Exploitation of Recombinant Techniques Robin Augustine

Exploitation of Recombinant Techniques

The Recombinant DNA technique was first proposed by Peter Lobban, a graduate student,
with A. Dale Kaiser at the Stanford University Department of Biochemistry. The technique was then
realized by Lobban and Kaiser; Jackson, Symons and Berg; and Stanley Norman Cohen, Chang,
Herbert Boyer and Helling., in 1972–74. They published their findings in papers including the 1972
paper "Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40:
Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of
Escherichia coli", the 1973 paper "Enzymatic end-to-end joining of DNA molecules" and the 1974
paper "Construction of Biologically Functional Bacterial Plasmids in vitro",all of which described
techniques to isolate and amplify genes or DNA segments and insert them into another cell with
precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the
discovery, isolation and application of restriction endonucleases by Werner Arber, Daniel Nathans,
and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine. Cohen and Boyer
applied for a patent on the Process for producing biologically functional molecular chimeras which
could not exist in nature in 1974. The patent was granted in 1980.

The major growth seen in the
biotechnology industry in recent decades has
largely been driven by the exploitation of
recombinant DNA techniques. The initial
benefits have been predominantly in the
biomedical area, with products such as
vaccines and hormones that have received
broad public approval. In the environmental
biotechnology and industrial ecology sectors,
biotechnology has the potential to make
significant advances through the use of
genetically modified (GM) microbial inoculants
that can reduce agri-chemical usage or
remediate polluted environments. Although
many GM inoculants have been developed
and tested under laboratory conditions,
commercial exploitation has lagged behind.
The development of GM rice containing
vitamin A precursors (‘golden rice’) is an
example of how this technology can be applied
to develop products that are of societal benefit
(Ye et al. 2000). Biotechnology is also poised
to make a major impact in the development of
improved microbial inoculants for use in
agriculture. Wild-type bacterial and fungal
species are currently employed as biological
control agents, biofertilisers or phyto-
stimulators. There are limitations that restrict
the efficacy of some of these inoculants. Such
limitations can include poor survival of the
inoculants in particular soils, and inconsistent
or low production of required metabolites (van
Veen et al. 1997). GM technology has the
capacity to allow the engineering of new
The most notable applications of the
recombinant technology having direct impact
on humanity have been:
1. Large scale production of therapeutic
protein such as insulin, hormones,
vaccine and interleukins using
recombinant microorganisms.
2. Production of humanized monoclonal
antibodies for therapeutic application
3. Production of insect resistant cotton
plant by incorporation of insecticidal
toxin of Bacillus thuringiensis (Bt
cotton plant).
4. Production of golden rice (rice having
vitamin A) by incorporating three
genes required for its synthesis in rice
plant.
5. Bioremediation by the use of
recombinant organisms and
6. Use of genetic engineering techniques
in forensic medicine.
1. Exploitation of rDNA Technology in
Industrial Microbiology
It is genetically possible to "tailor" the
microorganisms for the production of any
microbial metabolite - vitamin, amino acid or
enzyme. Gene cloning extends the genome of
the microorganism by allowing the introduction
of novel genes from comparatively unrelated
species. The cloning of genes from higher
eukaryotes, particularly from man and his
domestic animals, has been seen to offer even

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greater industrial potential. Which microbes
strains in which these problems are overcome. should then be used as universal recipients for
This requires a thorough understanding of the such genes and hence as production
molecular basis of action in wild-type strains organisms. The two most ideal are the
(Bloemberg & Lugtenberg 2001; Walsh et al. prokaryote, Escherichia coli and the
2001).
 Exploitation of Recombinant Techniques
eukaryote, Saccharomyces cerevisiae.
Some of the important products which
gene cloning may make available in near
future. The above proteins could be obtained
on large scale through fermentation by
methods, relatively cheaper than the
conventional ones. For example, human
growth hormone was previously extracted from
the pituitary gland of cadavers and was mostly
in short supply. Now, increase in supply
should help more patients. Equally important is
the development of new vaccines through
gene-cloning. Genes for single antigens can
be cloned and expressed by bacteria and a
purified antigen which has not been derived
directly from the pathogenic organism or virus
may be used as a vaccine. In this way,
vaccines for viral hepatitis and foot-and-mouth
disease have been developed.
Types of biomolecules produced through
recombinant DNA technology
 Recombinant Hormones
Insulin (and its analogs), growth
hormone, follicle stimulating hormone,
salmon calcitonin.
 Blood products
Albumin, thrombolytics, fibrinolytics,
and clotting factors ( Factor VII, Factor
IX, tissue plasminogen activator,
recombinant hirudin )
 Cytokines and growth factors
Interferons, interleukins and colony
stimulating factors (Interferon, α, β and
γ, erythropoietin, interlukin-2, GM-
CSF, GCSF )
 Monoclonal antibodies and related
products
Mouse, chimeric or humanized; whole
molecule or fragment; single chain or
bispecific; and conjugated (rituximab,
trastuzmab, infliximab, bevacizumab)
 Recombinant Vaccines
Recombinant protein or peptides, DNA
plasmid and anti-idiotype (HBsAg
vaccine, HPV vaccine)
 Recombinant Enzymes
Dornase– α (Pulomozyme), Acid
glucosidase (Myozyme), α –L-
iduronidase (Aldurazyme) and Urate
Oxidase
 Miscellaneous products
Bone morphogenic protein, conjugate
antibody, pegylated recombinant
proteins, antagonist
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1.1 Production of enzymes
The commercial production and use of
enzymes is already a well-established
part of the biotechnology industry. Enzymes
are used in brewing, food processing, textile
manufacture,the leather industry, washing
powders, medical applications, and basic
scientific research to name just a few
examples. In many cases the enzymes are
prepared from natural sources, but in recent
years there has been a move towards the use
of enzymes produced by recombinant DNA
methods,where this is possible. In addition to
the scientific problems of producing a
recombinant-derived enzyme,there are
economic factors to take into account, and in
many cases the cost–benefit analysis makes
the use of a recombinant enzyme unattractive.
Broadly speaking,enzymes are either high-
volume/low cost preparations for use in
industrial scale operations, or are low-
volume/high value products that may have a
very specific and relatively limited market.
