NEET Biotechnology and its Applications Previous Year Questions with Solutions

Bacillus thuringiensis produces intracellular protein crystals during sporulation containing toxic insecticidal Cry proteins. Because the crystals are insoluble at neutral/acidic pH, the protoxin remains non-toxic to the bacterium.

• • Harmless to bacteria and non-target mammals.

Susceptible insect larvae possess a strongly alkaline midgut environment (pH 9.0–10.5). Upon ingestion, the high alkaline pH solubilizes the crystal proteins into active monomers.

• • Midgut proteases then clip terminal residues to liberate the ~60 kDa active core.

The activated toxin binds to cadherin receptors on the surface of midgut epithelial cells, inserting into the membrane to create open pores that cause osmotic swelling, cell lysis, gut paralysis, and death.

• • Alkaline pH is the indispensable initial chemical trigger.

Insulin for diabetes was traditionally extracted from pancreas of slaughtered cattle and pigs, causing severe allergic reactions in some patients:

• • Human insulin is synthesized as a pro-hormone containing an extra C-peptide which is removed during maturation.
• • The major challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form.

The breakthrough approach devised by Eli Lilly:

• • Synthesized two separate DNA oligonucleotides encoding human chain A (21 amino acids) and chain B (30 amino acids).
• • Inserted these sequences into expression plasmids downstream of the $\beta$-galactosidase promoter.
• • Transformed these plasmids into *Escherichia coli* (*E. coli*), which is a bacterium.
• • Chains A and B were extracted from the bacterial cultures and linked in vitro by forming disulfide bonds to yield active mature human insulin.

*E. coli* belongs to Domain Bacteria (Eubacteria). Hence, the organism class used is a Bacterium.

RNA interference is an evolutionarily conserved gene-regulatory and cellular defence mechanism in all eukaryotes:

• • It mediates sequence-specific post-transcriptional gene silencing (PTGS) of targeted mRNA transcripts.
• • The trigger for RNAi is complementary double-stranded RNA (dsRNA).
• • An endoribonuclease called Dicer processes long dsRNA into short interfering RNAs (siRNAs, ~21–23 bp).
• • These siRNAs incorporate into the RNA-induced silencing complex (RISC), which unwinds the duplex and guides the single-stranded antisense strand to the complementary target mRNA, causing enzymatic cleavage and degradation.

Preventing infestation of tobacco roots by the nematode *Meloidogyne incognita*:

• • Using *Agrobacterium* vectors, nematode-specific genes were introduced into the host plant.
• • The introduction of DNA was such that it produced both sense and anti-sense RNA in the host cells.
• • These two RNAs, being complementary to each other, annealed to form a double-stranded RNA (dsRNA) that initiated RNAi and silenced the specific mRNA of the nematode.
• • Consequently, the parasite could not survive in a transgenic host expressing specific interfering RNA.

The specific molecular agent responsible for mRNA silencing in RNAi is Complementary dsRNA.

Human insulin is a small globular protein hormone synthesized by beta-cells of the pancreatic islets of Langerhans:

• • It comprises 51 amino acids distributed into two polypeptide chains: Chain A (21 residues) and Chain B (30 residues), cross-linked by two inter-chain disulfide bridges.
• • Its biological activity relies on an intact, three-dimensional folded conformation.

The digestive physiology of orally consumed proteins:

• • In the stomach, gastric juice contains hydrochloric acid (pH ~1.8) and pepsin, which denatures and cleaves protein polypeptide bonds.
• • In the small intestine, pancreatic proteases (trypsin, chymotrypsin, elastase, carboxypeptidases) and intestinal brush-border peptidases rapidly hydrolyze insulin into dipeptides, tripeptides, and free amino acids.
• • As a consequence, intact functional insulin cannot survive transit through the GI tract to be absorbed into the portal circulation.

Insulin cannot be administered orally because it undergoes enzymatic digestion in the gastrointestinal tract, matching Option (2).

The Bt toxin is coded by a gene named cry. There are a number of them; for example, the proteins encoded by the genes cryIAc and cryIIAb control cotton bollworms, that of cryIAb controls corn borer. Thus, Statement I is true.

