Biotechnology and its Applications

Statement I is false: During translation, tRNA binds mRNA via anticodon-codon base pairing, and rRNA aligns and binds mRNA. Statement II is true: RNAi is conserved across all eukaryotes as an innate defense against transposons and viral infection.

In man, insulin is synthesised as a pro-hormone (proinsulin) containing an extra stretch called the C-peptide. Functional insulin has A and B chains linked together by disulphide bridges, not hydrogen bonds – so this statement is not correct. Genetically engineered insulin is produced in $E.\ coli$.

Statement I: Bt toxins are insect-group specific and are coded by $cry$ genes, e.g. $cry$IAc – true. Statement II: Bt toxin exists as an inactive protoxin in $B.\ thuringiensis$; after ingestion, it is converted into the active form due to the alkaline pH of the insect gut, not acidic – false. So Statement I is true but Statement II is false.

Correct sequence: Bacillus thuringiensis produces crystals with toxic insecticidal proteins (B) → these toxin proteins exist as inactive protoxins in the bacteria (E) → the alkaline pH of the insect gut solubilises the crystals (C) → the inactive toxin is converted into active form due to the alkaline pH (A) → the activated toxin binds to midgut cells, creates pores, and kills the insect (D). So the order is B → E → C → A → D.

Of these, serum and urine analysis does not fulfil the purpose of early diagnosis of a disease. In early stages of infections, when the disease symptoms are not visible, amplification of pathogenic nucleic acid can be done by PCR, detection of antibodies produced by host against pathogen by ELISA and rDNA technology can serve the purpose of early diagnosis.

ADA deficiency is caused by the deletion of the gene for adenosine deaminase, an enzyme crucial for the immune system to function.
Enzyme replacement therapy (injecting functional ADA) and bone marrow transplantation are not completely curative.
In gene therapy, lymphocytes from the patient's blood are grown in culture, a functional ADA cDNA is introduced into them using a retroviral vector, and they are returned to the patient. Since these cells are not immortal, the patient requires periodic infusion of such lymphocytes; so this is not a permanent cure either.
However, if the gene isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.
Hence the permanent cure is introducing bone marrow cells producing ADA into cells at early embryonic stages.

Genetically engineered Human Insulin is produced using E. coli (A-II); GM Cotton (Bt cotton) uses toxin genes from Bacillus thuringiensis (B-IV); ADA Deficiency is treated using Gene therapy (C-I); ELISA is based on Antigen-antibody interaction (D-III).

The Genetic Engineering Approval Committee (GEAC) is set up by the Indian Government to make decisions regarding the validity of GM research and safety of GM organisms for public use, so Statement I is true. Biopiracy refers to the unauthorised use of bio-resources (and traditional knowledge) by other nations/organisations without compensating the native people/country, not the use of bio-resources by native people themselves, so Statement II is false.

The activated Bt toxin binds to the surface of midgut epithelial cells in the insect, creating pores that cause cell swelling and lysis, ultimately killing the insect.

Bioethics refers to the laws and rules established to prevent unauthorised exploitation of bio-resources and traditional knowledge (as opposed to biopiracy, which is the unauthorised exploitation itself).

RNA interference technology has been used to develop nematode-resistant tobacco plants (against Meloidogyne incognita), by silencing specific nematode genes essential for its survival within the host plant.

Gene therapy allows correction of a gene defect (A-IV); RNA interference provides cellular defence (B-III); ELISA is a diagnostic test for AIDS (C-II); Gel Electrophoresis is used for separation of DNA fragments (D-I).

Transgenic mice are being developed for use in testing the safety of vaccines before they are used on humans; transgenic mice are being used to test the safety of the polio vaccine. This statement is correct.
Transgenic models exist for many human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's disease, but cardiac diseases are not among those listed in NCERT.
Rosie, the first transgenic cow, produced human protein-enriched milk containing human alpha-lactalbumin (2.4 g per litre), not high-fat milk.
No 'super power' bulls have been produced by genetic engineering.
Hence genetic engineering has been used to produce transgenic mice for testing the safety of polio vaccine.

The mRNA transcribed from a gene is called the sense RNA.
In antisense technology, a piece of RNA (or a gene producing RNA) with a sequence complementary to a specific mRNA is introduced into the cell. This is the antisense RNA.
The antisense RNA pairs with the complementary sense mRNA to form double-stranded RNA.
This double-stranded RNA cannot be translated and is degraded, so the expression of that specific gene is stopped (the gene is silenced).
Examples: the Flavr Savr tomato, in which the production of the enzyme polygalacturonase is blocked to delay softening, and RNA interference used to protect tobacco plants against the nematode Meloidogyne incognita.
The other statements describe unrelated things; in particular RNA polymerase does not produce DNA, and somaclonal variation arises in tissue culture.
So antisense technology is when a piece of RNA that is complementary in sequence is used to stop the expression of a specific gene.