There is a nice twist to the gene manipulation
story in that some of the enzymes used in the
procedures are now they produced using
rDNA methods.
Many of the commercial suppliers list
recombinant variants of the common
enzymes,suc h as polymerases (particularly
for PCR) and others. Recombinant enzymes
can sometimes be engineered so that their
characteristics fit the criteria for a particular
process better than the natural enzyme, which
increases the fidelity and efficiency of the
process. In the food industry,one area that
has involved the use of recombinant enzyme
is the production of cheese. In cheese
manufacture, rennet (also known as rennin,c
hymase,or chymosin) has been used as part
of the process. Proteases and lipases are
commonly used to assist cleaning by
degradation of protein and lipid-based
staining. A recombinant lipase was developed
in 1988 by Novo Nordisk A/V (now known as
Novozymes). The company is the largest
supplier of enzymes for commercial use in
cleaning applications. Their recombinant
lipase was known as Lipolase,whic h was the
first commercial enzyme developed using
rDNA technology and the first lipase used in
detergents. A further development involved an
engineered avriant ofLipolase called Lipolase
Ultra,whihc gvi es enhanced fat removal at low
wash temperatures.
1.2 Therapeutic products for use in
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human healthcare
Recombinant DNA products for use in
medical therapy can be divided into three main
 Exploitation of Recombinant Techniques
categories. Firstly, protein products may be
used for replacement or supplementation of
human proteins that may be absent or
ineffective in with a particular illness.
Secondly, proteins can be used in specific
disease therapy,to alleviate a disease state
by intervention. Thirdly,the production of
recombinant vaccines is an area that is
developing rapidly and which offers great
promise.
The widespread condition diabetes
mellitus (DM) is usually caused by cells in the
islets of Langerhans in the pancreas failing to
produce adequate amounts of the hormone
insulin. Many millions of people worldwide are
affected by DM, and the World Health
Organization estimates that the global
incidence will double by 2025. Sufferers are
classed as having either insulin dependent
DM (IDDM) or non-insulin dependent DM
(NIDDB). Insulin-dependent patients
obviously require the hormone. As DM is
caused by a problem with a normal body
constituent (insulin), therapy falls into the
category of replacement or
supplementation. Banting and Best
developed the use of insulin therapy in
1921,and for the next 60 or so years diabetics
were dependent on natural sources of
insulin,with the attendant problems of supply
and quality. In the late 1970s and early 1980s
recombinant DNA technology enabled
scientists to synthesise insulin in bacteria,
with the first approvals granted by 1982.
Recombinant-derived insulin is now available
in several forms,and has a major impact on
diabetes therapy. One of the most widely
used forms is marketed under the name
Humulin by the Eli Lilly company. In an early
method for the production of recombinant
insulin,the insulin A- and B-chains were
synthesised separately in two bacterial strains.
The insulin A- and B-genes were placed under
the control of the lac promoter,so that
expression of the cloned genes could be
switched on by using lactose as the inducer.
Following purification of the A- and B-
chains,they were linked together by a chemical
process to produce the final insulin molecule.
A development of this method involves the
synthesis of the entire proinsulin polypeptide
from a single gene sequence. The product is
converted to insulin enzymatically.
2. Exploitation of rDNA technology in
Medicine and forensic medicine
Studies in bacteria and bacterial
viruses have led to methods to manipulate and
recombine DNA in unique and reproducible
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ways and to amplify these recombined
molecules millions of times. Once properly
identified, the recombinant DNA molecules
can be used in various ways useful in
medicine and human biology. There are many
applications for recombinant DNA technology.
Cloned complementary DNA has been used to
produce various human proteins in
microorganisms. Insulin and growth hormone
have been extensively and successfully tested
in humans and insulin has been licensed for
sale. Mass production of bacterial and viral
antigens with recombinant DNA technology is
likely to provide safe and effective vaccines for
some disorders for which there is no
prevention. The cloned probes for the human
α- and β-globin loci, for specific disease
genes, such as the Z allele of α-antitrypsin,
and for random genomic sequences are
proving useful for prenatally diagnosing
disorders and preventing their clinical
consequences. The diagnosis and treatment
of human is area in which genetic
manipulation is beginning to have a
considerable effect. Many therapeutic proteins
are now made by recombinant DNA methods,
and the number available is increasing
steadily. Thus the treatment of conditions by
recombinant-derived products is already well
established. Progress in both of these areas is
of course closely linked to our increasing
knowledge of the human genome, and thus
many new developments in medical and
forensic applications will appear as we
decipher genome.
2.1 Diagnosis and characterisation
of medical conditions
Genetically based diseases (often
called simply ‘genetic diseases’) represent one
of the most important classes of disease,par
ticularly in children. A disorder present at birth
is termed a congenital abnormality,and
around 5% of newborn babies will suffer from
a serious medical problem of this type. In most
of these cases there will be a significant
genetic component in the aetiology (cause)
of the disease state. It is estimated that about
a third of primary admissions to paediatric
hospitals are due to genetically based
problems,whilst some 70% of cases
presenting more than once are due to genetic
defects. In addition to genetic problems
appearing at birth or in childhood,it seems that
a large proportion of diseases presenting in
later life also have a genetic cause or
predisposition. Thus medical genetics,in its
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traditional non-recombinant form,has already
had a major impact on the diagnosis of
disease and abnormality. The development of
 Exploitation of Recombinant Techniques
molecular genetics and rDNA technology has
not only broadened the range of techniques
available for diagnosis,but has also opened up
the possibility of novel gene-based treatments
for certain conditions.
