l Continuous exposure to high levels of B. thuringiensis results in
development of resistance in some insects, e.g. Plodia interpunctella
and Cadra cautella. There was a> 250 fold in crease in re sis tance of
P. interpunctella af ter 36 gen er a tions and a seven fold in crease in
C. cautella af ter 21 gen er a tions. This re sis tance was as so ci ated with an
increased susceptibility to Cry IC toxin, i.e. increase in Cry IC binding sites
in the midgut epithelium of these insects.
l Most of the cases of resistance to Cry proteins may be due to changed
binding properties of midgut receptors.
l The transgenic maize inbreeds and their hybrids were completely
resistant to European corn borer Ostrinia nubilalis. Similarly, transgenic
rice plants were significantly resistant to leaf folder and stem borer,
while broccoli were completely resistant to diamond black moth.Applications in Food Improvement
l Biotechnology also promises approach towards improvement of seed
storage protein quality, e.g. cereal seed proteins are deficient in lysine
while those of pulses is deficient in sulphur containing amino acids.
l A new gene encoding a storage protein which is rich in deficient amino
acids is introduced into the crop to correct its amino acid deficiency.
And the transgene is linked to a seed specific promoter to ensure its
expression only in seeds.
l Vicillin is the major seed storage protein of pea, it contains 7% lysine
but no sulphur containing amino acid (methionine and cysteine).
Therefore, its protein is low in sulphur which needs to be ameliorated.
For this, the gene SFAS for storage protein of sunflower albumin is
extracted and fused with vicilin gene promoter. The promoter is known
to confer on genes that correct developmental and tissue specific
expression and accumulation of proteins in seeds.
l Seed storage protein quality is based on isolation and modification of
concerned protein encoding gene sequence, either by replacing one or
few codons with the selected codons or by inserting one or few selected
additional codons at appropriate sites.
l Zein, the prolamine storage proteins of cereals are deficient in essential
amino acids lysine and tryptophan. Single lysine replacements in
N-terminal coding sequence as well as within and between peptide
repeats and double lysine replacement constructs of prolamine genes
have been prepared.
l In another case, the 7S legume seed storage protein, β-phaseolin gene
promoter was transferred to rice. Such transgenic rice plants expressed
the gene in their endosperm and some showed at least 4% of their total
proteins to be β-phaseolin.
l Rice gt 1 gene encodes the major rice seed storage protein and has been
modified to encode higher levels of lysine, tryptophan and methionine
and is driven by its own promoter.
l Scientists have also synthesised and patented a gene encoding a
protein, called CP 3-5 containing 35% lysine and 22% methionine. The
gene CP 3-5 was coupled with seed specific promoters and transferred
into maize.
l An antisense gene is produced by inverting or reversing the orientation
of the protein encoding region of a gene in relation to its promoter. As a
result, the natural sense strand of the gene becomes oriented in 3 '→ 5 '
direction with reference to its promoter and is transcribed. The RNA
produced by this gene has the same sequence as the antisense strand of
normal gene (except for T in place of U) and is therefore, known as
antisense RNA.lThe antisense RNA technology is used to produce transgenic
slow ripening tomato called Flavr savr. The enzyme
polygalactouronase (PG) degrades pectin, the major component
of cell wall leading to softening of fruits and deterioration in fruit
quality. Transgenic tomatoes containing antisense construct of
gene encoding PG show reduced expression of PG, and therefore
slower ripening and fruit softening.Applications of Medical Biotechnology
lA recombinant vaccine contains either a protein or gene
encoding a protein of a pathogenic origin that is immunogenic
and critical to the pathogen function and produced using
recombinant DNA technology.
lSuch vaccines based on recombinant proteins are called
sub unit vaccines.
lThe genes encoding immunogenic proteins are identified and
isolated from a pathogen and expressed in suitable host for
mass production of proteins. The proteins are then extracted
and purified and mixed with suitable stabilisers and adjuvants,
before being ready for immunisation, e.g. Hepatitis B surface
antigen (HBsAg) against hepatitis B virus.
lDNA based vaccines are called DNA vaccines, in which the gene
encoding the relevant immunogenic protein is isolated, cloned
and integrated into a suitable expression vector. The
preparation is introduced into the individual to be immunised.
The gene ultimately gets expressed in the individual and
immunogenic protein produced invokes both humoral and
cell-mediated immunities.
lImmuno-PCR technique uses PCR amplification of a marker
DNA segment attached to an antibody for detection of antigen
for which this antibody is specific. The PCR products are
analysed by gel electrophoresis or labelled using fluorochromes
and haptens. The antigen antibody complex will be formed only
in those wells containing the target antigen.
lA large number of human genes encoding pharmaceutically
valuable proteins have been cloned and expressed in
microorganisms, particularly E. coli and yeast. Several of the
recombinant proteins are used in treatment of various diseases
such as diabetes (by insulin), dwarfism (by human growth
hormone), cancer (by interferons, interleukins, etc), thromosis
(streptokinase) and AIDS (by interferons, granulocyte
macrophage colony stimulating factor).
Gene therapy may be classified into two types :
lGerm line gene therapy, in which the sperms or eggs are
modified by the introduction of functional genes which
integrates into their genome. This change due to gene therapy is
heritable and highly effective in treating genetic disorders but
not applied in humans due to the various technical and ethical
reasons.
lSo matic cell gene ther apy, in which the gene is in tro duced in
so matic cells, es pe cially of those tis sues where ex pres sion of
con cerned gene is crit i cal for health. This approach is again
divided into two groups on the basis of end result of process.
lAug men ta tion ther apy is the type of so matic cell gene ther apy
in which the func tional gene is in tro duced in ad di tion to the
de fec tive gene en dog e nous to the cell. i.e. the mod i fied cell
con tains both the de fec tive as well as normal copies of gene.