Q.Do eukaryotic cells have restriction endonucleases? Justify your answer.

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 …

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 …

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 …

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 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 …

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. …

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. …

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. ✓ …