2.2 Diagnosis of infection
In addition to genetic conditions that
affect the individual,rDNA technology is also
important in the diagnosis of certain types of
infection. Normally,bacterial infection is
relatively simple to diagnose,once it has taken
hold. Thus the prescription of antibiotics may
follow a simple investigation by a general
practitioner. A more specific characterisation
of the infectious agent may be carried out
using microbiological culturing techniques,and
this is often necessary when the infection does
not respond well to treatment. Viral infections
may be more difficult to diagnose,although
conditions such as Herpes infections are
usually obvious. Despite traditional methods
being applied in many cases,there may be
times when these methods are not
appropriate. Infection by the human
immunodeficiency virus (HIV) is one case in
point. The virus is the causative agent of
acquired immune deficiency syndrome
(AIDS). The standard test for HIV infection
requires immunological detection of anti-HIV
antibodies. However,these antibodies may not
be detectable in an infected person until
weeks after initial infection,b y which time
others may have been infected. A test such as
this,where no positive result is obtained even
though the individual is infected,is a false
negative. The use of DNA probes and PCR
technology circumvents this problem by
assaying for the actual viral
DNA in the T-lymphocytes of the patient,thus
permitting a diagnosis before the antibodies
are detectable. Other examples of the use of
rDNA technology in diagnosing infections
include tuberculosis (caused by the
bacterium Mycobacterium tuberculosis),
human papilloma virus (HPV) infection,and
Lyme disease (caused by the spirochaete
Borrelia burgdorferi).
2.3 Patterns of inheritance
Although diagnosis of infection is an
important use of rDNA technology,it is in the
characterisation of genetic disease that the
technology has perhaps been most applied in
medicine to date. Before dealing with some
specific diseases in more detail,it may be
useful to review the basic features of
transmission genetics,and outline the range of
factors that may determine how a particular
disease state presents in a patient. Since it
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was rediscovered in 1900,the work of Gregor
Mendel has formed the basis for our
understanding of how genetic characteristics
are passed on from one generation to the
next.We have already seen that the human
genome is made up of some 3 billion base-
pairs of information. This is organised as a
diploid set of 46 chromosomes,ar ranged as
22 pairs of autosomes and one pair of sex
chromosomes. Prior to reproduction,the
haploid male and
female gametes (sperm and oocyte
respectively) are formed by the reduction
division of meiosis,whic h reduces the
chromosome number to 23. On fertilization of
the oocyte by the sperm,diploid status is
restored,with the zygote receiving one
member of each chromosome pair from the
father,and one from the mother. In males the
sex chromosomes are X and Y,in females
XX,and thus it is the father that demteinr es
the sex otfhe child. Traits may be controlled
by single genes,or by many genes acting in
concert. Single-gene disease traits are known
as monogenic disorders,whilst those involving
many genes are polygenic. Inheritance of a
monogenic disease trait usually follows a basic
Mendelian pattern,and can therefore often be
traced in family histories by pedigree analysis.
A gene may have alleles (different forms) that
may be dominant (exhibited when the allele is
present) or recessive (the effect is masked
by a dominant allele). With respect to a
particular gene,indi viduals are said to be
either homozygous (both alleles the same) or
heterozygous (the alleles are
different,perhaps one dominant and one
recessive). Patterns of inheritance of
monogenic traits can be associated with the
autosomes, as either autosomal dominant or
autosomal recessive, or may be sex-linked
(usually with the X chromosome,thus showing
X-linked inheritance). In addition to the
nuclear chromosomes, mutated genes
associated with the mitochondrial genome can
cause disease. As the mitochondria are
inherited along with the egg,these traits show
maternal patterns of inheritance.
2.4 Treatment using rDNA
technology – gene therapy
Once genetic defects have been
identified and characterised,the possibility of
treating the patient arises. If the defective
gene can be replaced with a functional copy
(sometimes called the transgene,as in
transgenic) that is expressed correctly,the
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disease caused by the defect can be
prevented. This approach is known as gene
therapy,and is one of the most promising
 Exploitation of Recombinant Techniques
aspects of the use of gene technology in
medicine. There are two possible approaches
to gene therapy: (i) introduction of the
transgene gene into the somatic cells of the
affected tissue,or (ii) introduction into the
reproductive (germ line) cells. These two
approaches have markedly different ethical
implications. Most scientists and clinicians
consider somatic cell gene therapy an
acceptable practice,
no more morally troublesome than taking an
aspirin. However,tink erring with the
reproductive cells,with the probability ofgerm
line transmission,is akin to altering the gene
pool of the human species,whic h is regarded
as unacceptable by most people. Thus genetic
engineering of germ cells is an area that is
likely to remain off limits at present. There are
several requirements for a gene therapy
protocol to be effective.
Firstly,the gene defect itself will have
been characterised,and the gene cloned and
available in a form suitable for use in a clinical
programme. Secondly,there must be a system
available for getting the gene into the correct
site in the patient. Essentially these are vector
systems that are functionally equivalent to
vectors in a standard gene cloning protocol –
their function is to carry the DNA sequence
into the target cells. This also requires a
mechanism for physical delivery to the
target,whic h may involve inhalation,injection
or other similar methods. Finally,if these
requirements can be satisfied,the inserted
gene must be expressed in the target cells if a
non-functional gene is to be ‘corrected’.
Ideally,the faulty gene would be replaced by a
functional copy. This is known as gene
replacement therapy,and requires
recombination between the defective gene and
the inserted functional copy. Due to technical
difficulties in achieving this reliably in target
cells,the alternative is to use gene addition
therapy. In addition therapy there is no
absolute requirement for reciprocal exchange
of the gene sequences,and the inserted gene
functions alongside the defective gene. This
approach is useful only if the gene defect is
not dominant,in that a dominant allele will still
produce the defective protein,whic h may
overcome any effect of the transgene. Therapy
for dominant conditions could be devised
using antisense mRNA,in which a reversed
copy of the gene is used to produce mRNA in
the antisense configuration. This can bind to
the mRNA from the defective allele and
effectively prevent its translation.
A further complication in gene therapy
is the target cell or tissue system itself. In
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some situations it may be possible to remove
cells from a patient and manipulate them
outside the body. The altered cells are then
replaced,with function restored. This approach
is known as ex vivo gene therapy. It is mostly
suitable for diseases that affect the blood
system. It is not suitable for tissue-based
diseases such as DMD of CF,in which the
problem lies in dispersed and extensive tissue
such as the lungs and pancreas (CF) or the
skeletal muscles (DMD). It is difficult to see
how these conditions could be treated by ex
vivo therapy,and therefore the technique of
treating these conditions at their locations is
used. This is known as in vivo gene therapy.
