Question 1. Crystals of Bt toxin produced by some bacteria do not kill the bacteria themselves because
- (a) bacteria are resistant to the toxin
- (b) toxin is immature
- (c) toxin is inactive
- (d) bacteria encloses toxin in a special sac.
Sol. (c) Crystals of Bt toxin produced by some bacteria do not kill the bacteria themselves because toxin is present in inactive form. Et toxin protein is produced by bacterium, Bacillus thuringiensis. It exists as inactive protoxins and is converted into an active form of toxin due to the alkaline pH of the gut ofan insect upon ingestion.
Question 2. What are transgenic bacteria? Illustrate using any one example.
- Transgenic bacteria are those bacteria whose genome are manipulated by introduction of foreign gene from another organism using recombinant DNA technology. This introduced foreign gene is made to express in the host bacteria for the production of various commercially important products.
- An example of transgenic bacteria is Escherichia coli which produces synthetic human insulin. Two DNA sequences corresponding to A and B chains of human insulin are prepared and introduced in the plasmids. These recombinant plasmids are introduced into E. coli. This E. coli (now called transgenic) produces insulin chains A and B which are later extracted and combined by creating disulphide bonds to form human insulin.
Question 3. Compare and contrast the advantages and disadvantages of production of genetically modified crops.
Sol. Plants whose genes have been altered by manipulation are called genetically modified crops. GM crops have been advantageous in several ways and at the same time have some disadvantages also.
Contrast and comparison of advantages and disadvantages of genetically modified crops are as follows:
Question 4. What are Cry proteins? Name an organism that produces it. How has man exploited this protein to his benefit?
- Cry proteins are crystal proteins encoded by cry genes.
- These proteins are endotoxins produced by bacterium, Bacillus thuringiensis during a particular phase of their growth. Bacillus thuringiensis is a common Gram positive, spore-forming, soil bacterium.
- Man has exploited Cry protein for the production of insect-resistant transgenic Et crops because this protein has insecticidal toxin which can kill specific insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). Specific Et toxin genes isolated from Bacillus thuringiensis are incorporated into the several crop plants such as cotton and corn. Et toxin protein exists as inactive protoxin. When an insect ingests inactive toxin crystals, these are converted into 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 creates pores that cause cell swelling and lysis and eventually cause death of the insect.
Note: When resources are limited, vegetative Bt cells undergo sporulation during which a spore is formed along with proteins accumulated in the cytoplasm of the cells as crystalline inclusions. Proteins in these crystals are called Cry (from Crystal). At the end of the sporulation process, crystals and spores are liberated concomitantly by lysis of the cells. Once Cry protein is ingested by a susceptible insect, the alkaline pH of its digestive tract denatures the insoluble crystals, making them soluble and thus amenable to being cut with proteases found in the insect gut, which liberate the cry toxin from the crystal. The Cry toxin is then inserted into the insect gut epithelial cell membrane, paralysing the digestive tract and forming a pore. The insect stops eating and starves to death; live Bt bacteria may also colonize the insect which can contribute to death.
Question 5. What is gene therapy? Illustrate using the example of adenosine deaminase (ADA) deficiency.
- Gene therapy is a collection of methods that uses genes to correct a gene defect diagnosed in a child or embryo. If a defective gene causes a necessary protein to be faulty or missing, gene therapy delivers a normal copy of the gene into the individual or embryo which restores the function of the protein by taking over the function of and compensating 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 deficiency causes SCID (Severe Combined Immuno Deficiency) in which B-lymphocytes and T-lymphocytes are not formed. The ADA gene provides instructions for producing the enzyme adenosine deaminase. This enzyme is produced in all cells, but the highest levels of adenosine deaminase occur in immune system cells called lymphocytes.
- In gene therapy for ADA deficiency,
- Lymphocytes or bone marrow cells from the blood of the patient are grown in a culture outside the body.
- A functional ADA cDNA is then introduced into these lymphocytes or marrow cells using a retroviral vector.
- The transformed lymphocytes or marrow cells containing ADA gene are subsequently returned to the patient’s bone marrow. Thus, the gene activated producing functional T-lymphocytes and activating the patients immune system.
Note: The adenosine deaminase (ADA) enzyme is important for the normal functioning of the immune system. The individual suffering from this disorder, arise due to deficiency of the ADA enzyme, can be cured by transplantation of bone marrow cells.
Question 6. Diagrammatically represent the experimental steps in cloning and expressing an human gene (say the gene for growth hormone) into a bacterium like E.coli?
- DNA cloning, as the starting point for many genetic engineering approaches to biotechnology research, is a method of producing multiple identical copies of specific template DNA. It involves the use of a vector to carry the specific foreign DNA fragment into the host cell.
- The mechanism of cloning and transfer of gene for growth hormone into E.coli is represented below:
- Human growth hormone (hGH) is the naturally occurring growth hormone of humans or a genetically engineered form that is used to treat children with growth hormone deficiencies.