Bt toxin exists as an inactive protoxin in the bacterium. Once an insect ingests the inactive toxin, it is converted into an active form of toxin due to the ALKALINE pH of the insect midgut which solubilizes the crystals. The activated toxin binds to the surface of midgut epithelial cells, creates pores, causes cell swelling and lysis, leading to death. Statement II erroneously claims acidic pH, making it false.

Statement I is true but Statement II is false, matching Option (1).

Transgenic animals are produced to yield biological products; human protein (α-1-antitrypsin) is used to treat emphysema (A -> III). Adenosine deaminase (ADA) deficiency can be treated in some children by bone marrow transplantation or enzyme replacement therapy (in which functional ADA is given to the patient by injection) (D -> II).

The proteins encoded by genes cryIAc and cryIIAb control cotton bollworms (C -> I), while that encoded by cryIAb controls corn borer (B -> IV).

A-III, B-IV, C-I, D-II corresponds to Option (1).

Conventional diagnostic techniques rely on gross biochemical changes or visible pathogen counts in blood/urine. By the time symptoms appear and serum/urine metabolites alter, the infection is already widely established:

NCERT states verbatim under Molecular Diagnosis: "Using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible. Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno-sorbent Assay (ELISA) are some of the techniques that serve the purpose of early diagnosis."

PCR can detect trace pathogens ($<10$ viral particles) by exponential target DNA amplification. ELISA exploits high-affinity antibody-enzyme conjugates to detect picogram antigen quantities before clinical symptom onset.

Gene therapy encompasses medical interventions designed to correct genetic defects diagnosed in a child or embryo:

In 1990, the first clinical gene therapy was administered to a 4-year-old girl suffering from adenosine deaminase ($ADA$) deficiency. Lymphocytes were harvested from the patient's blood, transformed in vitro using a retroviral vector carrying functional $cDNA$ for $ADA$, and re-infused into her body.

In molecular cancer diagnosis, single-stranded DNA from patient cell clones is immobilized on a nitrocellulose membrane and allowed to hybridize with a radioactively labelled ($^{32}\text{P}$) probe designed complementary to the normal gene:

As stated verbatim in NCERT: *"A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography. The clone having the mutated gene will hence not appear on the photographic film, because the probe will not have complimentarity with the mutated gene."*

Human insulin is synthesized by beta cells of the islets of Langerhans as an inactive pro-hormone (pro-insulin): - Pro-insulin structure: Consists of three polypeptide chains: Chain A (21 amino acids), Chain B (30 amino acids), and an intervening C-peptide (connecting peptide, 31-35 amino acids). - Maturation: Endopeptidases cleave out the C-peptide, leaving mature functional insulin consisting of only the A-chain and B-chain linked together by two interchain disulphide bridges (and one intrachain disulphide bond on Chain A). - Therefore: - Statement (a) is True: C-peptide is absent in mature insulin. - Statement (c) is True: Pro-insulin possesses the extra C-peptide segment. - Statement (d) is True: A-peptide and B-peptide are interconnected by disulphide bonds.

The major challenge in producing insulin using rDNA technology was getting insulin assembled into a mature form without cellular proteolytic processing machinery: - In 1983, the American pharmaceutical company Eli Lilly synthesized two independent DNA sequences corresponding to Chain A and Chain B of human insulin. - They inserted them separately into plasmids of *Escherichia coli*. - Chains A and B were produced separately, extracted, purified, and subsequently combined by creating disulfide bonds chemically *in vitro*. - Consequently, genetically engineered insulin never contains C-peptide. Thus, Statement (b) is False.

Using conventional methods of diagnosis (such as serum analysis, urine culture, or stool examination), early detection is impossible because pathogens are only detected after symptoms appear and pathogen numbers have proliferated to very high levels. When disease symptoms appear, pathogen concentration is already substantial in the body.

