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Somatic cell hybridization/ parasexual hybridization 1 First interspecific hybrid developed between Nicotiana glauca and Nicotiana langsdorfii in 1972.

First intergeneric hybrid developed between Solanum tuberosum and Lycopersicon esculentum and the hybrids are known as ‘Pomatoes or Topatoes’ .

Insertional inactivation

The sticky ends are made up of unpaired bases . They are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of enzyme DNA ligase.

a. BamHI should be used, as restriction site for this enzyme is present in tet^R region.

b. PvuI will not be used, as restriction site for this enzyme is present in amp^R region (not in tet^R). EcoRI will not be used, as restriction site for this enzyme is not present in selectable marker tet^R.

Correct answer is (c) Tracy

1. Restriction endonucleases have the ability to recognize specific sequences in DNA and cleave it. 

2. They are 4 to 8 nucleotides long and characterized by a particular type of internal symmetry.

3. The specific site at which restriction endonuclease cuts the DNA is called recognition site or restriction site. 

4. Each restriction endonuclease recognizes its specific recognition sequence. 

5. Restriction cutting may result in DNA fragments with blunt ends or cohesive or sticky ends or staggered ends (having short, single stranded projections).

For example, recognition sequence of by the enzyme EcoRI is 

3′ —– -CTTAA-G —–5′ 

5′ —– -G A A T T C —–3′ 

It is as palindrome, i.e. When read on opposite strand of DNA (3′ to 5′ or 5′ to 3′) it reads same. 

When the enzyme EcoRI recognizes this sequence, it breaks each strand at the same site in the sequence i.e. between the A and G residues.

The genetically engineered crop plants carrying desirable traits are called transgenic plants. 

Examples of transgenic plants:

1. Bt Cotton : Bt cotton is a transgenic plant. Bt toxin gene has been cloned and introduced in many plants to provide resistance to insects without the need of insecticides.

2. Golden rice : It is a genetically engineered rice with higher beta carotene (provitamin A) content. 

3. Flavr savr tomato : It is developed by inhibiting synthesis of polygalactournonase by inserting antisense gene. This type of tomato has a longer shelf life.

1. Transgenic plants are potential factories or bioreactors for biochemicals and biopharmaceuticals like starch, sugar, lipids, proteins, hormones, antibodies, vaccines or enzymes. 

2. Various fine chemicals, perfumes, adhesive compounds industrial lubricants, etc. can be isolated from plants. 

3. Plants can be the source of biodegradable plastic and ‘renewable’ energy to replace fossil fuels. 

4. Transgenic plants useful for production of novel drugs like interferons, edible vaccines, antibodies, amino acids, immunotherapeutic drugs, etc. Thus, they are like bioreactors for molecular farming.

Examples

1. The gene for Human growth hormone has been inserted into the chloroplast DNA of tobacco plants. 

2. Humanized antibodies against HIV, Respiratory syncytial virus (RSV), Herpes simplex virus (HSV), the cause of “cold sores” are developed using transgenic plants. 

3. Superglue’ is a biochemical glue for body repairs during surgery. It is produced by tobacco plants which contain genes encoding for adhesive proteins which allow marine mussels to stick to rocks. 

4. Protein antigens to be used in vaccines : e.g. Patient-specific antilymphoma vaccines. B-cell lymphomas are clones of malignant B cells expressing a unique antibody molecule on their surface. 

5. Transgenic plants cam be used as factories for producing oil having nutritional value like cod-liver oil. These plants are engineered with a oil encoding gene from marine algae. 

6. Transgenic plants produce the antimalarial drug, Artemisinin. 

7. Genetically engineered opium poppy can be used to produce powerful painkillers. 

8. Transgenic plants like potatoes, tomatoes, bananas, soybeans, alfalfa and cereals can be used as edible vaccine.

1. Recalcitrant seeds are those whose survival is educed upon drying (reduction in moisture below a certain level) and freezing and thus are difficult to store. 

2. It involves subcellular damage of seeds which results in loss of viability, when dried. 

3. Plants which produce recalcitrant sees could be conserved using tissue culture technique.

Superglue is a biochemical glue for body repairs during surgery.

Gene therapy is being used as follows:

1. Replacement of missing or defective genes. 

2. Delivery of genes that speed the destruction of cancer cells. 

3. Supply of genes that cause cancer cells to revert back to normal cells. 

4. Delivery of bacterial or viral genes as a form of vaccination. 

5. Delivery of DNA to antigen expression and generation of immune response. 

6. Supply of gene for impairing viral replication. 

7. Provide genes that promote or impede the growth of new tissue. 

8. Deliver genes that stimulate the healing of damaged tissue.

Genes can be delivered by three ways:

1. Ex vivo delivery : In this type of gene delivery, viral or non-viral vectors are used to introduce the desired gene in the cells isolated from patient, e.g. Parkinson’s disease, a neurological disorder. 