Features of these two types of gene therapy
are illustrated in Fig. 11.6,with both
approaches having been used with some
success.
Getting transgenes into patients-
The biology of the system must be established
and evaluated,and then the physical method
for Treatment using rDNA technology – gene
therapy getting the gene to the site of action
has to be considered. Deciding on the best
method for addressing these two aspects of a
therapeutic procedure is one important part of
the strategy. As with vectors for use in cloning
procedures,vir uses are an attractive option for
delivering genes into human cells. We can use
the term vector in its cloning context,as a
piece of DNA into which the transgene is
inserted. The viral particle itself is often called
the vehicle for delivery of the transgene,
although some authors describe the whole
system simply as a vector system. Three main
viral systems have been developed for gene
therapy protocols, based on retroviruses,
adenoviruses and adeno-associated
viruses. The advantage of viral systems is
that they provide a specific and efficient way of
getting DNA into the target cells.
Eg: Gene therapy for adenosine deaminase
deficiency, Gene therapy for cystic fibrosis,
2,5 Human monoclonal antibody
production
Monoclonal antibodies (Mab) are very
specific immunoglobulin that exhibit a wide
range of biological activities. In addition to use
in diagnostics, antigen binding sites of
antibody molecules have great potential for
developing bioactive peptides Because of their
specific ligand binding activity, they were
considered as the magic bullets as
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hypothesized by Paul Ehrlich. Hybridoma
technology, which used the fusion of
myoeloma and B cells, helped in the in vitro
 Exploitation of Recombinant Techniques
production of monoclonal antibody. However,
this technology developed by Kohler and
Milestein was not very helpful as most of the
antibodies were of murine origin and have the
problems of their low immunogenicity.
Application of recombinant DNA technology
resulted in development of chimeric and
humanized antibody with high efficiency and
activity. Because of their efficacy in cancer,
there have been tremendous activities in
developing monoclonal antibodies for human
therapy. In humans, antibodies are classified
as member of five family or isotypes. These
are named as immunoglobulin alpha, (IgA),
delta (IgD), epsilon ( IgE), gamma ( IgG) and
mu (IgM). Most of the isotypes have molecular
weight around 160-190 kD except IgM whose
molecular weight is around 1000kD due to its
pentameric nature. The most prevalent
antibody in human is IgG and majority of the
therapeutic antibodies are of IgG types. Even
though antibodies act on wide varieties of
pathways, therapeutic antibodies work on one
of the following four ways (a) as
immunotoxicotherapy where the antibody
prevent or reverse toxic effect of venom, toxin,
drug or ligand (b) destruction of target cell,
where the antibodies are used to destruct the
target cell such as lymphocytes, cancer cells
etc. (c) alteration of the cell function and finally
(d) antibody mediated drug delivery, where the
drug is conjugated with the antibody for
specific targeting. For the large-scale
production of monoclonal antibodies,
expression of monoclonal antibody genes is
accomplished through recombinant DNA
technology. More than 20 monoclonal
antibodies have been approved for human
uses. Table 5 lists some of the important
monoclonal antibodies as a result of
recombinant DNA technology. It is expected
that in near future, due to production of
humanized antibodies using recombinant DNA
technology, there will be many more Mab in
the market.
3. Exploitation of rDNA Technology in
Agriculture
The genetic manipulation of plants has
been going on since prehistoric times, when
early farmers began carefully selecting and
maintaining seeds from their best sow for the
next season. Plant breeders have cross
fertilized related plants to provide next
generation plants with new characteristics
such as higher yield, resistance to diseases
and better nutrient content long before the
science of genetics was developed.
Recombinant DNA technology can be used for
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insertion of genes in plants not only from
related plant species, but also from unrelated
species such as microorganisms. This process
of creation of transgenic plants is far more
precise and selective than traditional breeding.
Application of recombinant technology is
primarily for the production of transgenic
plants with higher yield and nutritional values,
increased resistance to stress and pests.
Several commercially important transgenic
crops such as maize, soybean, tomato, cotton,
potato, mustard, rice etc. have been
genetically modified. During the last couple of
decades, considerable progress has been
made to understand the function of genes,
isolation of novel genes and promoters as well
as the utilization of these genes for the
development of transgenic crops with
improved and new characters. There are many
potential application of plant genetic
engineering. In fact, in 2002, more than 5.5
million farmers worldwide cultivated about 58.7
million hectares (about 148 million acres)
crops that were genetically manipulated for
herbicide tolerance, insect resistance, delayed
fruit ripening and improved oil quality.
Application of recombinant DNA technology
has primarily helped in producing three major
types of transgenic plant having improved
performances. These are:
(1) Development of stress tolerant plant
(2) Development of plant having improved
yield
(3) Transgenic plant as a source of
biopharmaceuticals
3.1 Development of stress tolerant
plant
(a) Plant resistant to environmental stress:
Plants need to cope up with abiotic stresses
such as drought, cold, heat and soils that are
too acidic or salty to support plant growth.
While plant breeders have successfully
incorporated genetic resistance to biotic
stresses into many crop plants through
crossbreeding, their success at creating crops
resistant to abiotic stresses has been more
limited, largely because few crops have close
relatives with genes for resistance to these
stresses. Therefore rDNA technology is being
increasingly used to develop crops that can
tolerate difficult growing conditions.
Genetically modified tomato and canola plants
that tolerate salt levels 300 percent greater
than non-genetically modified varieties have
been developed. Other researchers have
identified many genes involved in cold, heat
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and drought tolerance found naturally in some
plants and bacteria. Scientists in Mexico have
produced maize and papaya that are tolerant
 Exploitation of Recombinant Techniques Robin Augustine

to the high levels of aluminum that significantly increase in yield along with dramatic reduction
impede crop plant productivity in many in pesticides use. The most notable example is
developing countries. Bt cotton (which contains Cry/Ac gene) that is
(b) Herbicide Resistant plant: Many effective resistant to a notorious insect pest Bollworm
broad spectrum herbicides do not distinguish (Hellicoperpa armigera) and only last year
between weeds and crops, but crop plants can (2002) Bt cotton was adopted in India.
be modified to make them resistant to Biotechnology has opened up new
herbicides, so as to eliminate weeds more avenues for natural protection for plants by
selectively. For example, the herbicide providing new biopesticides, such as
TM microorganisms, that are toxic to targeted crop
Roundup contains the active ingredient
pests but do not harm humans, animals, fish,
glyphosate, which kills plants by binding to the
birds or beneficial insects. As biopesticides act
active site of enzymes called
in unique ways, they can even control pest
enolpyruvalshikimate phosphate synthase
population that have developed resistance to
(EPSP synthase). This enzyme is critical for
conventional pesticides. Using recombinant
the synthesis of aromatic amino acids.
