Biotechnology and its Applications — Notes

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 genetically modified organisms (GMOs).

Uses of GM plants: tolerance to abiotic stresses (cold, drought, salt, heat); reduced reliance on chemical pesticides (pest-resistant crops); reduced post-harvest losses; increased efficiency of mineral use (prevents early exhaustion of soil fertility); enhanced nutritional value (golden rice, rich in vitamin A); tailor-made plants supplying alternative resources to industries (starches, fuels, pharmaceuticals).

Bt crops. Bacillus thuringiensis forms protein crystals with an insecticidal protein. The Bt toxin kills lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes).

• The toxin is an inactive protoxin, so the bacterium is not killed.
• In the insect gut the alkaline pH solubilises the crystals and activates the toxin.
• The active toxin binds to the surface of midgut epithelial cells and creates pores, causing swelling and lysis; the insect dies.
• The toxin is coded by cry genes: cryIAc and cryIIAb control cotton bollworms; cryIAb controls corn borer.

Pest-resistant plants by RNA interference (RNAi). The nematode Meloidogyne incognita infects tobacco roots and reduces yield. RNAi occurs in all eukaryotes as a method of cellular defence: it silences a specific mRNA because a complementary double-stranded RNA (dsRNA) binds to it and prevents translation. Natural sources of dsRNA are infection by RNA viruses and mobile genetic elements (transposons) that replicate via an RNA intermediate.

• Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant.
• The plant produced both sense and antisense RNA, which formed dsRNA and started RNAi.
• The specific mRNA of the nematode was silenced, so the parasite could not survive in the transgenic host.

Recombinant DNA technology allows mass production of safe and more effective therapeutic drugs that do not cause unwanted immunological responses, as similar products from non-human sources can. About 30 recombinant therapeutics have been approved for human use worldwide; 12 are marketed in India.

Genetically engineered insulin. Insulin from the pancreas of slaughtered cattle and pigs caused allergy or other reactions in some patients.

• Mature insulin has two short polypeptide chains, A and B, linked by disulphide bridges (chain A 21 and chain B 30 amino acids, 51 in all).
• In mammals it is made as a prohormone (proinsulin) with an extra stretch, the C peptide, which is removed during maturation. Bacteria cannot carry out this step.
• In 1983, Eli Lilly prepared two DNA sequences for chains A and B, introduced them into plasmids of E. coli, produced the chains separately, extracted them and combined them by creating disulphide bonds to form human insulin.

Gene therapy corrects a gene defect diagnosed in a child or embryo: genes are inserted into the person’s cells and tissues, and a normal gene compensates for the non-functional one.

• First clinical use: 1990, a 4-year-old girl with adenosine deaminase (ADA) deficiency. ADA is crucial for the immune system; the disorder results from deletion of the ADA gene.
• Other treatments: bone marrow transplantation; enzyme replacement therapy (functional ADA by injection) — neither is completely curative.
• Method: lymphocytes from the patient’s blood are grown in culture; a functional ADA cDNA is introduced using a retroviral vector; the cells are returned to the patient. Lymphocytes are not immortal, so periodic infusions are needed.
• 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.

• PCR amplifies very low concentrations of a pathogen’s nucleic acid: used to detect HIV in suspected AIDS patients and mutations in suspected cancer patients; also used to identify many other genetic disorders.
• A single-stranded DNA or RNA tagged with a radioactive molecule (probe) hybridises to its complementary DNA in a clone of cells and is detected by autoradiography; a clone with a mutated gene does not appear on the photographic film because the probe is not complementary to it.
• ELISA works on the principle of antigen–antibody interaction: it detects antigens of the pathogen, or antibodies made against it.

Stem cell technology. Stem cells are unspecialised cells that can divide and form many kinds of specialised cells (for example, bone-marrow stem cells form blood cells). They hold promise for repairing or replacing damaged tissues and, with gene therapy in early embryonic cells, for lasting cures.

Animals whose DNA has been manipulated to possess and express an extra (foreign) gene are transgenic animals. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, but over 95 percent of all existing transgenic animals are mice.

Uses:

• Normal physiology and development — study of how genes are regulated and how their products affect body functions and development, e.g. insulin-like growth factor.
• Study of disease — models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer’s disease, so that new treatments can be investigated.
• Biological products — medicines made from biological products can be expensive; transgenic animals that produce useful products can be made, e.g. human protein α-1-antitrypsin used to treat emphysema; attempts for products to treat phenylketonuria and cystic fibrosis.
• Vaccine safety — transgenic mice are used to test the safety of the polio vaccine before it is used on humans.
• Chemical safety (toxicity) testing — animals carrying genes that make them more sensitive to toxic substances give results in less time.

Rosie (1997): the first transgenic cow; produced human protein-enriched milk (2.4 grams per litre) containing human alpha-lactalbumin, nutritionally more balanced for human babies than natural cow milk.

Manipulation of living organisms needs regulation, because genetic modification may have unpredictable results when such organisms enter an ecosystem.

GEAC. The Indian Government set up the Genetic Engineering Approval Committee, which makes decisions on the validity of GM research and the safety of introducing GM organisms for public services.

Patents and biopiracy.

• There is growing public anger that certain companies are granted patents for products and technologies that use genetic materials, plants and other biological resources long identified, developed and used by farmers and indigenous people.
• Basmati rice: India has an estimated 200 000 varieties of rice, including at least 27 documented varieties of basmati. 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; it was claimed as an invention.
• Other traditional Indian products, such as turmeric and neem, have also been subjects of patent attempts.
• Biopiracy: the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned, without compensatory payment.
• Industrialised nations are rich financially but poor in biodiversity and traditional knowledge; developing and under-developed nations are rich in both. Some nations are developing laws to prevent unauthorised exploitation of bio-resources and traditional knowledge.
• 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.