SHORT ANSWER TYPE QUESTIONS 

Question. Name the vector used for introducing the nematode specific gene in tobacco plant.
Answer.Ti plasmid of Agrobacterium tumefaciens

Question. What is golden rice?
Answer.Golden rice is a transgenic rice having gene for beta- carotene.

Question. For which variety of Indian rice patent was made by a USA company?
Answer.Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention.

Question. Name the diagnostic test for AIDS when the concentration of HIV is (a) low (b) high.
Answer.PCR, ELISA

Question. State the principle used in ELISA?
Answer.Antigen-antibody interaction

Question. PCR is a powerful diagnostic technique. Why?
Answer.Early-stage diagnosis is possible before the onset of symptoms.

Question. Name the recombinant vaccine that is currently being used in vaccination program?
Answer.Hepatitis B recombinant vaccine is used for vaccination of hepatitis virus

Question. Expand GEAC
Answer.GEAC- Genetic Engineering Approval Committee

Question. Why does Bt toxin not kill the bacteria in which it is present?
Answer.Because Bt toxin exists as inactive pro-toxin.

Question. What is ‘Flavr Savr’?
Answer.It is a transgenic tomato variety which has blocked production of polygalacturonase

Question. State a method of cellular defence which works in all eukaryotic organisms.
Answer.RNA interference

Question. Name the first transgenic cow. Which gene was introduced in this cow?
Answer.Rosie. Human alpha- lactalbumin gene was introduced.

Question. Mention the source organism of the gene cryIAc and its target pest.
Answer.Source organism- Bacillus thuringiensis, Target pest- Cotton bollworms

Question. What is biopiracy?
Answer.It is the use of bioresources by multinational companies and other organizations without proper authorization or compensation payment to the concerned country or organization.

Question. State the purpose for which the Indian government has set up GEAC.
Answer.GEAC was set up to make decisions regarding the validity of GM Research and the safety of introducing GM- organisms for public services.

SHORT ANSWER TYPE 

Question. Mention two applications of biotechnology in medicine with examples.
Answer.Insulin production, treatment of genetic disorders with gene therapy as in ADA deficiency

Question. Why do the toxic insecticidal proteins secreted by Bacillus thuringiensis kill the insect and not the bacteria itself?
Answer.The Bt toxin protein exists as inactive protoxins but once an insect ingests the inactive toxin is converted into an active form due to the alkaline pH of the gut which solubilise the crystals. Therefore, it does not kill the bacteria.

Question. List any four beneficial effects of GM plants.
Answer.Increased tolerance against abiotic stresses (cold, drought, salt, heat)
Reduced reliance on chemical pesticides (pest-resistant crops)
Reduced post-harvest losses.
Increased efficiency of minerals used by plants (this prevents early exhaustion of fertility of soil

Question. Suggest two possible treatment that can be given to patients with ADA Deficiency.
Answer.Enzyme replacement therapy (in which functional ADA is injected), Bone marrow transplantations, culturing the lymphocytes followed by introduction of functional ADA cDNA into it and returning it into the patient’s body (any two)

Question. What are transgenic animals? How was the first transgenic cow found to be more useful than the normal cow, for humans?
Answer.Animals having undergone DNA manipulation (to express an extra / foreign gene) -(Rosie) produced human protein – enriched milk / 2.4 gm protein per litre which contained human alpha – lactalbumin, and was nutritionally more balanced (product) for human babies than natural cow milk.

Question. How does silencing of specific mRNA in RNA interference prevent parasitic infestation?
Answer.The parasite cannot survive where the dsRNA expresses itself and prevents the translation of the specific mRNA of the nematode.

Question. What is the cause of Adenosine deaminase deficiency in a person?
Answer.Adenosine deaminase deficiency is due to the deletion of the gene coding for the enzyme adenosine deaminase.

Question. GMO’s have been useful to mankind. List about five useful GMO’s created so far. and how they have been useful.
Answer.Production of Crops tolerant to abiotic stress, Crops with reduced post-harvest loss, enhanced nutritional value, physiological models, human lactalbumin producing cow.