- Human growth hormone (hGH) is a small, single chain peptide hormone encoded by the GHN gene. It is produced by the anterior pituitary gland and is essential for normal growth and development in humans. The mature structure of hGH contains 191 amino acid residues. Since genes in eukaryotes are split genes with exons or coding sequences interrupted by introns or non-coding sequences, for the expression of gene for human growth hormone, cDNA synthesised from mRNA of human growth hormone is cloned through polymerase chain reaction (PCR). Cloned cDNA and vector are cut with the same restriction enzyme and both are ligated by using DNA ligase. The vector carrying cDNA is transformed into host, E. coli for expression. Transformed E. coli are cultured in growth medium for the expression of human growth hormone gene. The experimental steps in cloning and expression of gene for human growth hormone into a bacterium such as E. coli are as follows:
Question 7. Can you suggest a method to remove oil (hydrocarbon) from seeds based on your understanding of rDNA technology and chemistry of oil?
Sol. Recombinant DNA technology (rDNA) is a technique used for manipulating the genetic material of an organism to obtain the desired result. The constituents of oil present in seed are glycerol and fatty acids.
The genes for the formation of glycerol or fatty acids which are the main constituents of oil, should be identified and removed from the seed by using enzyme restriction endonucleases. Such DNA molecule should then be treated with DNA ligases to join the broken ends. These cells when grown in a minimum nutrient medium, under aseptic conditions will differentiate into a new variety whose seeds will not have oil in them because the gene producing the oil has been removed.
Question 8. Find out from internet what is golden rice.
Sol. Golden rice is a variety of rice (Oryza sativa) produced through recombinant DNA technology to biosynthesise beta-carotene, a precursor of vitamin A, in the edible parts of rice. It was developed by Ingo Potrykus from Swiss Federal Institute of Technology in Zurich, Switzerland and Peter Beyer from University of Freiburg in 1990. Golden Rice gets its name because the high levels of beta-carotene make it a golden-yellow colour. It was developed as a fortified food to be grown and consumed in areas where rice is a major staple food. It is generally consumed in its milled form in which outer layers (pericarp, tegmen and aleurone layers) are removed. The main reason for milling is to remove the oil-rich aleurone layer, which turns rancid upon storage. As a result, the edible part of rice grains consists of the endosperm, filled with starch granules and protein bodies, but it lacks several essential nutrients for the maintenance of health, such as carotenoids exhibiting provitamin A-activity. Thus, reliance on rice as a primary food staple leads to vitamin A deficiency (VAD). The deficiency of vitamin A causes childhood blindness in the developing world, and weakens the immune system, increasing vulnerability to illnesses such as measles, respiratory infections and diarrhoea, often leading to death.
Question 9. Does our blood have proteases and nucleases?
- Yes, protease and nuclease are found in blood.
- Proteases are enzymes that catalyse cleavage of peptide bonds in proteins.
- They are involved in many physiological reactions from digestion of proteins of food to highly regulated cascades. Proteases such as thrombin, factor XII (Hageman factor) and plasmin are found in blood serum and carry out functions such as blood coagulation, dissolution of blood clot and induction of physiological immune responses. Other proteases are found in leucocytes (elastase, cathepsin G) and play several different roles in metabolic control.
- Nucleases are enzymes that cleave phosphodiester bonds in nucleic acids. Some nucleases are DNA specific (DNase), some are RNA specific (RNase), and some degrade both DNA and RNA. Nucleases (such DNase and RNase) are also found in blood serum and lysosomes of white blood cells (macrophages) and help in degradation of DNA and RNA of pathogens and apoptotic cells.
Note: Inblood, serum is the component that is neither a blood cell(serum does not contain white or red blood cells) nor a clotting factor. It is the blood plasma with the jibrinogen removed.
Question 10. Consult internet and find out how to make orally active protein pharmaceutical. What is the major problem to be encountered?
- Orally active pharmaceutical proteins are biologically active proteins or peptides which are adminisitered orally for the treatment of a variety of diseases, such as diabetes, cancer, infectious diseases, haemophilia, and anaemia. Common pharmaceutical proteins include antibodies, hormones, interleukins, enzymes and anticoagulants. Currently, most of all recombinant pharmaceutical proteins are produced in mammalian cells because mammalian cells are capable of producing high-quality proteins similar to the naturally occurring ones. In addition, many approved recombinant therapeutic proteins are generated in Escherichia coli.
- The major problem encountered in oral delivery of proteins and peptides is their degradation by proteolytic enzymes present in gastrointestinal tract, their poor stability in lower pH of gastric fluid, large molecular size and their inability to cross gastrointestinal membrane due to low lipophilicity. These characteristics lead to the low bioavailability of most orally administered peptides and short half-lives (<30 min) and failure of delivery of intact protein/peptide to the targeted site. Nano-carrier-based delivery is an appropriate choice of drug carriers owing to their property to protect proteins from degradation by the low pH conditions in stomach or by the proteolytic enzymes in the gastrointestinal tract
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