
double stranded DNA integrates into the plant genome. The T-DNA comprises
three genes including those for plant hormones and opines, as mentioned above.
Once the T-DNA is integrated into the host plant cell genome, these genes may
be expressed leading to the establishment of crown gall disease. Agrobacterium
tumefaciens then contains a very effective delivery system for bacterial genes into
a plant cell; a natural process which is utilised by genetic engineers to introduce
'foreign' genes into plants as described in Chapter 9.
Cauliflower mosaic virus
The study of plant viruses has, historically, lagged behind that of animal or
bacterial viruses due to difficulties in their culture and isolation. While some
may be grown in isolated plant cultures, many require the whole plant and some
also require an insect intermediate either simply as a means of transmission
between plants, or additionally as a site for virus replication. Transfer of
viruses between plants by insects is the most common means, especially by
insects which penetrate and suck plants, aphids being an example. However,
there are other routes such as transfer by plant parts through infected seed, tubers
or pollen. Other agents are those able to penetrate plant tissue which include
soil nematodes and parasitic fungi. The genetic material of most plant viruses
is RNA, either double or single stranded. Caulimovirus, or more commonly,
CaMV is unusual in having a DNA genome. This has proven very fortuitous
for genetic engineering as it possesses two very strong constitutive promoters,
the 35S and the 19S; for an explanation of the term 'promoter' see Chapter 2.
Since these promoters originated from a plant virus, any construct made with
the intention of expressing the 'foreign' gene in a plant has a higher likelihood
of the signals being recognised by the transcription machinery of the plant than
if they were derived from, for example bacterial promoters. These promoters
have proved very successful and particularly the 35S, often designated in
publications as '35S CaMV', has become almost the archetype promoter to
drive the expression of 'foreign' genes in plants. Examples of these are given
in Chapter 9. However, while genetic engineering, building on these and other
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