Question. Suggest any two possible treatments that can be given to a patient exhibiting adenosine deaminase deficiency.
Answer.Enzymes replacement therapy (in which functional ADA is injected) (ii) Bone marrow transplantation (iii) Gene therapy / Culturing the lymphocytes followed by introduction of functional ADA cDNA into it & returning it into the patient ‘s body (Any two)

Question. What is GEAC and what are its objectives?
Answer.GEAC (Genetic Engineering Approval Committee) is an Indian government organisation. Its objectives are to examine the validity of GM Research and to inspect the safety of introducing GM- organisms for public services and for their large-scale use.

Question. How can biotechnology help in increasing the yield of tobacco?
Answer.By the technique of RNA interference. Agrobacterium act as vectors. During this process nematode specific gene is introduced in the host plant which forms complementary RNA double strand specific to nematode DNA. This silence specific mRNA of nematode, makes tobacco pest resistant and thereby increasing the yield.

Question. How is a mature, functional insulin hormone different from its pro-hormone form?
Answer.Mature functional insulin is obtained by processing of pro-hormone which contains extra peptide called C-peptide. This C-peptide is removed during the maturation of pro-insulin to insulin.

Question. What is gene therapy?
Answer.Gene therapy is a collection of methods that allows collection of a gene defect that has been diagnosed.

Question. How are transgenic animals beneficial in chemical safety testing?
Answer.Transgenic animals carry the genes which make them more sensitive to toxic substances, so the results are obtained in very less time.

Question. How are transgenic animals used in vaccine safety?
Answer.Transgenic mice are used to test the safety of vaccines before they are tested on humans. If it is safe and reliable the drugs can be administrated in humans, so in that case transgenic animals can act as model for vaccine testing.

Question. Give a schematic representation of the transformation of a pro- insulin to insulin.
Answer.

Question. A method to present infestation of a nematode Meloidegyne incognitia on roots of tobacco is silencing the specific mRNA. What is the scientific name of the technique? How is this performed by ds- RNA?
Answer.RNA interference (RNAi) RNAi in all eukaryotic organisms involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to prevents translation of the mRNA (silencing). The source of this complementary could be virus having RNA genome or transposons. Through Agrobacterium vectors, nematode specific genes are introduced into the host plant. The DNA produces both sense and anti-sense RNA in host cells. The two RNA’s being complementary form double stranded RNA and silence the specific mRNA.

Question. How has the Bt toxin been effective against insects?
Answer.The larva ingests the leaves of Bt cotton containing the toxin, the toxin is activated in the gut of the insect by enzymes, which solubilize the toxin, the toxin binds to the lining of epithelial cells create pores cause swelling and death of the insect.

Question. What problem was faced in production of insulin by rDNA technology and how was it resolved?
Answer.The main challenge was getting the insulin assembled into a mature form.

Question. What are the three critical research areas of Biotechnology?
Answer.Three critical areas- Providing the best catalyst in the form of a microbe/ pure enzyme, creating optimal conditions for their action through engineering, downstream processing technologies.

SHORT ANSWER TYPE 

Question. Name the genus of baculovirus. Why are they consider good bio control agent?
Answer.Nucleopolyhadrovirus is a genus of baculovirus. They are species- specific, have narrow-spectrum insecticidal application and no negative impact on non- target organisms, hence they are considered good bio control agent.

Question. How has the bacterium Bacillus thuringiensis helped us in controlling caterpillars of insect pests?
Answer.Bacillus thuringiensis products are endotoxin which when ingested and release in the gut of the larvae of insect pests disrupts the insects gut lining thereby killing them.

Question. (a) Mention the cause and body system affected by ADA deficiency in humans. (b) Name the vector used for transferring ADA-DNA into the recipient cells in humans. Name the recipient cells.
Answer.(a) The cause is the defective gene not producing ADA.The immune system is affected.
(b) A retroviral vector is used, recipient cells are lymphocytes.

