Q.Which of the following enzymes catalyse the removal of nucleotides from the ends of DNA?

Step 1 — Checking Statement I

The cloning vector pBR322 has two antibiotic resistance genes, ampicillin resistance (ampR) and tetracycline resistance (tetR), each containing unique restriction sites usable for cloning. The BamHI site (along with SalI) lies within the tetR gene (PstI lies within ampR). So Statement I is true.

Step 2 — Checking Statement II

When foreign DNA is inserted at a restriction site located inside the tetR gene, it disrupts the gene's coding sequence — this is called insertional inactivation. As a result, a recombinant E. coli carrying an insert at the BamHI site loses tetracycline resistance and therefore cannot grow in a medium containing tetracycline (such colonies are identified by replica plating as tetracycline-sensitive). So Statement II, which claims it can grow, is false.

Step 1 — (a) Visualisation of DNA in gel electrophoresis

DNA fragments separated by gel electrophoresis are stained with ethidium bromide and viewed under UV light as bright orange-coloured bands. So a → iv.

Step 2 — (b) Precipitation of DNA

After isolating DNA in aqueous form, it is precipitated out of solution by adding chilled ethanol. So b → iii.

Step 3 — (c) Breaking the cell wall of bacteria

Bacterial cell walls (made largely of peptidoglycan) are digested using the enzyme lysozyme. So c → ii.

Step 4 — (d) Breaking the cell wall of fungus

Fungal cell walls (made of chitin) are digested using the enzyme chitinase. So d → i.

Step 5 — Assembling the code

Combining these: a-iv, b-iii, c-ii, d-i, which corresponds to option (A).

Step 1 — The malarial life cycle in the human host

1. An infected female Anopheles mosquito injects sporozoites while biting.
2. The parasites first multiply in the liver cells.
3. They then attack the red blood cells (RBCs), multiplying inside them.
4. The RBCs rupture, releasing new parasites and a toxic substance called haemozoin.

Step 2 — Why haemozoin causes the fever

Haemozoin is a waste product formed as Plasmodium digests the haemoglobin of the RBC (it polymerises the toxic free haem it cannot excrete). When the RBCs burst synchronously, a bolus of haemozoin enters the circulation and triggers the immune/inflammatory response responsible for the chill and high fever recurring every three to four days.

Step 3 — Complete the cycle

When another mosquito bites the infected person, the gametocytes it takes up develop and fuse in the mosquito's gut, forming sporozoites that migrate to its salivary glands — so the mosquito is the vector, and the parasite is a digenic organism (needing two hosts).

Step 4 — Eliminate the other options

• (A) Typhoid — Salmonella typhi, a bacterium; sustained high fever, but no haemozoin (it is not an RBC parasite).
• (B) Dengue — a virus spread by Aedes; no haemozoin.
• (C) Pneumonia — Streptococcus pneumoniae / Haemophilus influenzae, a lung infection. (Note the trap: Haemophilus merely sounds like haemozoin.)

ANSWER: D

The key here is not to memorise blindly, but to connect each drug to its biological function and the disease or condition it treats. Each of these is a product of microbial fermentation or extraction, and their names often hint at their use.

Let’s go through each one.

1. Cyclosporin-A (i)

   This is a cyclic peptide produced by the fungus Trichoderma polysporum. Its primary medical use is as an immunosuppressive agent — it suppresses the immune system to prevent rejection of transplanted organs. So it matches with c.

2. Streptokinase (ii)

   Produced by the bacterium Streptococcus, this enzyme dissolves blood clots by converting plasminogen into plasmin. It is used in emergency medicine to treat heart attacks and strokes — it is a clot buster. So it matches with a.

3. Statins (iii)

   These are produced by the fungus Monascus purpureus (and also synthesised chemically). They work by inhibiting the enzyme HMG-CoA reductase, which is key in cholesterol synthesis. Their effect is to lower blood cholesterol levels. So they match with d.

4. Penicillin (iv)

   The classic antibiotic discovered by Alexander Fleming from the mould Penicillium notatum. It kills bacteria by interfering with cell wall synthesis. It is an antibiotic. So it matches with b.

Now, putting the matches together:

i → c, ii → a, iii → d, iv → b.

The concept: What is $\alpha$-1 antitrypsin, and why does it matter?

$\alpha$-1 antitrypsin (AAT) is a serine protease inhibitor — a protein made primarily in the liver and secreted into the bloodstream. Its main job is to neutralize neutrophil elastase, an enzyme released by white blood cells during inflammation. If left unchecked, neutrophil elastase chews up the elastic fibres in lung tissue, leading to emphysema (a form of chronic obstructive pulmonary disease, COPD).

So AAT is not an enzyme itself — it inhibits an enzyme. It is also not an antacid (that neutralizes stomach acid), and it has no role in arthritis treatment. The key clinical link: people born with a genetic deficiency of AAT are at high risk of early-onset emphysema. The therapy? Purified AAT from human plasma is infused intravenously to raise blood levels and protect the lungs.