DNA technology, the gene that makes these
Roundup is an extremely effective herbicide
microorganisms lethal to certain insects can
but it kills almost all species of plants,
be transplanted into the plants on which that
including most crop plants. On the other hand,
insect feeds. The plant that once was a food
it is very safe for humans and animals
source for the insect now kills it, lessening the
because they do not have EPSP synthase. By
need to spray crops with chemical pesticides
using rDNA technology, modified EPSP
to control infestation. One such microorganism
synthase gene (that produced enzymes that
is commonly found soil bacterium Bacillus
were still functional but were not inhibited by
thuringiensis. The spores of Bacillus
glyphosate) have been introduced into crop
thuringiensis (Bt) contain a crystalline protein
plants such as cotton and soyabean. These
(Cry) which breaks down to release a toxin,
genetically modified plants were found to be
known as delta-endotoxin which is highly toxic
highly resistant to treatment with Roundup.
to lepidopteran larvae. This toxin binds the
Genes that provide resistance to other
intestinal lining and creates pores resulting in
herbicides such as sulfonyl ureas,
an ion imbalance, paralysis of the digestive
gluphosinates etc. have also been developed
system, and consequent deathof the insect. Bt
and transferred to produce various transgenic
toxin sprays and powders have been in use for
plants.
many years. Different Cry genes, also known
(c) Insect resistant plant: To minimize crop
as Bt genes have been identified, cloned and
damage by insects, mites and nematodes,
characterized. Effective gene constructs have
farmers use synthetic pesticides extensively
made it possible to deliver these genes into
which cause severe effects on human health
plant tissues so that they are expressed at
and environment. The transgenic technology
levels high enough to kill the insects. The Bt
provides an alternative and innovative method
genes are effective against different orders of
to improve pest control management which is
insects. Bt cotton and maize which have
eco friendly, effective, sustainable and
increased resistance to boll worms have been
beneficial in term of yield. This involves
developed and cultivated since 1996. Farmers
genetic incorporation of toxic gene ( product of
get benefited by saving costs by using less of
which is lethal to insect ) in to the plant. This
traditional pesticides. However, one of the
kill the insects without use if dangerous
major concerns about Bt based transgenics is
insecticide thus has double benefit in crop
the possibility of development of toxin resistant
improvement. The first genes available for
insects. Efforts are also underway to identify
genetic engineering of crop plats for pest
and transfer other genes from Bt, which can
resistance were Cry genes (popularly known
impart insecticidal properties to the plants.
as Bt genes) from a bacterium Bacillus
One example in this is transfer of vip gene i.e.
thuringiensis. These are specific to particular
vegetative insecticidal proteins, for which the
group of insect pests, and are not harmful to
trials are being conducted in some countries.
other useful insects like butter flies and silk
(d) Disease resistance plant: Plants are
worms. Transgenic crops (e.g. cotton, rice,
maize, potato, brinjal, cauliflower, cabbage
etc.) with Bt genes have been developed and
susceptible to viral, bacterial and fungal
diseases. Much progress has been made in 7
evolving transgenic plants resistant to viruses.
such transgenic varieties proved effective in
For example, expression of a gene that
controlling the insect pests and it has been
encodes the coat protein of tobacco mosaic
claimed worldwide that it has led to significant
virus (TMV) in transgenic tobacco plants has
 Exploitation of Recombinant Techniques
been shown to cause the plants to resist TMV
infection. A number of other viral resistant
plants species have been developed including
squash and potatoes. Genetic engineering of
crop plants for resistance to fungal and
bacterial infections has been more difficult.
However, by studying the protective genes
that are expressed in naturally disease-
resistant plants, an encouraging progress has
been made. The proteins encoded by these so
called pathogenesis related proteins (PR
proteins) can, in some cases, provide limited
disease protection in transgenic plants. There
are several strategies for engineering plants
for viral resistance and these utilizes the
genes from virus itself (e.g. the viral coat
protein gene). The virus-derived resistance
has given promising results in number of crop
plants such as tobacco, tomato, potato, alfalfa,
and papaya. Some viral resistant transgenic
plants like papaya resistance to papaya ring
spot virus have been commercialized in some
countries. Plants respond to pathogens by
inducing a variety of defense responses like
pathogenesis-related proteins (PR proteins),
enzymes that degrade/destroy fungal cell wall
(chitinase), antifungal proteins and
compounds, phytoalexins, etc. Several
transgenic crop plants showing increased
resistance to fungal pathogens are being
raised with genes coding for the different
compounds mentioned above.
3.2 Development of plant having
improved yield
(a) Increasing yield
In addition to increase crop productivity by
using built-in protection against diseases,
pests, environmental stresses and weeds to
minimize losses, attempts are being made to
use biotechnology to improve crop yields
directly. Researchers at Japan's National
Institute of Agrobiological Resources added
maize photosynthesis genes to rice to
increase its efficiency of converting sunlight to
plant starch and increased yields by 30
percent. Other scientists are altering plant
metabolism by blocking gene action in order to
shunt nutrients to certain plant parts. Yields
increase as starch accumulates in potato
tubers and not leaves, or oilseed crops, such
as canola, allocate most fatty acids to the
seeds. Crops that have better accessibility to
the micronutrients they need are also being
developed. Mexican scientists have genetically
modified plants to secrete citric acid, a
naturally occurring compound, from their roots.
In response to the slight increase in acidity,
minerals bound to soil particles, such as
calcium, phosphorous and potassium, are
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released and made available to the plant.