Enzyme-Linked Immunosorbent Assay (ELISA) is based on the principle of antigen-antibody interaction: - Infection by a pathogen can be detected either by the presence of minute amounts of antigens (proteins, glycoproteins of pathogen origin) or by detecting antibodies synthesized by the host immune system against the pathogen. - Because enzyme-substrate colorimetric amplification yields immense sensitivity, ELISA detects sub-clinical infections far earlier than conventional laboratory tests.

- Southern Blotting / Hybridization: Used primarily for DNA profiling, gene mapping, or detecting mutated genes in research/forensics, but is not the front-line clinical diagnostic tool highlighted in NCERT for infectious pathophysiology. - Western Blotting: Used as a confirmatory test (e.g. for HIV), but ELISA is the canonical front-line screening and early diagnostic technique highlighted in the NCERT syllabus.

Adenosine Deaminase (ADA) is a housekeeping enzyme in the purine salvage pathway that catalyzes the irreversible deamination of adenosine and deoxyadenosine into inosine and deoxyinosine: - In the absence of functional ADA (due to autosomal recessive loss-of-function mutations/deletions on chromosome 20), its substrate deoxyadenosine accumulates intracellularly in lymphoid tissues. - Deoxyadenosine is phosphorylated into cytotoxic levels of deoxyadenosine triphosphate (dATP), which allosterically inhibits ribonucleotide reductase, stalling DNA replication and repair. - Immature thymocytes, mature T lymphocytes, and B lymphocytes undergo accelerated apoptosis, completely crippling both cell-mediated and humoral immunity (Severe Combined Immunodeficiency, SCID).

- Parkinson’s disease: A neurodegenerative motor disorder caused by loss of dopaminergic neurons in the substantia nigra of the basal ganglia. - Digestive disorders: Involve gastric, hepatic, or pancreatic enzyme deficiencies (e.g. celiac disease, lactase deficiency). - Addison’s disease: An endocrine disorder caused by autoimmune destruction of the adrenal cortex, leading to hyposecretion of glucocorticoids and mineralocorticoids.

In humans and mammals, insulin is synthesized as a pro-hormone (proinsulin) that requires proteolytic processing before it becomes fully active and functional.

Proinsulin contains an extra polypeptide segment called the C-peptide (connecting peptide). During maturation into functional insulin, this C-peptide is cleaved and removed; it is completely absent in mature active insulin.

Mature functional insulin consists of two polypeptide chains: Chain A (21 amino acids) and Chain B (30 amino acids). These two chains are covalently linked together by two interchain disulphide bonds (and one intrachain bond in Chain A), NOT by weak hydrogen bonds.

In 1983, American company Eli Lilly prepared two DNA sequences corresponding to chains A and B of human insulin, introduced them into plasmids of E. coli to produce chains A and B separately, extracted them, and combined them by creating disulphide bonds in vitro.

Bacillus thuringiensis is a soil bacterium that forms intracellular crystalline protein endotoxins (Bt toxins) encoded by cry genes during sporulation.

Bt toxins have insecticidal activity specific to insect pests belonging to Lepidoptera (cotton bollworms, armyworms), Coleopterans (beetles), and Dipterans (flies, mosquitoes).

Insect pests are herbivorous insects that damage and feed on the cotton bolls. Insect predators are natural beneficial enemies of pests. Bt cotton specifically targets the herbivorous insect pests that ingest the cotton plant tissue.

Resistance to root-knot nematodes (Meloidogyne incognita in tobacco) is achieved through RNA interference (RNAi), not Bt endotoxin genes.

Bt cotton is an insect-resistant transgenic crop created by introducing endotoxin cry genes from the soil bacterium Bacillus thuringiensis. Thus, (a) matches (iv).

The first clinical gene therapy was given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency, where functional ADA cDNA was introduced into her lymphocytes using a retroviral vector. Thus, (b) matches (i).

RNAi takes place in all eukaryotic organisms as a method of cellular defence involving silencing of a specific mRNA due to a complementary dsRNA molecule. Thus, (c) matches (ii).

PCR is a routine molecular diagnostic technique used to detect pathogens like HIV and cancer mutations at very low concentrations even before symptoms appear. Thus, (d) matches (iii).