Step-by-step reasoning

1. Eliminate (A) — an antacid

   Antacids are simple bases like magnesium hydroxide or calcium carbonate that neutralize stomach acid. $\alpha$-1 antitrypsin is a protein, not a base, and has nothing to do with gastric pH. So (A) is wrong.

2. Eliminate (B) — an enzyme

   AAT is a protease inhibitor, not a protease. It binds to and inactivates the enzyme neutrophil elastase. Calling it an enzyme would be like calling a lock a key — it blocks the action, not catalyses a reaction. So (B) is wrong.

3. Consider (D) — used to treat arthritis

   Arthritis (especially rheumatoid arthritis) involves inflammation, but AAT therapy is not a standard treatment for it. Some research has explored AAT for other inflammatory conditions, but the established, approved use is for emphysema due to AAT deficiency. So (D) is not correct in the exam context.

4. Confirm (C) — used to treat emphysema

   This is the direct application. Patients with AAT deficiency receive weekly intravenous infusions of purified AAT (brand name: Prolastin, among others). This slows the progression of emphysema and reduces lung damage. The therapy is specifically indicated for emphysema caused by AAT deficiency.

Step 1 — The problem a selectable marker solves.

Transformation is very inefficient: only a tiny fraction of the host cells actually take up the plasmid. We need a way to make only those cells grow.

Step 2 — How the resistance gene does it.

The vector carries, say, $amp^R$ (ampicillin resistance). Plating the transformation mixture on ampicillin-containing medium kills every cell that did not take up the vector; survivors necessarily carry it. This is selection.

Step 3 — Selecting recombinants specifically.

pBR322 carries two markers, $amp^R$ and $tet^R$. A foreign gene ligated into the $tet^R$ site inactivates it (insertional inactivation). So:

• grows on ampicillin and tetracycline $\Rightarrow$ non-recombinant (vector re-circularised),
• grows on ampicillin but not tetracycline $\Rightarrow$ recombinant.

Step 4 — Reject the others.

• (B) Cleaving of the vector is done by restriction endonucleases, a property of the enzyme, not of a marker gene.
• (C) Transfer of the foreign gene into the host is achieved by the vector's ori + transformation/vector machinery, not by the resistance gene.
• (D) Competence is induced by divalent cations such as $\text{CaCl}_2$ (or heat shock), again nothing to do with the marker.

ANSWER: A

Step 1 — The problem the method solves.

Plant cells have a rigid cellulose cell wall, so naked DNA cannot simply diffuse in. Direct gene-transfer methods must physically breach the wall and membrane. The biolistic method does exactly that: DNA is precipitated onto inert, dense microparticles of gold or tungsten, which are then accelerated to high velocity by a gene gun so they pierce the cell wall and deliver the DNA into the cell/nucleus.

Step 2 — Why the other three are not this method.

• (A) Microinjection — DNA is injected directly into the nucleus with a fine glass micro-needle; used for animal cells (e.g. producing transgenic animals). No particles, no bombardment.
• (C) Heat-shock method — competent bacterial cells held in divalent Ca²⁺ are given a brief 42 °C shock so plasmid DNA passes through transient pores. Chemical/thermal, not mechanical.
• (D) Disarmed pathogen vector — Agrobacterium tumefaciens, the "natural genetic engineer of plants", whose Ti-plasmid is disarmed (tumour genes removed) and used to carry the gene in. This is vector-mediated, not particle bombardment.

Step 3 — Commit.

Only the biolistic/gene-gun method matches every element of the stem (high velocity + gold/tungsten microparticles + DNA coating + plant cells).

ANSWER: B

Step 1 — What REN means and how REs are named.

A restriction endonuclease cuts DNA at a specific palindromic recognition sequence. Its name is built from its bacterial source: first letter = genus (capital), next two letters = species, then the strain and a Roman numeral for the order of isolation.

Step 2 — Test each option.

• (A) Haemophilus influenzae → HindII (the first restriction endonuclease ever isolated) and HindIII. A source. ✓
• (B) Escherichia coli → EcoRI, the workhorse enzyme cutting $\mathrm{5'\text{-}G\!\downarrow\!AATTC\text{-}3'}$ to give sticky ends. A source. ✓
• (D) Bacillus amyloliquefaciens → BamHI. A source. ✓
• (C) Agrobacterium tumefaciens → this soil bacterium is the "natural genetic engineer of plants": its Ti (tumour-inducing) plasmid is disarmed and used as a cloning vector to deliver genes into plant cells. It lends its name to no restriction enzyme. ✗

Step 3 — Commit.

The question asks for the bacterium that is not a source of a restriction endonuclease, so the odd one out is Agrobacterium tumefaciens.

ANSWER: C