Nitrogen is the critical limiting element for plant
growth and researchers from many scientific
disciplines are tearing apart the details of the
symbiotic relationship that allows nitrogen-
fixing bacteria to capture atmospheric nitrogen
and provide it to the plants that harbor them in
root nodules as given below:
(1) Plant geneticists in Hungary and
England have identified the plant
gene and protein that enable the
plant to establish a relationship with
nitrogen-fixing bacteria in the
surrounding soil.
(2) Microbial geneticists at the University of
Queensland have identified the
bacterial gene that stimulates root
nodule formation.
(3) Collaboration among molecular
biologists in the European Union,
United States and Canada yielded
the complete genome sequence of
one of the nitrogen-fixing bacteria
species.
(4) Protein chemists have documented the
precise structure of the bacterial
enzyme that converts atmospheric
nitrogen into a form the plant can
use.
(b) Increase in quality of plant products
One of the most successful research
efforts to change the characteristics of a plant
produce was carried out with tomatoes.
Tomatoes need to be picked while still green
so that they are firm enough to withstand
mechanical handling and transport.
Unfortunately, they do not develop the same
flavor and texture of vineripened tomatoes.
Softening of tomatoes and many other fruits is
caused by the enzyme pectinase or
polygalacturonase (PGA). This enzyme
digests the pectin polysaccharide that cements
the plant cells together. Softening of the fruit is
caused, in part by this breakdown of pectin. In
order to reduce the levels of PGA in ripening
tomatoes, researchers placed the PGA gene
in reverse orientation relative to the CaMV 35S
promoter. This results in transcription of an
antisense RNA that is complementary to the
normal sense PGA mRNA. Although the exact
mechanism is unknown, antisense RNA is
able to arrest the translation of the
endogenous PGA mRNA in the tomato fruit.
Transgenic tomato plants that express an
antisense PGA gene only have about 5 to 10%
8
of normal PGA levels. Fruits of these plants
have normal color and flavor but they soften
more slowly and can be picked and processed
 Exploitation of Recombinant Techniques
after they are ripe. They also have a higher
content of soluble solids and are therefore
better than normal tomatoes for processed
tomato products. Transgenic lines of potato
having increased levels of starch also have
been developed by introducing a gene
construct that expresses a gene from bacteria
that produce an enzyme that enhances starch
biosynthesis. A promoter from a potato gene
that encodes the major protein in potato tubers
has been used, so that the expression of the
introduced gene is limited to the tuber. Tubers
accumulate approximately 3 to 5% more
starch than normal potatoes and when they
are deep fried absorb less oil and yield chips
having fewer calories. Some of the other value
added transgenic crops include:
(a) Golden rice: containing beta carotene
to overcome vitamin A deficiency in
regions where rice is the staple food
(b) Canola containing high levels of oleic
acids and laurate
(c) Barley containing feed enzymes
(d) tomatoes which does not rot in room
temperature
(e) Other vegetables and fruits with
delayed ripening as well as modified
flavour characteristics
Transgenic crops with improved nutrition
quality have already been produced by
introducing genes involved in the metabolism
of vitamins, minerals and amino acids. Few
examples of genetic modification of nutritional
quality are described below.
Vitamin A: Vitamin A deficiency can lead to
night blindness and skin disorders, among
others. About 124 million children worldwide
are deficient in vitamin A and a quarter of a
million go blind each year due to vitamin A
deficiency. The staple food rice is extremely
low in vitamin A, and therefore the
improvement of vitamin A content is very
important. In a remarkable example of genetic
engineering, Prof. Ingo Potrykus and Dr. Peter
Beyer developed genetically engineered rice
(popularly known as ‘Golden Rice’), which is
enriched in pro-vitamin A by introducing three
genes involved in the biosynthetic pathway for
carotenoid, the precursor for vitamin A. The
seeds of Golden Rice are yellow in colour
because of pro-vitamin A is produced in the
entire grain.
Seed Protein Quality: The Nutritional quality
of cereals and legumes are limited because of
deficiency of the essential amino acids, i.e.
lysine in cereals, and methionine and
tryptophan in pulses. Two genetic engineering
approaches have been used to improve the
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seed protein quality. In the first case, a
transgene (e.g. gene for protein containing
sulphur rich amino acids) was introduced into
pea plant (which is deficient in methionine and
cysteine, but rich in lysine) under the control of
seed specific promoter. In the second
approach, the endogenous genes are modified
so as to increase the essential amino acids
like lysine in the seed proteins of cereals.
The gas hormone, ethylene is involved in the
regulation of fruit ripening. Therefore, ripening
can be slowed down by blocking or reducing
ethylene production. This can be achieved by
introducing ethylene forming gene(s) in a way
that will suppress its own expression in the
crop plants. Such fruits ripen very slowly
(however, they can be ripen by ethylene
application) and are very important for export
to longer distances without spoilage as they
show longer-self life due to slow ripening. A
notable example of this kind is the ‘Flavr Savr’
transgenic tomatoes, which were
commercialized in U.S about 6 year ago.
3.3 Transgenic plant as a source of
bio pharmaceuticals
Plants are among the most efficient
bioreactors which produce quantities of
material with sunlight and soil based nutrients
as inputs. Attempts are being made to replace
the traditional fermentation procedure for the
production of biopharmaceuticals to plant
based production. The benefits of using plants
are the ability to increase production at low
cost by planting more acres, rather than
building fermentation capacity, lower capital
and operating cost, simplified downstream
processing etc. Therapeutic drugs to treat
cancer, infectious diseases, autoimmune
diseases, cardiovascular diseases and other
conditions and several vaccines can
potentially be grown in plants. Plant transgenic
technology is being used to produce a plant
that will generate a seed that expresses a
desired therapeutic protein. This seed can
propagate under the right growing conditions
to yield plants and seed stock for producing
the desired protein. The desired protein can be
extracted from the seed to make a
biopharmaceutical. Plant based therapeutics
are expected to be much more cost effective.
For example, Dow Plant Pharmaceuticals is
using corn to grow pharmaceuticals by
designing and selecting the plant which will
contain the active pharmaceutical within the
endosperm seed compartment. Benefits of
producing the pharmaceuticals in the corn
9
include long term storage advantage, easier
purification in view of limited number of soluble
seed proteins in a corn seeds, low microbial
 Exploitation of Recombinant Techniques
load, low proteolytic activity and specialized
promoters to enable expression of the protein
in specific parts of the plants.
