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IQ City Medical College - Biochemistry Pre-Professional Exam

Complete Model Answers

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QUESTION 1 [1+3+6+3+2 = 15 marks]

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i) Provisional Diagnosis [1 mark]

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ii) Inhibitors of Eukaryotic Transcription [3 marks]

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iii) Post-Transcriptional Modifications [6 marks]

1. 5' Capping

• A 7-methylguanosine (7-mG) cap is added to the 5' end of the pre-mRNA
• Linked by an unusual 5'-5' triphosphate bond
• Functions:
  • Protects mRNA from 5'-exonuclease degradation
  • Required for ribosome recognition and initiation of translation
  • Facilitates nuclear export of mRNA

2. 3' Polyadenylation

• A poly-A tail of 150-200 adenylate residues is added to the 3' end
• Catalyzed by poly-A polymerase after cleavage at a consensus signal sequence AAUAAA
• Functions:
  • Protects mRNA from 3'-exonuclease degradation
  • Facilitates nuclear export
  • Promotes translation efficiency

3. RNA Splicing (Removal of Introns)

• Introns (non-coding intervening sequences) are removed; exons are joined
• Occurs in the spliceosome - a large ribonucleoprotein complex containing snRNAs (U1, U2, U4, U5, U6) and proteins
• Mechanism: Two transesterification reactions via a lariat intermediate
• Alternative splicing allows one gene to produce multiple protein isoforms

4. RNA Editing

• Post-transcriptional alteration of nucleotide sequence (see Q1-v below)

Summary diagram:

5'cap - Exon1 - Exon2 - Exon3 - Poly-A tail
        (Introns removed by splicing)

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iv) Eukaryotic RNA Polymerase [3 marks]

• Largest and most complex - 12 subunits
• Has a unique C-terminal domain (CTD) on its largest subunit that is phosphorylated during transcription initiation
• Requires general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) to form the pre-initiation complex at TATA box

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v) RNA Editing [2 marks]

1. Adenosine-to-Inosine (A-to-I) editing:
   • Most common in humans
   • Enzyme: ADAR (Adenosine Deaminase Acting on RNA)
   • Inosine behaves like guanosine in translation
   • Example: Editing of GluR-B (glutamate receptor subunit) mRNA - changes CAG (Gln) to CIG (Arg), altering calcium channel permeability
   • Also mediates editing of apolipoprotein B mRNA
2. Cytosine-to-Uracil (C-to-U) editing:
   • Enzyme: APOBEC (Apolipoprotein B mRNA Editing Catalytic Polypeptide)
   • Classic example: Apolipoprotein B mRNA
     • In liver: unedited → ApoB-100 (4536 aa) for VLDL/LDL
     • In intestine: edited CAA (Gln) → UAA (STOP codon) → ApoB-48 (2152 aa) for chylomicrons

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QUESTION 2 [5×3 = 15 marks]

i) Insulin and Glucose Used to Treat Hyperkalaemia

• Insulin activates the Na+/K+-ATPase pump on skeletal muscle and hepatocyte cell membranes
• This pump drives K+ into cells in exchange for Na+
• Result: Serum K+ falls by 0.5-1.5 mEq/L within 30-60 minutes
• Glucose (50 mL of 50% dextrose) is co-administered to prevent hypoglycaemia from the insulin
• This is a temporizing measure - it shifts K+ intracellularly but does NOT remove it from the body
• Used in emergency treatment of hyperkalaemia (K+ > 6.5 mEq/L or with ECG changes)

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ii) Consumption of Goitrogens Leads to Goitre

1. Goitrogens (e.g., thiocyanates from cabbage, broccoli, cassava; glucosinolates) inhibit thyroid peroxidase (TPO) enzyme
2. TPO is required for: (a) oxidation of iodide to iodine, and (b) organification of iodine onto tyrosine residues on thyroglobulin
3. Inhibition → decreased T3/T4 synthesis → low serum thyroid hormones
4. Low T3/T4 → negative feedback is removed → increased TSH secretion from anterior pituitary
5. TSH stimulates thyroid follicular cell growth (hypertrophy + hyperplasia) → Goitre (enlarged thyroid)

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iii) Warfarin and Phenobarbital Can Cause Drug Interactions

• Warfarin is an anticoagulant metabolized by CYP2C9 (hepatic cytochrome P450 enzyme)
• Phenobarbital is a potent inducer of CYP450 enzymes (CYP1A2, CYP2C9, CYP3A4) via nuclear PXR/CAR receptors
• When phenobarbital is co-administered: it upregulates CYP450 enzymes → increased warfarin metabolism → decreased warfarin plasma levels → reduced anticoagulant effect → risk of thrombosis