Question. Describe the various stage involved in gene transfer for the commercial production of human insulin by Eli Lilly.
Answer.(a) Eli Lilly prepared two DNA sequences corresponding to the A and B chains of human insulin. (b) Sticky ends were produced in the Escherichiacoli plasmid and the insulin gene by treating them both with the same restriction endonucleases. (c) These two are then joined together by the enzyme DNA ligase. (d) The bacteria are then grown in sterilised bioreactors in the appropriate growth medium. (e) The chains A and B are produced separately, extracted and purified. (f) These two chains are then combined by creating disulphide bonds to form human insulin.

Question. (a) Give the scientific name of the soil bacterium which produces crystal (Cry) proteins. (b) How are these proteins useful in agriculture? (c) What do the differently written terms ‘Cry’ and ‘cry’ represent respectively?
Answer.(a) Bacillus thuringiensis. (b) These Cry proteins are toxic to certain larvae of insects and thus provide resistance against them. The gene encoding Cry proteins are used in several crop plants (Bt toxin). Such a crop plant is resistant to the particular insect pest. (c) Cry represents crystal protein while cry refers to the gene encoding the Cry protein.

Question. List the three molecular diagnostic techniques that help detect pathogens from suspected patients. Mention one advantage of these techniques over conventional method
Answer.The three molecular diagnostic techniques that help to detect pathogens from suspected patients are: (a) Recombinant DNA technology. (b) Polymerase chain reaction (PCR). (c) Enzyme-linked immuno-sorbent assay (ELISA). These techniques are better than the conventional method because they help in early diagnosis of the disease even when the bacteria or virus concentration is very low.

LONG ANSWER TYPE QUESTIONS

Question. (i) Name the nematode that infests and damages tobacco roots.
(ii) How are transgenic tobacco plants produced to solve this problem?
Answer.(i). Meloidogyne incognita
(ii) Using Agrobacterium vectors, nematode specific genes are introduced into the host plant. The introduction of DNA is done such that it produces both sense and anti-sense RNA in the host cells. These two RNAs being complementary to each other forms a double stranded RNA (ds RNA) that initiate RNAi and thus silences the specific mRNA of the nematode. The consequence is that the parasite is not able to survive in a transgenic host expressing specific interfering RNA. The transgenic plant therefore is protected from the parasite.

Question. One of the main objectives of biotechnology is to minimize the use of insecticides on cultivated crops. Explain with the help of a suitable example how insect resistant crops have been developed using techniques of biotechnology.
Answer.
An example of use of biotechnology to minimize the use of insecticides in agriculture is production of transgenic Bt cotton. Soil bacterium, Bacillus thuringiensis produces proteins that kills certain insects like, lepidopterans (tobacco budworm, armyworm) Coleopterans (beetles) and dipterans (flies, mosquitoes) etc. B. thuringiensis form some protein crystals. These crystals contain a toxic insecticidal protein. This toxin does not kill the Bacillus bacterium because it exists as inactive protoxin in them. But, once an insect ingests the crystals, it’s converted into an active form of toxin due to the alkaline pH of the alimentary canal. Through genetic engineering Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into several crop plants such as cotton. The choice of genes depends upon the crop and the targeted pest, as most Bt toxins are insect-group specific. The toxin is coded by a gene named cry. Two cry genes, cryIAc and cryIIAb have been incorporated in cotton. The genetically modified crop is called Bt cotton as it contains Bt toxin genes against cotton bollworms.

Question. What is gene therapy? Illustrate using the example of adenosine deaminase (ADA) deficiency?
Answer.
Gene therapy is the technique of genetic engineering to replace a faulty gene by a normal healthy functional gene. The first clinical gene therapy was given in 1900 to a 4-year-old girl with adenosine deaminase deficiency (ADA deficiency). This enzyme is very important for the immune system to function. Severe combined immunodeficiency (SCID) is caused due to defect in the gene for the enzyme adenosine deaminase. SCID patients lack functional T-lymphocytes and, therefore, fails to fight the infecting pathogens. Lymphocytes are extracted from the patient’s bone marrow and a normal functional copy of human gene coding for ADA is introduced into these lymphocytes with the help of retroviral vector. The cells so treated are reintroduced into the patient’s bone marrow. The lymphocytes produced by the cell contain functional ADA gene which reactivate the victim’s immune system.
Though these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the genes isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

Question. Digrammatically represent the experimental steps in cloning and expressing an human gene (say the gene for growth hormone) into a bacterium.
Answer.

