Biotechnology and its Applications

In this chapter: applications in agriculture (GM crops, Bt cotton, RNAi); applications in medicine (insulin, gene therapy, molecular diagnosis); transgenic animals; ethical issues, GEAC, patents and biopiracy.

Biotechnological applications in agriculture

There are three options for increasing food production: agrochemical-based agriculture, organic agriculture, and genetically engineered crop-based agriculture. Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called genetically modified organisms (GMOs).

Benefits of GM plants

• More tolerant to abiotic stresses (cold, drought, salt, heat).
• Reduced reliance on chemical pesticides (pest-resistant crops).
• Reduced post-harvest losses.
• More efficient use of minerals by plants, preventing early exhaustion of soil fertility.
• Enhanced nutritional value, e.g. Golden rice, rich in vitamin A.
• Tailor-made plants supply alternative resources to industries: starches, fuels and pharmaceuticals.

Bt cotton

• Some strains of Bacillus thuringiensis produce proteins that kill certain insects: lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes).
• The bacterium forms protein crystals during a particular growth phase. These contain a toxic insecticidal protein that exists as an inactive protoxin, so it does not kill the bacterium itself.
• Once an insect ingests it, the alkaline pH of the gut solubilises the crystals and converts the protoxin into an active toxin. The toxin binds to the surface of midgut epithelial cells, creates pores, and causes cell swelling and lysis, leading to the insect's death.
• The toxin is coded by genes named cry. Proteins encoded by cryIAc and cryIIAb control cotton bollworms; cryIAb controls corn borer.

Gene names are written in lower-case italics (cryIAc); the proteins they code are written with a capital letter and not italicised (Cry IAc).

Pest-resistant plants: RNA interference (RNAi)

• The nematode Meloidogyne incognita infects the roots of tobacco plants and reduces yield.
• RNAi is a method of cellular defence in all eukaryotic organisms: it silences a specific mRNA by a complementary dsRNA molecule that binds to it and prevents translation.
• The source of this complementary RNA can be an infection by viruses with 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. They produced both sense and anti-sense RNA in the host cells. These two complementary strands formed dsRNA, which started RNAi and silenced the specific mRNA of the nematode. The parasite could not survive in the transgenic host.

Biotechnological applications in medicine

Genetically engineered insulin

• Insulin used for diabetes was earlier extracted from the pancreas of slaughtered cattle and pigs; this caused allergies and other reactions to the foreign protein in some patients.
• Insulin has two short polypeptide chains, A and B, linked by disulphide bridges.
• In mammals it is made as a pro-hormone with an extra stretch called the C peptide. The C peptide is absent in mature insulin and is removed during maturation.
• In 1983, Eli Lilly, an American company, prepared two DNA sequences corresponding to the A and B chains of human insulin, introduced them into plasmids of E. coli, produced the chains separately, extracted them and combined them by creating disulphide bonds to form human insulin (Humulin).

Gene therapy

• A collection of methods to correct a gene defect diagnosed in a child or embryo: genes are inserted into a person's cells and tissues to treat a disease. A normal gene is delivered to 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.
• ADA is essential for the immune system to function; the disorder is caused by deletion of the gene for ADA.
• Some children can be cured by bone marrow transplantation; others by enzyme replacement therapy (functional ADA given by injection). Neither is completely curative.
• Gene therapy method: lymphocytes from the patient's blood are grown in culture, a functional ADA cDNA is introduced using a retroviral vector, and the cells are returned to the patient. These cells are not immortal, so the patient needs periodic infusions.
• If the gene isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

Molecular diagnosis

• Conventional methods (serum and urine analysis) cannot detect a disease early. Early detection is possible with recombinant DNA technology, PCR and ELISA.
• PCR detects very low concentrations of a pathogen by amplifying its nucleic acid, e.g. in suspected AIDS patients to detect HIV, and to detect mutations in genes of suspected cancer patients, and many other genetic disorders.
• A single-stranded DNA or RNA tagged with a radioactive molecule (probe) hybridises with its complementary DNA in a clone of cells, followed by detection using autoradiography. A clone with a mutated gene will not appear on the photographic film, because the probe does not complement it.
• ELISA works on the principle of antigen-antibody interaction. It detects infection by the presence of antigens (proteins, glycoproteins of the pathogen) or by antibodies synthesised against the pathogen.

Transgenic animals

Transgenic animals are those whose DNA has been manipulated to possess and express an extra (foreign) gene. Rats, rabbits, pigs, sheep, cows and fish have been produced; over 95 per cent of all existing transgenic animals are mice.

Ethical issues

• The Indian Government has set up the GEAC (Genetic Engineering Appraisal Committee), which makes decisions on the validity of GM research and the safety of introducing GM organisms for public services.
• Patents: in 1997 an American company got patent rights on Basmati rice through the US Patent and Trademark Office, for a "new" variety derived from Indian farmers' varieties crossed with semi-dwarf varieties. Other Indian products such as turmeric and neem have also been patented or attempted.
• India has 27 documented varieties of Basmati.
• Biopiracy: the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned, and without compensatory payment.
• The Indian Parliament has cleared the second amendment of the Indian Patents Bill, which takes such issues into consideration, including patent terms, emergency provisions and research and development initiatives.

Common traps: (1) Bt toxin is an inactive protoxin in the bacterium; it is activated by alkaline gut pH, not acidic. (2) cryIAc and cryIIAb: bollworm; cryIAb: corn borer. (3) RNAi target is the nematode's mRNA, and the pest is Meloidogyne incognita on tobacco roots. (4) Mature insulin has no C peptide. (5) The ADA gene therapy used lymphocytes and a retroviral vector, and is not permanent. (6) Rosie's milk had human α-lactalbumin, not insulin or antitrypsin.

NEET focus

• Mechanism of Bt toxin; cry genes and their targets.
• RNAi steps; sense and anti-sense RNA.
• Structure of insulin; Eli Lilly, 1983.
• ADA deficiency gene therapy, 1990.
• PCR, probes and ELISA in diagnosis.
• Transgenic animals, Rosie (1997), α-1-antitrypsin; GEAC; biopiracy.

Practice questions