• If phenobarbital is started while patient is on stable warfarin dose → INR falls → inadequate anticoagulation
• If phenobarbital is stopped → CYP450 activity returns to baseline → warfarin accumulates → bleeding risk
• Dose adjustment of warfarin is essential; close INR monitoring required

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iv) Mutations Occur Quite Frequently at G-C Sequences

1. In mammals, cytosine residues in CpG dinucleotides are frequently methylated at the 5-position by DNA methyltransferases → forming 5-methylcytosine (5mC)
2. 5-Methylcytosine undergoes spontaneous deamination → converts to thymine (not uracil, as in unmethylated C)
3. This creates a G:T mismatch; if not repaired before replication → C→T transition mutation
4. Because 5mC is not recognized as readily by mismatch repair systems as uracil, repair is less efficient
5. Result: CpG sites mutate at a rate 10-fold higher than other dinucleotides

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v) In Cancer, Hypoxia Stimulates Neoangiogenesis

1. Rapidly growing tumour cells outpace their blood supply → hypoxic microenvironment (pO2 < 10 mmHg)
2. Hypoxia stabilizes HIF-1α (Hypoxia-Inducible Factor-1 alpha): Under normoxia, HIF-1α is hydroxylated by prolyl hydroxylase (PHD) → recognized by VHL protein → ubiquitinated → proteasomal degradation
3. Under hypoxia, PHD is inactive → HIF-1α accumulates, translocates to nucleus, dimerizes with HIF-1β
4. HIF-1 complex binds Hypoxia Response Elements (HRE) in DNA → activates transcription of:
   • VEGF (Vascular Endothelial Growth Factor) - primary angiogenic driver
   • PDGF, bFGF, angiopoietins
5. VEGF binds VEGFR on endothelial cells → proliferation, migration, new vessel formation (neoangiogenesis/angiogenesis)
6. New blood vessels supply oxygen and nutrients to tumour → tumour growth, invasion, and metastasis

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QUESTION 3 [3×6 = 18 marks]

i) Positive Walker's Sign, Positive Steinberg Sign, Ectopia Lentis - Hallmark Features of Marfan's Syndrome

• Autosomal dominant disorder caused by mutations in FBN1 gene (chromosome 15q21) encoding fibrillin-1
• Fibrillin-1 is a glycoprotein essential for formation of microfibrils in the extracellular matrix
• Defective fibrillin → abnormal elastic fibers → multisystem involvement

• Fibrillin-1 also normally sequesters TGF-β; loss of fibrillin → increased free TGF-β → abnormal tissue remodeling

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ii) PCR - Types and Clinical Applications

• DNA template, two specific primers (forward and reverse), Taq DNA polymerase (thermostable), dNTPs, Mg²+ ions, buffer

1. Denaturation (94-96°C): double-stranded DNA is denatured
2. Annealing (50-65°C): primers bind to complementary sequences
3. Extension (72°C): Taq polymerase synthesizes new strand from 5'→3' Each cycle doubles the target DNA → after 30 cycles: 2³⁰ ≈ 10⁹ copies

• Diagnosis of infectious diseases (HIV, TB, hepatitis, COVID-19)
• Prenatal diagnosis (genetic disorders, chromosomal anomalies)
• Cancer diagnosis (BCR-ABL in CML, EGFR mutations)
• Forensic medicine and paternity testing
• HLA typing for organ transplantation
• Detection of minimal residual disease in leukemia

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iii) Superoxide Dismutase and Glutathione Peroxidase are Enzymatic Antioxidants

Superoxide Dismutase (SOD)

• Reaction: 2 O₂•⁻ + 2H⁺ → H₂O₂ + O₂
• Converts superoxide radical to less reactive H₂O₂
• Types:
  • SOD1 (Cu/Zn-SOD): cytoplasm and nucleus
  • SOD2 (Mn-SOD): mitochondrial matrix - most important
  • SOD3 (Cu/Zn-SOD): extracellular
• Clinical significance: SOD1 mutations → ALS (amyotrophic lateral sclerosis)

Glutathione Peroxidase (GPx)

• Reaction: H₂O₂ + 2 GSH → 2 H₂O + GSSG
• Also reduces lipid hydroperoxides (LOOH)
• Cofactor: Selenium (selenocysteine at active site) - dietary selenium deficiency impairs GPx activity
• Requires glutathione (GSH) as electron donor
• Regeneration: GSSG is reduced back to 2 GSH by glutathione reductase using NADPH (from HMP shunt)
• Clinical significance: G6PD deficiency → reduced NADPH → reduced GSH regeneration → impaired GPx activity → hemolytic anemia with oxidant drugs