Question. List five recombinant proteins which are used in medical practice. Find where they are used as therapeutics.
Answer.The five recombinant proteins which are used in medical practice and their use in therapeutics are:
(i) Human insulin-diabetes
(ii) Human growth hormone-Dwarfism
(iii) Interferons-Hepatitis B, cancer
(iv) Prednisolone-Rheumatoid arthritis
(v) Erythropoietin- Stimulation of production of erythrocytes

Question. (i) What is plasmid?
(ii) Plasmid is a boon to biotechnology. Justify this statement using the production of human insulin as an example.
Answer.(i) Plasmid is a structure in bacterial cells consisting of DNA that can exist and replicate independent of the chromosome. Plasmids provide genetic instructions for certain cell activities (like resistance to antibiotics). They can be transferred from cell to cell in a bacterial colony. Plasmids are widely used as vectors to produce recombinant DNA for gene cloning.
(ii) DNA sequences for the two chains of human insulin were prepared and introduced into plasmid to produce insulin chains. As plasmids have the power of replication, it will increase the number of insulin chains, thus producing large amount of insulin. In 1983, Eli Lilly an American company, first 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. These chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin (humulin).

Question. (i) How is mature insulin different from proinsulin secreted by pancreas in humans?
(ii) Why is the functional insulin thus produced considered better than the ones used earlier by diabetic patients?
(iii) Describe the responsibility of GEAC set up by the Indian government.
Answer.(i). The proinsulin has A, B and C polypeptide strands. It is non-functional. The mature insulin has only A and B polypeptide strands. It is functional.
(ii) The insulin produced by recombinant DNA (rDNA) technology does not produce sensitive allergic reactions and other immunological reactions whereas those used earlier produced allergic reactions and other complications because they were foreign proteins extracted from pancreas of slaughtered cattle or pigs.
(iii) GEAC is Genetic Engineering Approval Committee. It takes decisions regarding the validity of GM research and the safety of introducing GM organisms for public services.

Question. Name the source of Taq polymerase. Explain the advantage of its use in biotechnology.
(ii) List four applications of genetically modified plants
Answer.(i). Source of Taq polymerase is Thermus aquaticus, a bacterium. Taq polymerase is a thermostable enzyme which remains active in high temperature induced denaturation of dsDNA. It is used in repeated amplification of a gene.
(ii) Applications of genetically modified plants are:
• GM plants can tolerate adverse environmental conditions
• GM plants are resistant to diseases resulting from viral, bacterial and fungal infections
• They are resistant to pesticides and pests such as nematodes and insects
• They can produce pharmaceutically important compounds such as insulin, interferons, hormones, blood clotting factors, antibodies and vaccines

Question. (i) Give the scientific name of the soil bacterium which produces crystal (Cry) proteins. How are these proteins useful in agriculture?
(ii) What does the term ‘Cry’ and ‘cry’ represent respectively?
(iii) What is special of ‘Flavr Savr’ variety of tomato? Why is it preferred to its normal native variety?
Answer.
Crystal (Cry) proteins are produced by Bacillus thuringiensis. Crystal proteins contain a toxic insecticidal protein. When cry genes are introduced into the crops, they produce crystal (Cry) proteins which are toxic for insects, thus making the crop insect-pest resistant.
(ii) The gene encoding the Cry protein is called cry gene. The Cry is protein and written in Roman letters in which the first letter is always capital whereas cry is a gene which is written in italics and all letters are small.
(iii) Flavr savr, a variety of tomato is a genetically modified plant. In this variety one gene which produces polygalactouronase enzyme is inactivated. The non-availability of the enzyme prevents over ripening. Thus fruit remains fresh for a long time. And it also retains flavor, superior taste and higher quantity of total soluble solid. So, it prevents post-harvest losses. Thus, it is preferred over normal native variety.