2. In vivo delivery : In this method, therapeutic genes are directly delivered at the target sites of the cells of diseased tissue. Intravenous infusion genes are injected directly into tumor in the treatment of cancer. 

3. Use of virosomes (Liposome + inactivated HIV), bionic chips.

1. Bt cotton is well known example of insect resistant transgenic plant which is engineered with a gene from B. thuringiensis. 

2. ‘cry’ gene present in B. thuringiensis produces a protein that forms crystalline inclusions in bacterial spores. 

3. When insect ingests it, because of high pH and the proteinase enzymes present in insect’s midgut, the crystalline inclusions are hydrolyzed to release the core toxic fragments. 

4. This toxin causes midgut paralysis and disruption of midgut cells of insect. 

5. Bt toxin acts against many species of Lepidoptera, Diptera and Coleoptera insects.

Superglue is produced by tobacco plants which contain genes encoding for adhesive proteins which allow marine mussels to stick to rocks.

Human growth hormone gene is introduced in sheep for promoting growth and meat production.

Oil is synthesized by the condensation of 3 fatty acid with a molecule of glycerol. Fatty acids are formed through an enzyme complex called fatty acid synthetase. The oil from the seeds can be removed by preventing the synthesis of either glycerol or the enzyme lipase which catalyses the synthesis of oil. It can be achieved by knocking out the genes coding for the enzyme lipase or the enzyme fatty acid synthetase.

Introduction of required genes into cells and tissues to treat diseases / by delivery of normal gene to take over the function of tron-functional gene/by gene therapy, First gene therapy was given to four year old girl with Adenosine deaminase deficiency. 

Detailed Answer:

Gene therapy is an attempt to deal with hereditary or genetic or congenital diseases. This aims at correction of a genetic defect by delivery of a normal gene into an individual or embryo to take over or compensate the function for a non-functional gene. The first disease to have a gene therapy is ADA (Adenosine deaminase) deficiency. In this, the gene coding for enzyme ADA gets deleted leading to deficiency of ADA and problems in immune system. Gene therapy for ADA deficiency includes Isolation of lymphocytes from patient's blood and culturing them in-vitro.

Functional ADA cDNA are then introduced into the cultured lymphocytes. These lymphocytes are returned back to the patient's body. 

Permanent cure for this problem is the introduction of gene isolated from bone marrow cells producing ADA into cells at early embryonic stages.

Gene Therapy

ADA (Adenosine deaminase) deficiency Lymphocytes from the blood of the patient are grown in a culture, a functional ADA cDNA is introduced into these lymphocytes, which are subsequently returned to the patient. The permanent cure is done by introducing ADA cDNA into cells at early embryonic stages.

1. Determine the sequences of the 3 billion base pairs that makes up human DNA. 

2. Store this information in data base. 

3. Develop improvised tools for data analysis. 

4. Transfer related technologies to other sectors, such as industries. 

5. Address the ethical, legal and social issues (ELSI) that may arise from the project.

Plasmid and Bacteriophage.

(a) Insulin production is 100 mg/L;

so fermentor volume needed for 1 Kg of insulin is 1 Kg / 100mg = 1000, 000mg/100,g = 10,000mg = 10,000L.

So we need 10,000-litre fermentor to produce 1 Kilogram of insulin in one batch.

(b) In this case the cell concentration is increased to 50 g/L;

so insulin production per liter will be 50 X 100 = 5000 mg = 5 g / L;

Thus, to produce 1 Kilogram of insulin we need 1 Kilogram / 5 g = 1000 g / 5g = 200 g. 

So, if the cell concentration is increased 50 times, we need 200-litre reactor to produce 1 Kilogram of insulin.

Causes: 

1.presence of proteins in the culture medium 

2. some molecules produced by the microbes. 

Control: Antifoams such as fatty acids(olive oil, sunflower oil) or silicone 

Somaclonal variations 

(a)It is associated with chromosomal variation 

(b) It helps in production of mutants e.g. disease resistant in Potato

The two components were—antibiotic resistance gene and plasmid vector of Salmonella typhimurium.

Selectable marker helps in the identification and elimination of non-transfomants and permitting the growth of the transformants. Therefore, they are considered essential in cloning vector.

Selectable markers are essential to identify and eliminate non-transformants, by selectively permitting the growth of the transformant.

1. Genes of interest can be inserted into animals, so as to produce better farm animals.

2. Desired proteins like insulin, hormones, interferons, vitamins can be manufactured in bacteria on an industrial basis.

3. Recombinant vaccines, improved antibiotics can be produced by gene cloning.

In biolistics method, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.

The host that produces a foreign gene product is called competent host. The product is called recombinant protein.

Cloning vectors are used for transferring fragments of foreign DNA into a suitable host. They are also used to select recombinants from non-recombinants.

The small, extra chromosomal double stranded, circular forms of DNA which are capable of autonomous replication are called plasmids.