In this chapter we deals with BIOTECHNOLOGY AND ITS APPLICATIONS.It is a important chapter for NEET,BOARD,AFMC examinations
12.1 BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE
the three options that can be thought for increasing food production
(i) agro-chemical based agriculture;
(ii) organic agriculture; and
(iii) genetically engineered crop-based agriculture.
The Green Revolution succeeded in tripling the food supply but yet
it was not enough to feed the growing human population. Increased yields
have partly been due to the use of improved crop varieties, but mainly
due to the use of better management practices and use of agrochemicals
(fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive, and further increases in yield with
existing varieties are not possible using conventional breeding.
Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO). GM
plants have been useful in many ways. Genetic modification has:
(i) made crops more tolerant to abiotic stresses (cold, drought, salt, heat).
(ii) reduced reliance on chemical pesticides (pest-resistant crops).
(iii) helped to reduce post harvest losses.
(iv) increased efficiency of mineral usage by plants (this prevents early
exhaustion of fertility of soil).
(v) enhanced nutritional value of food, e.g., golden rice, i.e., Vitamin ‘A’
In addition to these uses, GM has been used to create tailor-made
plants to supply alternative resources to industries, in the form of starches,
fuels and pharmaceuticals.
Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt for short). Bt toxin gene has
been cloned from the bacteria and been expressed in plants to provide
resistance to insects without the need for insecticides; in effect created a bio-pesticide. Examples are Bt cotton, Bt corn, rice, tomato, potato and
Bt Cotton: Some strains of Bacillus thuringiensis produce proteins that
kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). B. thuringiensis
forms protein crystals during a particular phase of their growth. These
crystals contain a toxic insecticidal protein. Actually, the Bt toxin protein exist as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.
Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into the several crop plants such as cotton (Figure 12.1).
The choice of genes depends upon the crop and the targeted pest, as most Bt toxins are insect-group specific. The toxin is coded by a gene
cryIAc named cry. There are a number of them, for example, the proteins encoded by the genes cryIAc and cryIIAb control the cotton bollworms,
that of cryIAb controls corn borer.
Resistant Plants: Several nematodes parasitise a wide variety of plants and animals including human beings. A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great reduction
in yield. A novel strategy was adopted to prevent this infestation which
was based on the process of RNA interference (RNAi). RNAi takes place
in all eukaryotic organisms as a method of cellular defense. This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing).
The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons)
that replicate via an RNA intermediate.
Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA was such that it produced both sense and anti-sense RNA in the host cells. These two RNA’s being complementary to each other formed a double stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the nematode. The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.
12.2 BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE
The recombinant DNA technological processes have made immense impact
in the area of healthcare by enabling mass production of safe and more
effective therapeutic drugs. Further, the recombinant therapeutics do not
induce unwanted immunological responses as is common in case of
similar products isolated from non-human sources. At present, about
30 recombinant therapeutics have been approved for human-use the
world over. In India, 12 of these are presently being marketed.
12.2.1 Genetically Engineered Insulin
Management of adult-onset diabetes is possible by taking insulin at
regular time intervals.Would the insulin isolated from other animals be just as effective as that secreted by the human body itself and would it not elicit an immune response in the human body? Now, imagine if bacterium were available that could make human insulin. Suddenly the whole process becomes so simple. You can easily grow a large quantity of the bacteria and make as much insulin as you need.Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs. Insulin from an
animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein. Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by
disulphide bridges (Figure 12.3). In mammals, including humans, insulin is synthesised as a pro-hormone (like a pro-enzyme, the pro-hormone also needs to be processed before it becomes a fully mature and functional hormone).which contains an extra stretch called the C peptide.This C peptide is not present in the mature insulin and is was getting insulin assembled into a mature form. In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.
12.2.2 Gene Therapy
Gene therapy is a collection of methods that allows correction of a
gene defect that has been diagnosed in a child/embryo. Here genes
are inserted into a person’s cells and tissues to treat a disease.
Correction of a genetic defect involves delivery of a normal gene into
the individual or embryo to take over the function of and compensate
for the non-functional gene.The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function. The disorder is caused due to the deletion of the gene for adenosine deaminase. In some children ADA deficiency can be cured by bone marrow transplantation; in others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. But the problem with both of these approaches that they are not completely curative. As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture outside the body. A functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes, which are subsequently returned to the patient. However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure
12.2.3 Molecular Diagnosis
for effective treatment of a disease, early diagnosis and understanding its pathophysiology is very important. Using conventionaln methods of diagnosis (serum and urine analysis, etc.) early detection is
not possible. Recombinant DNA technology, Polymerase Chain Reaction
(PCR) and Enzyme Linked Immuno-sorbent Assay (ELISA) are some of
the techniques that serve the purpose of early diagnosis.
