Biotechnology, as you would have learnt essentially deals with industrial scale production of biopharmaceuticals and biological using genetically modified microbes, fungi, plants and animals. The applications of biotechnology include therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment and energy production.
(A) BIOTECHNOLOGICAL APPLICATION IN AGRICULTURE :
Let us take a look at 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 batter 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 is there any alternative path that our undestanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Use of genetically modified crops is a possible solution.
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. Is there any alternative path that our understanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Uses genetically modified crops is possible solution.
Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO).
GM has been used to create tailor made plants to supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.
Use of genetically modified (GM) plant:
1. To enhance nutritional quality of food
eg. Golden rice - Vitamin A enriched rice (In this rice gene of B-carotene is transfered).
2. Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).
3. Helped to reduce post harvest losses eg. Flavr Savr Tomato : Transgenic variety of Tomato - Flavr Savr due to the inhibition of polygalacturonase enzyme which degrades pectin. So that tomato variety remains fresh and retain flavour much longer. Flavr Savr Tomato develop by anti-sense technology.
4. Increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).
5. To produce pharmaceutical product
eg. Production of Hirudin : Hirudin is a protein that prevents blood clotting. The gene incoding hirudin wes chemically synthesized and transfered into Brassica napus. Where hirudin accumulates in seeds. The hirudin is purified and used as medicine.
6. To produce herbicide resistant plant eg. First transgenic plant was tobacco. It contains resistant gene against weedicide (Glyphosate).
7. Pest-resistant crops reduced reliance on chemical pesticides.
i. Insect resistant plant
eg. 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.
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.
Bacillus thuringiensis, produces crystal [Cry] protein. This Cry protein is toxic to Larvae of certain insects. Each Cry protein is toxic to a different group of insects. The gene encoding cry protein is called "cry gene". This Cry protein isolated and transferred into several crops. A crop expressing a cry gene is usually resistant to the group of insects for which the concerned Cry protein is toxic. There are a number of them, for example, the proteins encoded by the genes crylAc and cryllAb control the cotton bollworms, that of crylAb controls corn borer.
However, gene symbol italics, e.g., cry. The first letter or the protein symbol, on the other hand is always capital and the symbol is always written in roman letters. e.g., Cry.
ii. Nematode resistant plant:
Several nematodes parasitise a wide variety of plants and animals including human beings. A nematode Meloidogyne 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 antisense 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.
BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE
A. Genetically Engineered Insulin
It is a proteinaceous hormone having 51 Amino acids arranged in two polypeptides A and B having 21 and 30 Amino Acids respectively and joined by S-S disulphide
bridges.
Sir Edward sharpy-Shafer (1916) was the first to note that diabetes of some persons was because of failure of some islands of pancreas to produce a substance which he called insulin (Derived from the latin, insula, meanning island).
Banting and best (1921) were the first to isolate insulin from dog's pancreas and used it to cure diabetes in man. The first genetically engineered insulin obtained by recombinant DNA technique with the help of E-Coli was produced by the American firms, Eli-Lilly on July 5, 1983. It has been given the trade name humulin and has been approved for clinical use.
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. In mammals, including humans, insulin is synthesised as a prohormone (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 removed during maturation into insulin. The main challenge for production of insulin using rDNA techniques 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.
B.Gene Therapy:
A new system of medicine gene therapy, may develop to treat some hereditary diseases such as SCID, haemophilia etc.
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 involved 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.
C. Medical Diagnosis of Disease (Molecular diagnosis)
You know that for effective treatment of a disease, early diagnosis and understanding its pathophysiology is very important. Using conventional 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 technique to identify any 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 complimentarity 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.
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 percent of all existing transgenic animals are mice.
1. 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 intoducing 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.
2. 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 humane 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.
3. 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 that natural cow-milk.
4. 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.
5. 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.
Note:-
1. First transgenic animal was mouse contain gene for growth hormone. This enlarged mouse was known as supermouse.
2. First introduced transgenic crop in India (2002) is Bt-cotton.
3. Charles weismann of University of Zurich, obteined interferon through recombinant E. Coli (1980).
4. Microbe have been engineered to produce Human growth Hormone (HGH) for curing dwarfism.
5. Vaccines which are produced by genetic engineering e.g., for Hepatitis-B and Herpes virus.
6. Nitrogen fixation genes may be transferred from bacteria to the major food crops to boost food production without using expensive fertilizers.
7. Bioremediation: in pollution control, microbes have been engineered to break up the crude oil spills. Dr. Ananda Mohan chakarborthi introduced plasmids from different strains into a single cell of Pseudomonas putida. The result was new genetically engineered bacterium which would clean the oil spills called "Superbug" (Oil eating bug). He transferred four types of genes/ plasmids in this bacteria. These are OCT, XYL, CAM and NAH.
8. Genetic modified food- the food is prepared from genetically modified crop (transgenic) is called genetically modified food or G.M. Food.
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