BioTechnology and Its Applications

Syllabus:

1. Biotechnological applications in agriculture

2. Biotechnological Applications in Medicine

3. Transgenic Animals 

4. Ethical issues

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: - 

a. made crops more tolerant to abiotic stresses (cold, drought, sale, heat).

b. reduced reliance on chemical pesticides (pest-resistant crops).

c. helped to reduce post harvest losses.

d. increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil)

e. enhanced nutritional value of food e.g. golden rice is vitamin A enriched rice

Pest resistant plant (decrease amount of pesticide used): 

Bt stands for Bacillus thuringiensis. 

Bt toxin is produced by a bacterium called Bacillus thuringiensis.

Bt toxin gene has been cloned from the bacteria and been expressed in plants to provide resistance to insects without the need for insecticides (bio-pesticide) examples: Bt cotton, Bt corn, rice, tomato, potato and soyabean etc. 

Bt Cotton: 

-Some strains of Bacillus thuringiensis produce proteins that kill certain insects like lepidopterans (tobacco budworm, armyworm), coleopterans (bettles) and dipterans (flies, mosquitoes)

-B. thuringiensis forms protein crystals contain a toxic insecticidal protein.

-Bt toxin protein exist as inactive protoxins

-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

-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 Bt and incorporated into several crop plants like cotton.

-most Bt toxins are insect-group specific.

-Choice of genes depends on crop and targeted pest

-Toxin is coded by a gene cryIAc named cry

-proteins encoded by the genes cryIAc and cryIIAb control the cotton bullworms, that of cryIAb controls corn borer.

Pest Resistant Plants:

-nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great reduction in yield.

-RNA interference (RNAi) strategy was adopted to prevent this infestation

-RNAi take place in all eukaryotic organisms as a method of cellular defence

-Specific mRNA are silenced due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing)

-Source of this complementary RNA could be from an infection by viruses having RNA genomes / mobile genetic elements (transposons) that replicate via an RNA intermediate.

-nematode-specific genes were introduced into the host plant using Agrobacterium vectors

-DNA was introduced such that it produced both sense and anti-sense RNA to host.

-two RNA's being complementary to each other formed a dsRNA that initiated RNAi and silenced specific mRNA of nematode.

-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:

-30 recombinant therapeutics have been approved for human-use. In India, 12 of these are presently being marketed.

Genetically Engineered Insulin:

-genetically modified bacteria produces Insulin for us

-Insulin consists of two short polypeptide chains : Chain A and Chain B, that are linked together by disulfide bridges.

-In mammals insulin is synthesised as a pro-hormone and need to be processed before it becomes a fully mature and function hormone which contain extra stretch called the C peptide.

-C peptide is not present in mature insulin and removed during maturation into insulin

-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.

-Chain A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

Gene Therapy: 

-It is a collection of methods that allows correction of a gene defect that has been diagnosed in a child/ embryo.

-His genes are inserted into a person's cells & tissues to treat a disease.

-Correction of a genetic defect involves delivery of a normal gene into the individual / embryo to take over the function of and compensate for the non-functional gene

-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.

-ADA deficiency can be cured by bone marrow transplantation

- ADA deficiency can be cured by enzyme replacement therapy, in which functional ADA is given to the patient by injection.

-Both above methods are not completely curative.

-lymphocytes from blood of the patient are grown in a culture outside the body and a functional ADA cDNA is introduced into these lymphocytes, which are subsequently returned to the patient

-as cells are not immortal, patient requires periodic infusion of genetically engineered lymphocytes.

-if gene isolate from marrow cells producing ADA is introduced into the cells at early embryonic stages, it becomes a permanent cure.

Molecular Diagnosis:

-rDNA, PCR & ELISA techniques serve early diagnosis

-Presence of pathogen is suspected after disease symptoms and that point concentration of pathogen is too high 

-low concentration of pathogen (bacteria/virus) can be detected by amplification of their nucleic acid by PCR

-PCR is used to detect HIV and mutations in genes in cancer cases

-a single stranded DNA/RNA, tagged with a radioactive molecule (probe) is hybridised to its complementary DNA in a clone of cells followed by detection using autoradiography

-clone having mutated gene will not appear on photographic film because 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.

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 and 95% of all existing transgenic animals are mice.

Benefits from transgenic animals:
-Normal physiology and development: gene regulation, effect of genes on normal functions of the body and its development

-study of complex factors involved in growth such as insulin like growth factor

-Study of disease: how genes contribute to the development of disease. Today transgenic models exist for many human diseases like cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's

-Biological products: 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 like human protein (alpha -1-antitrypsin) used to treat emphysema and others like phenylketonuria (PKU) & cystic fibrosis.

-1997, first transgenic cow, Rosie produced human protein-enriched milk (2.4 g / L). Milk contained human alpha-lactalbuin & was nutritionally a more balanced product ofr human babies than natural cow-milk

-Vaccine safety: Transgenic mice developed for use in testing the safety of vaccines before human trial.

-Transgenic mice used for polio vaccine later on on monkeys

Chemical safety testing (toxicity/ safety):

- Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals.

Ethical issues & Biopiracy

What is Biopiracy? 

Ans. Use of bioresources by MNCs  and their organisations without proper authorisation from the countries and people concerned without compensatory payment.