Complementary system:

O₂•⁻ --SOD--> H₂O₂ --GPx--> H₂O
                        ↑
                      2GSH → GSSG (→ recycled by glutathione reductase + NADPH)
Catalase also converts H₂O₂ → H₂O + O₂ (in peroxisomes)

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iv) ABG Report: pH = 6.7, HCO₃⁻ = 36 mmol/L, pCO₂ = 55 mmHg

• HCO₃⁻ = 36 mEq/L (elevated; normal: 22-26) → suggests metabolic alkalosis OR compensation
• pCO₂ = 55 mmHg (elevated; normal: 35-45) → suggests respiratory acidosis OR compensation

• pH is severely acidotic (6.7)
• pCO₂ is elevated → consistent with respiratory acidosis causing the low pH
• HCO₃⁻ is elevated → metabolic compensation (kidneys retaining bicarbonate)
• However, even with HCO₃⁻ compensation, pH is still very low → severe respiratory acidosis

• Type: Mixed Metabolic and Respiratory Acidosis (severe, pH 6.7 is incompatible with simple respiratory acidosis alone)

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• Ureteroenterostomy, Small bowel fistula, Extra chloride (TPN), Diarrhea (loss of HCO₃⁻)
• Carbonic anhydrase inhibitors (acetazolamide), Adrenal insufficiency, Renal tubular acidosis, Pancreatic fistula

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QUESTION 4 [4×5 = 20 marks]

i) Mechanisms of Hormone Action of Group II Hormones

• Steroid hormones: glucocorticoids (cortisol), mineralocorticoids (aldosterone), sex steroids (estrogen, progesterone, testosterone)
• Thyroid hormones (T3, T4)
• Vitamin D (calcitriol)
• Retinoic acid (Vitamin A)

1. Entry into cell: Being lipophilic, these hormones diffuse freely through plasma membrane
2. Receptor binding:
   • Steroid hormones: bind to receptors in cytoplasm (glucocorticoids, mineralocorticoids) or nucleus
   • Thyroid hormones: bind to receptors already in nucleus
3. Receptor activation:
   • Unbound receptors are associated with heat shock proteins (Hsp90, Hsp70) that maintain them in inactive state
   • Hormone binding → conformational change → dissociation of Hsp → receptor activation
4. Dimerization: Activated receptor forms homodimers (two same receptors) or heterodimers
5. Nuclear translocation (for cytoplasmic receptors): Hormone-receptor complex translocates to nucleus
6. DNA binding: Dimeric complex binds to specific DNA sequences called Hormone Response Elements (HRE):
   • GRE - Glucocorticoid Response Element
   • ERE - Estrogen Response Element
   • TRE - Thyroid Response Element
   • VDRE - Vitamin D Response Element
7. Transcriptional regulation:
   • Binding to HRE activates or represses gene transcription
   • Recruit co-activators (HATs - histone acetyltransferases) or co-repressors
   • Altered mRNA → altered protein synthesis
8. Response: Biological effect occurs after hours to days (delayed compared to Group I hormones)

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ii) Tumour Suppressor Genes

• Both alleles must be inactivated for loss of suppressor function
• First hit: inherited germline mutation (familial) or somatic mutation
• Second hit: loss of the remaining wild-type allele (LOH - Loss of Heterozygosity)

• Point mutations, deletions, chromosomal loss
• Promoter methylation (epigenetic silencing)
• Protein sequestration (e.g., MDM2 binds and degrades p53; HPV E6 protein targets p53)

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iii) Southern Blotting - Definition, Procedure, Applications

• Genomic DNA extracted from cells/tissue
• Digested with restriction endonucleases (e.g., EcoRI, HindIII) → DNA fragments of varying sizes

• Fragments separated by size on agarose gel (smaller fragments migrate faster toward anode)
• Run in presence of ethidium bromide or SYBR Green for visualization

• Gel treated with alkali (NaOH) → DNA denatured into single strands
• Neutralized with buffer

• Single-stranded DNA fragments transferred from gel to nitrocellulose or nylon membrane (positively charged)
• Transfer methods: Capillary action (original), vacuum blotting, electroblotting
• DNA fragments bind to membrane in same position as in gel (Southern transfer)

• DNA baked onto membrane at 80°C (nitrocellulose) or UV crosslinked (nylon)

• Membrane blocked with non-specific DNA/protein
• Labeled probe (radioactive ³²P or chemiluminescent) added - probe is complementary to sequence of interest
• Probe hybridizes to target DNA on membrane (base pairing)

