Big picture
The tools of recombinant DNA technology (restriction enzymes, vectors, PCR) let us move genes between organisms. This chapter is about what we do with those tools: crops that protect themselves from pests, human insulin made by bacteria, treatment of genetic disorders, early diagnosis of diseases, and animals that make useful human proteins.
These powerful techniques also raise questions of safety, ethics and ownership, so India has regulatory bodies and laws, and the chapter ends with biopiracy and patents. Exam questions often ask about Bt toxin's mechanism, RNAi, how insulin is produced, and the first gene therapy.
Learning goals
Explain the benefits of genetically modified (GM) crops.
Explain how Bt cotton resists insects, including how the Bt toxin works.
Explain how RNA interference makes tobacco resistant to nematodes.
Explain the production of genetically engineered insulin.
Explain gene therapy with the example of ADA deficiency.
Explain molecular diagnosis techniques.
Explain the uses of transgenic animals.
Explain the ethical issues, the role of GEAC, and biopiracy.
Core ideas
1. Biotechnology in agriculture: GM crops
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).
Less reliance on chemical pesticides (pest-resistant crops).
Help reduce post-harvest losses.
More efficient use of minerals by plants, preventing early exhaustion of soil fertility.
Enhanced nutritional value of food, for example Golden Rice, enriched with vitamin A.
Use as alternative resources to industries for starches, fuels and pharmaceuticals.
2. Bt cotton
Bt stands for *Bacillus thuringiensis, a soil bacterium that produces proteins that kill certain insects: lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans* (flies, mosquitoes).
How Bt toxin works:
B. thuringiensis forms protein crystals containing a toxic insecticidal protein, which exists as an inactive protoxin (so it doesn't harm the bacterium itself).
When an insect eats it, the alkaline pH of the insect's gut solubilises the crystals and converts the protoxin into the active toxin.
The active toxin binds to the surface of midgut epithelial cells and creates pores, causing the cells to swell and burst (lyse), and the insect dies.
The cry genes. The toxin is coded by genes called *cry*. Specific cry genes are introduced into crops:
Gene | Protein controls |
|---|---|
*cryIAc and cryIIAb* | Cotton bollworms |
*cryIAb* | Corn borer |
The gene is isolated from B. thuringiensis and incorporated into the crop, which then produces its own insecticide.
3. Pest-resistant plants through RNA interference
The nematode *Meloidogyne incognita infects the roots of tobacco* plants, causing a great reduction in yield.
RNA interference (RNAi) is a method of cellular defence in all eukaryotes that silences a specific mRNA using a complementary double-stranded RNA (dsRNA), which binds to the mRNA and prevents its translation.
How the resistant tobacco was made:
Using *Agrobacterium vectors, nematode-specific genes* were introduced into the host plant.
The genes were introduced so that they produced both sense and anti-sense RNA in the host cells.
These two complementary RNAs formed dsRNA, which started RNAi and silenced the specific mRNA of the nematode.
The parasite could not survive in the transgenic host expressing the RNA, so the plant was protected.
4. Biotechnology in medicine: genetically engineered insulin
Insulin used for diabetes was earlier extracted from the pancreas of slaughtered cattle and pigs. Some patients developed allergies to this animal insulin.
Structure. Human insulin has two short polypeptide chains, chain A and chain B, linked by disulphide bridges. In the body, it is made as a pro-hormone (pro-insulin) with an extra stretch, the C peptide, which is removed during maturation.
The challenge was to obtain insulin in its mature, assembled form.
The solution (1983): Eli Lilly, an American company, prepared two DNA sequences corresponding to chains A and B of human insulin and introduced them into plasmids of *E. coli to produce the chains separately. The chains were then extracted and combined by creating disulphide bonds to form human insulin (Humulin*).
5. Gene therapy
Gene therapy is a collection of methods to correct a gene defect diagnosed in a child or embryo, by delivering a normal gene into the person's cells to take over the function of the non-functional gene.
The first clinical gene therapy was given in 1990 to a four-year-old girl with adenosine deaminase (ADA) deficiency.
ADA is crucial for the immune system. Its deficiency is caused by the deletion of the gene for ADA.
Conventional treatments: bone marrow transplantation, or enzyme replacement therapy (injecting functional ADA), but these are not completely curative.
Gene therapy approach: lymphocytes from the patient's blood are grown in culture; a functional ADA cDNA is introduced into them using a retroviral vector; the lymphocytes are returned to the patient. Because lymphocytes are not immortal, the patient needs periodic infusions.