3.4 Therapeutic proteins, enzymes
and diagnostics
Transgenic plants can also produce a
variety of proteins used in diagnostics for
detecting human diseases and therapeutics for
curing human and animal diseases in large-
scale with low cost. The monoclonal
antibodies, blood plasma proteins, peptide
hormones and cytokinins are being produced
in transgenic plants and their parts such as
tobacco (in leaves), potato (in tubers),
sugarcane (in stems) and maize (in seed
endosperm). Plants are amazing and cheap
chemical factories that need only water,
minerals, sun light and carbon dioxide to
produce thousands of sophisticated chemical
molecules with different structures. Given the
right genes, plants can serve as bioreactors to
modified or new compounds such as amino
acids, proteins, vitamins, plastics,
pharmaceuticals (peptides and proteins),
drugs, and enzymes for food industry and so
on. Some of the potential and remarkable
examples of this kind are described here.
3.5 Edible vaccines
Crop plants offer cost-effective
bioreactors to express antigens which can be
used as edible vaccines. The genes encoding
antigenic proteins can be isolated from the
pathogens and expressed in plants and such
transgenic plants or their tissues producing
antigens can be eaten for
vaccination/immunization (edible vaccines).
The expression of such antigenic proteins in
crops like banana and tomato are useful for
immunization of humans since banana and
tomato fruits can be eaten raw. The edible
vaccines that are produced in transgenic
plants have great advantages like the
alleviation of storage problems, easy delivery
system by feeding and low cost as compared
to recombinant vaccines produced by bacterial
fermentation. Vaccinating people against
dreadful diseases like cholera and hepatitis B
by feeding them banana/ tomato, and
vaccinating animals against important
diseases such as foot and mouth disease by
feeding them sugar beets could be a reality in
the near future.
3.6 Metabolic engineering and
Secondary products
Plant biotechnology will lead to
improved plant sources for the production of
valuable secondary metabolites mentioned in
previous section on cell culture products.
Over-expression of the gene which encode for
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the first enzyme in a pathway generally results
in higher levels of the desired end product,
and this has been successfully done in the
enhancement of taxol production from the
transformed tissue cultures of Taxus sp.
Another strategy involves use of
Agrobaccterium rhizogenes to induce the
excessive formation of secondary roots in
plants that normally produce useful secondary
metabolites in this region. Transgenic plants
can be used as factories to produce
polyhydroxy butyrate (PHB, biodegradable
plastic). Genetically engineered Arabidopsis
plants produced PHB globules exclusively in
their chloroplasts without effecting plant
growth and development The large scale
production of PHB may be easily achieved in
tree plants like populus, where PHB can be
extracted from leaves. Industry has already
begun to explore the production of
biodegradable plastics from transgenic plants.
4. Exploitation of rDNA technology in
Veterinary
Applications to animal agriculture can
be expected in animal health management,
improved crops and feeds, manipulation of
animal physiology, and genetic improvement
of livestock species, Improved diagnostic
reagents and vaccines that will improve herd
health are currently under development. Yield
of crop plants such as corn will be increased
and the nutritional value of these feeds
improved through applications of recombinant
DNA technology. Administration of exogenous
hormones synthesized by bacteria holds great
promise for increasing the yield of milk and
possibly meat. Research on the transfer of
cloned genes into animals has progressed
rapidly and has recently been accomplished in
sheep and swine. Tissue-specific and
developmentally regulated expression of
transferred genes now seems possible with
defined gene promoter sequences. Several
applications of these biotechnologies can be
expected within 5 to 10 yr, whereas others
may require longer periods of research. The
21st century will herald a new era in animal
science research and applications, with
recombinant DNA and gene transfer playing
major roles.
4.1 In Animal Health Management
Improved diagnostic kits are being
detect disease conditions (Kobbe,
10
developed that utilize monoclonal antibodies to
1985).
These kits will allow veterinarians to diagnose
quickly (and economically) and design
treatment regimens on the farm rather than
relying on more expensive and time-
 Exploitation of Recombinant Techniques
consuming laboratory tests. Because these
new tests often rely on the detection of an
antibody produced in response to a disease
agent, it is difficult to distinguish between the
antibodies stimulated
by a vaccine or by the disease itself. Despite
these problems, tests will soon be available for
bovine brucellosis, avian bronchitis and
porcine pseudorabies. Neonatal calves have
been protected against
fatal enteric colibacillosis with a monoclonal
antibody to the K99 pilus antigen (Sherman et
al., 1983).
With the molecular cloning of the
FMD(foot-and-mouth disease) genome and
analysis of the viral proteins, it was possible to
determine that only one protein (VP1) was
immunogenic (Rowlands and Brown, 1985).
The VP1 was produced by genetic engineering
techniques in the form of a chimeric protein
containing part of the trp E protein from
Escherichia coli (Kleid et al., 1981). This
protein produced an immune response that
protected cattle and swine from contracting
this disease.
4.2 Improved Crops and Feeds for
Animal Production
An application of rDNA technoloy in
plant breeding and crop improvement leads to
the improvement of animal husbandary.
4.3 Manipulation of Animal
Physiology
Expression of eukaryotic genes in
bacteria has led to the production of pure
protein products such as hormones in
quantities sufficient for physiological
experiments (Seeburg et al., 1983).
recombinantly-derived bovine growth hormone
(rbGH) can increase milk production by as
much as 41% when injected daily, while not
affecting milk composition (Bauman et al.,
1985a). The effects of exogenous GH on the
growth of meat animals also are being actively
investigated. The control and regulation of
meat animal growth is complex and involves a
number of hormones (Etherton and Kensinger,
1984). Recent studies with porcine GH
indicated a 10% increase in growth rate when
administered daily to young swine (Chung et
al., 1985).
