Molecular Basis of Inheritance – Comprehensive Study Notes

Chapter Orientation & Big Picture

• Focus: Molecular Basis of Inheritance – how information is stored, copied, expressed and regulated; technological milestones (HGP, DNA-fingerprinting) included.
• Key learning threads
  • Chemistry & architecture of nucleic acids (DNA/RNA)
  • Experimental journey to identify genetic material
  • Mechanistic detail of replication, transcription, translation
  • Regulatory logic (operons, chromatin state)
  • Genomics & applied molecular forensics

6.1 The DNA – Chemical & Structural Fundamentals

• DNA = long polymer of deoxyribonucleotides
  • Length expressed in nucleotides or base-pairs (bp)
  • Characteristic lengths
  • $\varphi X174$ bacteriophage: $5386$ nt
  • Bacteriophage lambda: $48502\,\text{bp}$
  • E. coli: $4.6\times10^6\,\text{bp}$ (≈1.36 mm)
  • Human haploid: $3.3\times10^9\,\text{bp}$ (≈2.2 m)

6.1.1 Structure of a Polynucleotide Chain

• Each nucleotide = nitrogenous base + pentose sugar + phosphate
  • Bases
  • Purines: Adenine (A), Guanine (G)
  • Pyrimidines: Cytosine (C), Thymine (T – DNA only), Uracil (U – RNA only)
• Linkages & orientation
  • Base–sugar: N-glycosidic bond (→ nucleoside)
  • Nucleoside 5'-phosphorylated → nucleotide
  • 3'→5' phosphodiester bonds connect nucleotides → backbone
  • Free 5'-phosphate = 5'-end; free 3'-OH = 3'-end
• RNA vs DNA
  • Ribose has extra 2'-OH → increased reactivity/lability
  • U replaces T (T = 5-methyl-uracil)

Double-Helix Model (Watson–Crick, 1953)

• Empirical bases: Chargaff ratios (A=T, G=C) & Franklin/Wilkins X-ray diffraction
• Hallmarks
  • Two antiparallel strands: 5'→3' opposite 3'→5'
  • Complementary base-pairing
  • A··T (2 H-bonds); G··C (3 H-bonds) → uniform 1 nm diameter
  • Right-handed helix; pitch $=3.4\,\text{nm}$ ≈ $10$ bp/turn ⇒ $0.34\,\text{nm}$ between successive bp
  • Base-stacking + H-bonds = stability
• Conceptual fallout: Central Dogma $\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}$ (reverse in retroviruses = reverse transcription)

6.1.2 Packaging of the Helix

• Problem: metres-long DNA fits into $\approx10^{-6}\,\text{m}$ nucleus
• Prokaryotes: nucleoid – DNA loops anchored to basic proteins
• Eukaryotes
  • Core unit = nucleosome
  • Histone octamer (2×H2A, H2B, H3, H4) + ~$200\,\text{bp}$ DNA wrap
  • EM: "beads-on-string"; further coiling → chromatin fibre → metaphase chromosome
  • Euchromatin (light, transcription-active) vs heterochromatin (dark, inactive)
  • Higher-order compaction depends on non-histone chromosomal (NHC) proteins

6.2 The Search for Genetic Material

Transforming Principle (Griffith, 1928)

• Smooth (S, virulent, polysaccharide coat) vs Rough (R, non-virulent) Streptococcus pneumoniae
• Heat-killed S + live R → mice died; live S recovered ⇒ heritable "transforming principle"

Biochemical Characterisation (Avery, MacLeod, McCarty 1933-44)

• Purified macromolecules; only DNA fraction transformed R→S
• DNase destroyed activity; protease/RNase did not ⇒ DNA responsible

Hershey–Chase Blender Experiment (1952)

• Bacteriophage labelled with $^{32}\text P$ (DNA) or $^{35}\text S$ (protein)
• Post-infection centrifugation
  • Radioactive DNA entered bacteria; radioactive protein stayed in capsid
• Conclusive proof: DNA = genetic material (except some RNA viruses)

Desirable Properties of Genetic Material

1. Self-replication
2. Chemical & structural stability
3. Amenable to slow mutations (evolution)
4. Expressible as Mendelian traits

• DNA superior: deoxy sugar + T confers stability; double-strand enables repair. RNA excels at expression but is labile.

6.3 RNA World Hypothesis

• Early life likely used RNA for both genetic storage & catalysis (ribozymes)
• Instability drove evolution of more stable DNA; repair mechanisms followed.

6.4 DNA Replication

Semiconservative Model (Watson–Crick insight)

• Parental strands act as templates → each daughter duplex = 1 parental + 1 new strand

Experimental Proof – Meselson & Stahl (1958)

1. Grow E. coli in $^{15}\text N$ medium → heavy DNA
2. Shift to $^{14}\text N$, sample every generation, CsCl gradient
   • 1st gen: single hybrid band
   • 2nd gen: hybrid + light bands (50:50)
   • Prediction: After 3rd gen light : hybrid $=3:1$ etc.