Permanent cure: if the gene isolated from marrow cells producing ADA is introduced into cells at an early embryonic stage, it could be a permanent cure.
6. Molecular diagnosis
Conventional methods (serum and urine analysis) can't detect diseases early, when the pathogen is present in very low concentrations. Molecular techniques can.
Technique | How it helps |
|---|---|
PCR (polymerase chain reaction) | Amplifies very small amounts of a pathogen's nucleic acid, so infections can be detected early. Used to detect HIV in suspected AIDS patients and mutations in genes in suspected cancer patients |
Probe and autoradiography | A single-stranded DNA or RNA probe tagged with a radioactive molecule hybridises with its complementary DNA in a clone of cells; detected by autoradiography. A clone with a mutated gene won't appear on the film, because the probe doesn't match |
ELISA (enzyme-linked immunosorbent assay) | Based on antigen–antibody interaction. Detects infection by the presence of the pathogen's antigens (proteins, glycoproteins) or by the antibodies made against the pathogen |
7. Transgenic animals
Transgenic animals have had their DNA manipulated to possess and express an extra (foreign) gene. Over 95% of existing transgenic animals are mice; others include rats, rabbits, pigs, sheep, cows and fish.
Use | Example |
|---|---|
Studying normal physiology and development | Studying how genes regulate body functions, such as growth factors like insulin-like growth factor |
Studying disease | Transgenic animals serve as models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's |
Biological products | Transgenic animals that produce useful products: α-1-antitrypsin, used to treat emphysema. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk containing human alpha-lactalbumin (2.4 g per litre), nutritionally better for babies than normal cow milk |
Vaccine safety testing | Transgenic mice are used to test the safety of the polio vaccine before use on humans |
Chemical safety testing (toxicity testing) | Animals with genes that make them more sensitive to toxic substances give results in less time |
8. Ethical issues
GEAC. The Indian Government set up the Genetic Engineering Appraisal Committee (GEAC) to decide on the validity of GM research and the safety of introducing GM organisms for public services.
Patents and biopiracy.
Companies have obtained patents on products and technologies that use genetic materials, plants and other biological resources long identified and used by farmers and indigenous people.
Basmati rice: India has about 27 documented varieties of basmati, known for its unique flavour and aroma. 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 traditional knowledge that has faced patent attempts includes turmeric and neem.
Biopiracy is the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned, and without compensatory payment.
Developing countries, rich in biodiversity and traditional knowledge, need laws to prevent unauthorised exploitation. The Indian Parliament amended the Indian Patents Bill to address patent terms, emergency provisions and research and development initiatives.
A reliable approach to questions
For Bt toxin, describe the sequence: protoxin → activated by alkaline gut pH → binds midgut cells → pores → cell lysis → death.
Name cry genes with their target pests correctly.
For RNAi, mention Meloidogyne incognita, Agrobacterium, sense and anti-sense RNA, dsRNA, and silencing of the nematode's mRNA.
For insulin, mention chains A and B made separately in E. coli and joined by disulphide bonds.
For gene therapy, mention ADA deficiency, 1990, lymphocytes, retroviral vector and periodic infusion.
Connections that matter
Biotechnology: principles and processes explains the tools (restriction enzymes, vectors, PCR) used here.
Microbes in human welfare introduced Bacillus thuringiensis as a biocontrol agent.
Molecular basis of inheritance explains mRNA, translation and the genetic code that RNAi interferes with.
Common mistakes
Saying the Bt toxin is active inside the bacterium. It is an inactive protoxin until activated in the insect's alkaline gut.
Saying RNAi kills the plant's own genes. It silences the nematode's mRNA.
Saying insulin was made as one chain in bacteria. Chains A and B were made separately and then joined.
Saying gene therapy for ADA deficiency is a one-time cure. Lymphocytes die, so infusions are periodic.
Confusing GEAC with a patent office. GEAC appraises GM research and safety.
Revision checklist
I can list the benefits of GM crops.
I can explain how Bt toxin works and name the cry genes.
I can explain RNAi in nematode-resistant tobacco.
I can explain the production of genetically engineered insulin.
I can explain gene therapy for ADA deficiency.
I can explain PCR, probes and ELISA in diagnosis.
I can explain the uses of transgenic animals and the issues of GEAC and biopiracy.