4.5 Genetic Improvement of
Livestock Species
Recent advances suggest the
possibility of improving livestock species by
the direct transfer of cloned genes to early
embryos. Since the first reports of successful
gene transfer to mice (Gordon et al., 1980;
Brinster et al., 1981; Costantini and Lacy,
1981; Gordon and Ruddle, 1981; Wagner et
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al., 1981a,b), astounding progress has been
made (see reviews of Gordon, 1983; Gordon
and Ruddle, 1985; Palmiter and Brinster,
1985; Petters, 1985). Production of transgenic
swine, sheep and rabbits has recently been
reported (Hammer et al., 1985b). These
experiments are possible through refinements
in experimental mammalian embryology that
include embryo recovery, in vitro culture, and
transfer (Mapletoft, 1984). Micromanipulations
of embryos have been improved to allow direct
microinjection of individual nuclei within the
embryo (Seidel, 1982; Gordon and Ruddle,
1983). Continued rapid progress should be
expected.
5. Exploitation of rDNA technology in
Environment
A vast majority of applications of
environmental biotechnology use naturally
occurring microorganisms (bacteria, fungi,
etc.) to identify and filter manufacturing waste
before it is introduced into the environment.
Bioremediation program involving the use of
microorganisms are currently in progress to
clean up contaminated air, tracks of land,
lakes and waterways. Recombinant
technology helps in improving the efficacy of
these processes so that their basic biological
processes are more efficient and can degrade
more complex chemicals and higher volumes
of waste materials. Recombinant DNA
technology also is being used in development
of bioindicators where bacteria have been
genetically modified as 'bioluminescors' that
give off light in response to several chemical
pollutants. These are being used to measure
the presence of some hazardous chemicals in
the environment. Other genetic sensors that
can be used to detect various chemical
contaminants are also undergoing trials and
include sensors that can be used to track how
pollutants are naturally degrading in ground
water. For example when gene such as the
mercury resistance gene (mer) or the toluene
degradation (tol) gene is linked to genes that
code for bioluminescence within living
bacterial cells, the biosensor cells can signal
extremely low levels of inorganic mercury or
toluene that are present in contaminated
waters and soils by emitting visible light, which
can be measured with fiber-optic fluro meters.
Conventional pesticides
fungicides are extensively used in modern
agriculture and some are now considered to
contribute to environmental pollution. In
11and
addition, some of these chemicals are
potentially hazardous, and residues can
 Exploitation of Recombinant Techniques
constitute a risk to food quality and human
health. The replacement of these with efficient
biological inoculants will make a significant
contribution to sustainable agriculture. The
second area where microbial inoculants will
have an impact is the decontamination of soil
and water polluted with heavy metals, organo-
chemicals and other toxic man-made
pollutants, a process termed bioremediation
(reviewed in Gadd 2000; Pieper & Reineke
2000). This approach has been further
developed using plants as delivery/support
systems for microbial inoculants in the
rhizosphere (rhizoremediation). Although
wildtype inoculants can be used to remediate
contaminated sites, there are major
bottlenecks in the process, including
bioavailability of the pollutant. In addition,
many man-made pollutants are xenobiotic
chemicals, containing structural elements not
typically found in nature. Bioremediation of
these recalcitrant pollutants can be best
achieved through the construction of strains
containing enzymes with new specificities or
new degradative pathways (Timmis & Pieper
1999; de Lorenzo 2001). The primary
requirements for the successful exploitation of
a GM inoculant are the development of an
effective strain that is robust in the
environment, and securing of the necessary
regulatory approval from the appropriate
competent authority. With regard to improving
the efficacy of biocontrol inoculants, the initial
focus has been on understanding the
molecular basis of biocontrol with the aim of
reprogramming genetic pathways. This work
established that synthesis of biocontrol
metabolites is under complex control in many
bacteria. For example, production of
secondary metabolites in Pseudomonas
species can be subject to quorum sensing,
transcriptional and post-transcriptional controls
(Aarons et al. 2000; Bloemberg & Lugtenberg
2001; Haas et al. 2000). Re-regulation or
bypassing these controls can lead to improved
biocontrol performance under laboratory
conditions (Delany et al. 2000). Approaches to
engineer improved inoculants for
bioremediation have recently been reviewed
and often involve the introduction of
heterologous genes into suitable host strains
(Timmis & Pieper 1999; Pieper & Reineke
2000; de Lorenzo 2001).With inoculants
developed for biocontrol and bioremediation,
as well as for other uses in Agbiotech such as
biofertilisation or phytostimulation it is
essential to carry out field-based trials in order
to accurately assess the efficacy of the strain.
In an open environment, inoculants are
Robin Augustine
competing and interacting with a diverse
community of organisms that can have
profound effects on the survival and
performance of the introduced strain (Sayler&
Ripp 2000;Walsh et al. 2001). Furthermore,
field-trial studies allow researchers to carry out
important impact assessment, gene flow and
persistence studies (Gilbert et al. 1993; de Leij
et al. 1995; Moënne-Loccoz et al. 2001)
required by regulatory bodies prior to the
commercial release of GM (and in some cases
non-GM) inoculants.
References
John P. Morrissey1, Ultan F. Walsh1, Anne
O’Donnell1, Yvan Moënne-Loccoz2 &
Fergal O’Gara1(2002)., Exploitation of
genetically modified inoculants for
industrial ecology Applications, Antonie
van Leeuwenhoek 81: 599–60
Bloemberg GV & Lugtenberg BJ (2001)
Molecular basis of plant growth
promotion and biocontrol by
rhizobacteria. Curr. Opin. Plant. Biol. 4:
343–350.
Robert M. Agriculture,Recombinant DNA,
Gene Transfer and the Future of Animal, J
Anim Sci 1986. 62:1759-1768.
Amulya K. Panda, RECOMBINANT DNA
TECHNOLOGY AND BIOTECHNOLOGY,
Application of Biotechnology.
Fermentation Microbiology and
Biotechnology Charles FA Bryce and
Mansi El-Mansi, Tylor and Francis , USA
(1999).
Mahaffee WF & Kloepper JW (1997)
Bacterial communities of the rhizosphere
and endorhiza associated with field-
grown cucumber plants inoculated with a
plant growth-promoting rhizobacterium
or its genetically modified derivative. Can.
J. Microbiol. 43: 344–353.
***
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