Question. What are transgenic animals? Explain any four ways in which such animals can be beneficial for humans.
Answer.
Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are called transgenic animals. Transgenic rats, rabbits, pigs and cows have been produced. Uses of transgenic animals for humans are:
(i) To study gene regulation, their effect on the normal functions of the body and its development.
(ii) Study of genes, which are responsible for diseases in human and their treatment, e.g. cancer.
(iii) Useful biological products can be produced by introducing the portion of DNA, which codes for a particular product into transgenic animals.
(iv)Transgenic mice are developed to test the safety of vaccines before being used in humans.

Question. In the given figure, Form (A) and Form (B) represents different forms of a proteinaceous hormone secreted by pancreas in mammals.

(a)What type of bonding is present between chains of this hormone? (b)What are these form (A) and form (B). How these forms differ from each other? (c) Explain how this hormone was produced by Eli Lilly, an American company, using rDNA technology.
Answer.
(a) Disulphide bonds
(b) Form (A) – Proinsulin Form (B) – Mature insulin. Proinsulin contains an extra stretch called C peptide which is absent in mature insulin. (c) Eli Lilly company prepared two DNA sequences corresponding to A and B peptide chains of human insulin and introduced them in plasmid E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulphide bonds to form insulin.

Question. Explain five areas where biotechnology has influenced human lives.
Answer.Biotechnology has influenced human lives as follows:
• Genetically modified crops with high nutritive value are provided by biotechnology.
• It has helped in the production of recombinant vaccines.
• It has devised techniques such as gene therapy for the treatment of genetic diseases.
• Genetically engineered microbes are produced to control environmental pollution.
• Transgenic animals are developed that can produce human proteins

Question. What are orally active protein pharmaceutical. What is the major problem to be encountered?
Answer. Orally active protein pharmaceuticals contain biologically active materials such as peptides or proteins, antibodies, and polymeric beads. It is administrated orally into the body through various formulations. It involves the encapsulation of protein or peptide in liposomes or formulations using penetration enhancers. These proteins or peptides are used for treatment of various diseases and are also used as vaccines. However, the oral administration of these peptides or proteins has some problems related to it. Once these proteins are ingested, the proteases present in the stomach juices denature the protein. As a result, their effect will be nullified. Hence, it is necessary to protect the therapeutic protein from digestive enzymes, if taken orally. This is the reason for the proteins to be injected directly into the target site.
This can be overcome by putting the proteins (eg–insulin) in a tablet whose coating is made of a material that does not get digest in stomachs acidic medium but dissolves in the intestines thus proteins get absorbed in intestine by villi. The way around this is to protect the therapeutic protein with a covering that will dissolve after it has passed through the stomach.

Question. What is golden rice?
Answer.Golden rice is a genetically modified variety of rice, Oryza sativa, which has been developed as a fortified food for areas where there is a shortage of dietary vitamin A. It contains a precursor of pro-vitamin A, called beta-carotene, which has been introduced into the rice through genetic engineering. The rice plant naturally produces beta-carotene pigment in its leaves. However, it is absent in the endosperm of the seed. This is because beta-carotene pigment helps in the process of photosynthesis while photosynthesis does not occur in endosperm. Since beta-carotene is a precursor of pro-vitamin A, it is introduced into the rice variety to fulfil the shortage of dietary vitamin A. It is simple and a less expensive alternative to vitamin supplements. However, this variety of rice has faced a significant opposition from environment activists. Therefore, they are still not available in market for human consumption.

Question. Compare and contrast the advantages and disadvantages of production of genetically modified crop
Answer.The production of genetically modified (GM) or transgenic plants has several advantages.
(i) Most of the GM crops have been developed for pest resistance, which increases the crop productivity and therefore, reduces the reliance on chemical pesticides.
(ii) Many varieties of GM food crops have been developed, which have enhanced nutritional quality. For example, golden rice is a transgenic variety in rice, which is rich in vitamin A.
(iii) These plants prevent the loss of fertility of soil by increasing the efficiency of mineral usage.
(iv) They are highly tolerant to unfavorable abiotic conditions.
(v) The use of GM crops decreases the post harvesting loss of crops. However, there are certain controversies regarding the use of genetically modified crops around the world. The use of these crops can affect the native biodiversity in an area.
For example, the use of Bt toxin to decrease the amount of pesticide is posing a threat for beneficial insect pollinators such as honey bee. If the gene expressed for Bt toxin gets expressed in the pollen, then the honey bee might be affected. As a result, the process of pollination by honey bees would be affected. Also, genetically modified crops are affecting human health. They supply allergens and certain antibiotic resistance markers in the body. Also, they can cause genetic pollution in the wild relatives of the crop plants. Hence, it is affecting our natural environment.