The Bt toxin protein exists in the bacterium as an inactive protoxin (crystals).
When an insect ingests it, the crystals are solubilised by the alkaline pH of the gut and the protoxin is converted into the active toxin. So it is activated by alkaline, not acid, pH.
The activated toxin binds to the surface of the midgut epithelial cells and creates pores, which cause the cells to swell and lyse, eventually killing the insect.
The bacterium is not killed because the protein is inactive inside it, not because the bacterium is resistant to the toxin.
The toxin is coded by a gene named $cry$ (different cry genes such as cryIAc, cryIIAb, cryIAb code for different toxins), not by 'several genes including cry'.
Hence the protein binds with epithelial cells of the midgut of the insect pest, ultimately killing it.

The nematode $Meloidogyne\ incognita$ infects the roots of tobacco plants and reduces the yield.
To prevent this, nematode-specific genes were introduced into the host plant using $Agrobacterium$ vectors.
The introduced DNA was such that it produced both sense and anti-sense RNA in the host cells.
These two RNAs, being complementary to each other, formed a double-stranded RNA (dsRNA) that initiated RNA interference (RNAi).
RNAi silenced the specific mRNA of the nematode, so the parasite could not survive in the transgenic host.
A toxic protein is the strategy of Bt crops, not of this nematode-resistant tobacco.
Hence the introduced DNA produced both sense and anti-sense RNA.

(A) has a mistake: Rosie was the first transgenic cow, not a buffalo; her milk contained human alpha-lactalbumin (2.4 g per litre).
(B) has a mistake: restriction enzymes cut DNA at specific recognition sequences. DNA is isolated from other macromolecules by using enzymes such as lysozyme, cellulase or chitinase, ribonuclease and protease, followed by precipitation with chilled ethanol.
(C) is correct: downstream processing (separation and purification of the product) is one of the steps of recombinant DNA technology.
(D) is correct: disarmed pathogen vectors such as the Ti plasmid of $Agrobacterium$ and retroviruses are used to transfer recombinant DNA into the host.
Hence the two statements with mistakes are (A) and (B).

Weeds compete with crop plants for nutrients, water and light, and their removal by hand is costly and labour intensive.
Broad-spectrum herbicides such as glyphosate kill weeds, but they also kill ordinary crop plants.
Herbicide resistant (tolerant) GM crops carry a gene that makes them unaffected by the herbicide.
When the field is sprayed with the herbicide, only the weeds are killed and the crop survives, so the weeds are eliminated from the field without manual weeding.
These crops are used along with herbicides, so the objective cannot be to eliminate weeds without the use of herbicides.
Encouraging eco-friendly herbicides or reducing herbicide accumulation in food is not the main objective.
So the main objective is to eliminate weeds from the field without the use of manual labour.

A transgene is a foreign gene introduced into an organism. For it to be expressed, it must be joined to a promoter that the host's RNA polymerase can recognise.
The promoter is the DNA sequence where RNA polymerase binds to start transcription. A tissue-specific promoter is active only in certain cells, so it decides in which tissue (and when) the transgene is expressed.
Example: a seed-specific promoter makes the transgene act only in seeds; the gene for human alpha-1-antitrypsin in Rosie the transgenic cow was placed under a milk-specific promoter.
A reporter gene only helps to identify transformed cells; an enhancer increases the level of transcription but the target tissue is fixed by the promoter.
So the expression of a transgene in the target tissue is determined by the promoter.

ELISA (Enzyme Linked Immuno-Sorbent Assay) is based on the antigen-antibody reaction. It detects the presence of viral antigens or of antibodies made against the virus (e.g. for HIV).
The antibody used is linked to an enzyme. After binding, a colourless substrate is added; the enzyme converts it into a coloured product, and the colour shows a positive result.
The enzymes commonly linked are alkaline phosphatase and horseradish peroxidase, so the enzyme (alkaline phosphatase) is the key reagent.
Southern blotting and DNA probes are used in DNA-based techniques, not in ELISA; catalase is not the enzyme used.
So in ELISA, alkaline phosphatase is the key reagent.

Viruses multiply only inside living cells. Animal cell cultures (e.g. monkey kidney cells, chick embryo fibroblasts, Vero cells) provide such cells on a large scale.
Most viral vaccines, such as those for polio, measles, mumps, rubella and rabies, are produced by growing the virus in animal cell cultures.
Insulin is now produced mainly by recombinant bacteria (E. coli) or yeast, and edible proteins (single cell protein) by microbes. Interferons are also made, but on a much smaller scale than vaccines.
So the maximum application of animal cell culture today is in the production of vaccines.

In somatic cell nuclear transfer (as used for Dolly the sheep), the nucleus of a donor cell is put into an egg cell whose own nucleus has been removed (enucleated).
So the nuclear genes of the organism come from the donor cell.
The cytoplasm, with its mitochondria (and their mitochondrial DNA), comes from the recipient egg cell. So the extranuclear genes come from the recipient cell.
The organism does not have nuclear genes of the recipient, because its nucleus was removed.
So the organism will have extranuclear genes of the recipient cell.