• Membrane washed to remove non-specific binding
• Autoradiography (X-ray film) or chemiluminescence detection
• Dark bands on X-ray film indicate position of target sequence

1. Restriction Fragment Length Polymorphism (RFLP) analysis - genetic mapping
2. Detection of gene rearrangements (e.g., IgH/TCR rearrangements in lymphoma)
3. Diagnosis of genetic diseases (sickle cell anemia, thalassemias - detects specific mutations)
4. Forensic DNA fingerprinting
5. Detection of viral DNA integration (e.g., HPV in cervical cancer)
6. Gene copy number analysis (amplification or deletion)
7. Diagnosis of Duchenne muscular dystrophy (dystrophin gene deletions)

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QUESTION 5 [4×5 = 20 marks]

i) Mechanism of Protein Sorting and Targeting via Rough Endoplasmic Reticulum (RER) Pathway

• mRNA for secretory proteins encodes an N-terminal hydrophobic signal peptide (15-30 amino acids) with a hydrophobic core
• Ribosomes begin translation in cytoplasm
• When signal peptide emerges from ribosome, it is recognized by SRP (Signal Recognition Particle) - a complex of 6 proteins + 7SL RNA

• SRP binding pauses translation (elongation arrest)
• SRP-ribosome-mRNA complex docks at SRP receptor (also called docking protein) on RER membrane

• SRP is released (requires GTP hydrolysis)
• Ribosome docks on translocon (protein-conducting channel = Sec61 complex) in RER membrane
• Translation resumes

• Growing polypeptide chain is threaded through the translocon directly into the RER lumen as it is synthesized
• For transmembrane proteins: stop-transfer sequences (hydrophobic segments) halt translocation and anchor protein in membrane laterally

• Signal peptide is cleaved from the polypeptide in the RER lumen by signal peptidase

• N-linked glycosylation: Oligosaccharyltransferase adds preformed oligosaccharide (Glc₃Man₉GlcNAc₂) to Asn residues (N-X-S/T motif)
• Disulfide bond formation: Protein disulfide isomerase (PDI) catalyzes
• Protein folding: Chaperones (BiP/GRP78, calnexin, calreticulin) assist
• Misfolded proteins → ERAD (ER-associated degradation) via ubiquitin-proteasome system

• Correctly folded proteins exit RER in COPII-coated vesicles
• Travel to cis-Golgi → medial-Golgi → trans-Golgi Network (TGN)
• Further modifications: O-linked glycosylation, phosphorylation, sulfation, proteolytic processing

• Secretory proteins: Packaged in secretory vesicles → exocytosis
• Plasma membrane proteins: Targeted to plasma membrane
• Lysosomal proteins: Tagged with mannose-6-phosphate (M6P) → M6P receptor → late endosomes → lysosomes

mRNA → Ribosome (cytoplasm) → Signal peptide synthesized
→ SRP recognizes signal → SRP receptor on RER
→ Translocation through translocon
→ Signal cleavage → Folding + Glycosylation in RER lumen
→ COPII vesicle → cis-Golgi → trans-Golgi → Secretory vesicle/Lysosome/Plasma membrane

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ii) Protein Energy Malnutrition (PEM)

• Severe calorie restriction → body catabolizes fat stores and muscle protein for energy
• Gluconeogenesis from amino acids, lipolysis → marked wasting
• Insulin levels low → lipolysis predominates

• Adequate carbohydrate → insulin secretion maintained → fat mobilization inhibited
• Protein deficiency → low albumin → reduced oncotic pressure → oedema
• Reduced apoprotein B → impaired VLDL assembly → hepatic fat accumulation → fatty liver
• Deficiency of antioxidant proteins → skin and hair changes

• Low serum albumin, transferrin, prealbumin (rapid turnover - best marker)
• Hypoglycaemia, hypothermia (impaired gluconeogenesis)
• Anaemia (reduced Hb synthesis)
• Immune deficiency (low IgA, T-cell dysfunction) → susceptibility to infections
• Reduced liver enzymes and detoxification capacity

• Phase 1 (Stabilization): Treatment of infections, electrolyte correction (K+, Mg2+), cautious refeeding
• Phase 2 (Rehabilitation): Gradual protein and calorie refeeding
• Warning: Refeeding Syndrome - rapid refeeding → hypophosphatemia, cardiac arrhythmia
• F-75 (stabilization) and F-100 (rehabilitation) WHO therapeutic feeds
• Micronutrient supplementation (zinc, Vitamin A, folic acid)

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