CASE BASED QUESTIONS

Case 1: Genetically Engineered Insulin

Read the following and answer the questions that follows:

Insulin is prepared by recombinant DNA (rDNA) technology for medicinal purposes on a large scale. It was first produced in 1983 by an American Biotech company. The trademark name is Humulin and it is licensed to Eli Lilly, the company which manufactured it for the first time.
Genes, which code for functional A and B peptides of insulin, were inserted in the plasmids of non-pathogenic E.coli strains. Both the chains are produced separately and joined afterwards by disulphide linkages. Earlier insulin was extracted from the pancreas of killed cattle and pigs. It had shortcomings. It used to stimulate allergic reactions and other immune responses due to its foreign origin in some people. Another challenge was to cater to the ever increasing demand and large scale production. Biosynthetic insulin produced by rDNA technology is purer than animal insulin. It reduces the formation of antibodies against it.

Question. The two polypeptides of human insulin are linked together by
(a) Hydrogen bonds
(b) Covalent bonds
(c) Disulphide bonds
(d) Peptide bonds

Question. C-peptide of human insulin is:
(a) A part of mature insulin molecule
(b) Responsible for formation of disulphide bridges
(c) Removed during maturation of pro-insulin to insulin.
(d) Responsible for its biological activity.

Question. Humulin is the term used for
(a) A powerful antibiotic
(b) A new digestive enzyme
(c) New Research integrated to human welfare
(d) Human insulin

Question. Which technology is used to produce human insulin in E. coli?
(a) RNA interference
(b) Polymerase chain reaction
(c) Gene therapy
(d) Recombinant DNA technology

Question. Production of human protein in bacteria by genetic engineering is possible because
(a) The genetic code is universal
(b) The mechanism of gene regulation is identical in humans and bacteria
(c) The human chromosome can replicate in bacterial cell
(d) Bacterial cells can carry out the RNA splicing reactions

Case 2: Bt Cotton

Read the following and answer the questions that follows:

Bacillus thuringiensis (often referred to as simply “Bt”) is a common, naturally occurring bacterium found in soils and on plant leaves worldwide. The secret to Bt’s success is a family of proteins that these bacteria produce that specifically target insect digestive tracts. These proteins are shaped like crystals, so they are commonly called “Crystalline toxins” or “Cry toxins.” These Cry toxins remain inactive until consumed by an insect. Once digested, the protein is activated and then binds to specific receptors in insect guts. Once bound, the Cry toxins pierce holes in the insect’s gut, ultimately causing the contents to leak and the insect to starve. Many types of Cry toxins exist with varying specificity to different insects (primarily moths and butterflies, beetles, and flies). The diverse Cry toxins can be mixed and matched to control several pests at once.

Question. The genetically modified brinjal in India has been developed for
(a) Drought resistance
(b) Enhancing mineral content
(c) Enhancing shelf life
(d) Insect resistance

Question. Cry-I endotoxins obtained from Bacillus thuringiensis are effective against
(a) Nematode
(b) Boll worms
(c) Mosquitoes
(d) Flies

Question. A protoxin is
(a) A primitive toxin
(b) A denatured toxin
(c) Toxin produced by protozoa
(d) Inactive toxin

Question.The trigger for activation of toxin of Bacillus thuringiensis is
(a) Acidic pH of stomach
(b) High temperature
(c) Alkaline pH of gut
(d) Mechanical action in the insect gut

Question. Bt cotton is not
(a) A GM plant
(b) Insect resistant
(c) A bacterial gene expressing system
(d) Resistant to all pesticides