Presence of a pathogen (bacteria, viruses, etc.) is normally suspected
only when the pathogen has produced a disease symptom. By this time
the concentration of pathogen is already very high in the body. However,
very low concentration of a bacteria or virus (at a time when the symptoms
of the disease are not yet visible) can be detected by amplification of their
nucleic acid by PCR.PCR is now routinely used to detect HIV in suspected
AIDS patients. It is being used to detect mutations in genes in suspected
cancer patients too. It is a powerful techqnique to identify many other
genetic disorders. A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography. The clone having the mutated gene will hence not appear on the photographic film, because the probe will not have complementarity with the mutated gene.
ELISA is based on the principle of antigen-antibody interaction.
Infection by pathogen can be detected by the presence of antigens
(proteins, glycoproteins, etc.) or by detecting the antibodies synthesised
against the pathogen.
12.3 TRANSGENIC ANIMALS
Animals that have had their DNA manipulated to possess and express an
extra (foreign) gene are known as transgenic animals. Transgenic rats,
rabbits, pigs, sheep, cows and fish have been produced, although over
95 per cent of all existing transgenic animals are mice.
(i) Normal physiology and development: Transgenic animals can
be specifically designed to allow the study of how genes are
regulated, and how they affect the normal functions of the body
and its development, e.g., study of complex factors involved in growth
such as insulin-like growth factor. By introducing genes from other
species that alter the formation of this factor and studying the
biological effects that result, information is obtained about the
biological role of the factor in the body. (ii) Study of disease: Many transgenic animals are designed to increase our understanding of how genes contribute to the development of disease. These are specially made to serve as models for human diseases so that investigation of new treatments for diseases is made possible. Today transgenic models exist for many human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer’s.
(iii) Biological products: Medicines required to treat certain human diseases can contain biological products, but such products are often expensive to make. Transgenic animals that produce useful biological products can be created by the introduction of the portion of DNA (or genes) which codes for a particular product such as human protein (α-1-antitrypsin) used to treat emphysema. Similar attempts are being made for treatment of phenylketonuria (PKU) and cystic fibrosis. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4 grams per litre). The milk contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies than natural cow-milk.
(iv) Vaccine safety: Transgenic mice are being developed for use in testing the safety of vaccines before they are used on humans. Transgenic mice are being used to test the safety of the polio vaccine.
If successful and found to be reliable, they could replace the use of monkeys to test the safety of batches of the vaccine.
(v) Chemical safety testing: This is known as toxicity/safety testing.
The procedure is the same as that used for testing toxicity of drugs. Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals. They are
then exposed to the toxic substances and the effects studied. Toxicity testing in such animals will allow us to obtain results in less time.
12.4 ETHICAL ISSUES
The manipulation of living organisms by the human race cannot go on
any further, without regulation. Some ethical standards are required to evaluate the morality of all human activities that might help or harm living organisms. Going beyond the morality of such issues, the biological significance of such things is also important. Genetic modification of organisms can have unpredicatable results when such organisms are introduced into the ecosystem. Therefore, the Indian Government has set up organisations such as GEAC (Genetic Engineering Approval Committee), which will make decisions regarding the validity of GM research and the safety of introducing GM-organisms for public services. The modification/usage of living organisms for public services (as food
and medicine sources, for example) has also created problems with patents granted for the same.
Rice is an important food grain, the presence of which goes back
thousands of years in Asia’s agricultural history. There are an estimated
200,000 varieties of rice in India alone. The diversity of rice in India is
one of the richest in the world. Basmati rice is distinct for its unique
aroma and flavour and 27 documented varieties of Basmati are grown
in India. There is reference to Basmati in ancient texts, folklore and
poetry, as it has been grown for centuries. In 1997, an American
company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed the company to sell a ‘new’ variety of
Basmati, in the US and abroad. This ‘new’ variety of Basmati had actually
been derived from Indian farmer’s varieties. Indian Basmati was crossed
with semi-dwarf varieties and claimed as an invention or a novelty. The
patent extends to functional equivalents, implying that other people
selling Basmati rice could be restricted by the patent. Several attempts
have also been made to patent uses, products and processes based on
Indian traditional herbal medicines, e.g., turmeric neem. If we are not
vigilant and we do not immediately counter these patent applications,
other countries/individuals may encash on our rich legacy and we may
not be able to do anything about it.
Biopiracy is the term used to refer to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned without compensatory payment.
Most of the industrialised nations are rich financially but poor in biodiversity and traditional knowledge. In contrast the developing and
the underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources. Traditional knowledge related to bio-resources can be exploited to develop modern applications and can
also be used to save time, effort and expenditure during their commercialisation.