Enzymology & Mechanics

• Key enzyme: DNA-dependent DNA polymerase
  • Adds dNTPs 5'→3'; needs template + primer
  • E. coli rate ≈ $2000\,\text{bp·s}^{-1}$; high fidelity
• Replication fork
  • Leading strand (template 3'→5') → continuous
  • Lagging strand (template 5'→3') → Okazaki fragments, joined by DNA ligase
• Other components: helicase, single-strand binding proteins, primase, topoisomerase
• Origin of replication (ori) critical; plasmid vectors supply ori for cloning.
• Eukaryotes: S-phase, multiple ori; tight coupling to cell cycle (failure → polyploidy)

6.5 Transcription – DNA → RNA

Basic Principles

• Only one DNA strand (template, 3'→5') copied; other = coding strand (identical to RNA except T↔U)
• Entire transcription unit = promoter + structural gene + terminator (plus enhancers/silencers)

RNA Polymerases

• Prokaryotes: single RNA pol + σ (initiation) + ρ (termination) factors
• Eukaryotes
  • Pol I → rRNA (28S,18S,5.8S)
  • Pol II → hnRNA (pre-mRNA)
  • Pol III → tRNA, 5S rRNA, snRNA

Processing (Eukaryotes)

1. Capping – 5' $\text{m}^7\text GTP$ cap
2. Splicing – remove introns, join exons (spliceosome, snRNPs)
3. Tailing – 3' poly-A (≈200-300 A’s)
4. Resulting mRNA exported to cytoplasm

• Split genes (introns) suggest ancient RNA-world heritage.

Coupling

• Prokaryotes: transcription & translation simultaneous in cytosol.

6.6 Genetic Code

Deciphering History

• Gamow’s triplet idea: $4^3 = 64$ possible codons > 20 aa
• Khorana: synthetic RNAs (polymer sequences)
• Nirenberg: cell-free translation assay
• Ochoa: polynucleotide phosphorylase for random polymers

Salient Features

1. Triplet codons – 64 total; 61 sense + 3 stop (UAA, UAG, UGA)
2. Unambiguous – one codon → one aa
3. Degenerate – aa may have multiple codons (synonyms)
4. Comma-less/continuous – no punctuation
5. Nearly universal – mitochondrial & protozoan exceptions
6. AUG – codes Met & serves as start/initiator

Frameshift Illustration

• Sentence analogy: "RAM HAS RED CAP"
  • +1 insertion → nonsense string
  • +3 insertion/deletion keeps reading frame → concept of frameshift vs in-frame mutations

Point Mutation Example

• β-globin: Glu→Val single base change ⇒ sickle-cell anaemia

Adapter Hypothesis – tRNA

• Cloverleaf (2°) → L-shaped (3°) molecule
  • Anticodon loop pairs with mRNA codon
  • 3' CCA amino acid acceptor site
  • Specific aminoacyl-tRNA synthetases charge tRNA with correct aa
  • No tRNAs for stop codons

6.7 Translation – mRNA → Polypeptide

• Occurs on ribosomes (rRNA + ≈80 proteins); rRNA 23S acts as ribozyme for peptide bond formation.

Stages

1. Initiation
   • Small subunit binds mRNA at 5' UTR/start codon
   • Initiator tRNA (Met-tRNA) joins; large subunit forms complete ribosome
2. Elongation
   • Aminoacyl-tRNA enters A-site, peptide bond formed to P-site chain
   • Ribosome translocates 5'→3'
3. Termination
   • Stop codon recruits release factor → chain release, subunit dissociation

• Translational unit = between start and stop codons; flanking 5' & 3' UTRs enhance efficiency/stability.

6.8 Regulation of Gene Expression

Levels in Eukaryotes

1. Transcription initiation
2. RNA processing (alternative splicing)
3. mRNA export
4. Translation control

Prokaryotic Paradigm – lac Operon (Jacob & Monod)

• Components
  • Regulatory i gene → repressor protein
  • Promoter (P), Operator (O)
  • Structural genes: z (β-galactosidase), y (permease), a (transacetylase)
• Mechanism (Negative regulation)
  1. No lactose → repressor binds O, blocks RNA-pol
  2. Lactose/allolactose (inducer) inactivates repressor → transcription ON
  3. When lactose consumed, inducer levels fall → operon OFF
• Also subject to positive regulation via cAMP-CAP (glucose effect)

6.9 Human Genome Project (HGP)

• 13-year international mega-project (1990-2003); cost initially estimated $\approx9$ billion USD
• Goals
  • Sequence $\approx3\times10^9$ bp, identify $20-25\,000$ genes
  • Develop databases, analytical tools, tech transfer & address ELSI issues
• Approaches
  1. ESTs – sequence expressed tags
  2. Shotgun sequencing + sequence annotation
  • Vectors: BAC, YAC; hosts: bacteria, yeast; Sanger dideoxy sequencing; bioinformatics essential
• Key findings
  • $3164.7\,\text{million}$ bases; $\approx30\,000$ genes (far fewer than expected)
  • $<2\%$ genome = coding; $>50\%$ gene functions still unknown
  • 99.9 % sequence identical among humans → $\approx3\times10^6$ variant bp/person
  • Repetitive DNA abundant; Chromosome-1 most genes (2968); Y fewest (231)
  • Identified $1.4\,\text{million}$ SNPs – basis for disease mapping, ancestry

Future & Applications

• Shift from one-gene studies to systems biology, high-throughput omics
• Medical genetics, pharmacogenomics, agriculture, energy, environmental biotechnology

6.10 DNA Fingerprinting

• Relies on polymorphic repetitive DNA (satellite DNA)
  • Mini-satellites → VNTRs (Variable Number Tandem Repeats)
  • Micro-satellites → STRs (Short Tandem Repeats)
• Properties
  • High copy number variability among individuals; inherited in Mendelian fashion
  • Same pattern in every tissue of an individual (except monozygotic twins share profile)
• Classical Technique (Jeffreys)
  1. DNA isolation
  2. Restriction digest
  3. Electrophoresis
  4. Southern blot to nylon/nitrocellulose
  5. Hybridise radiolabelled VNTR probe
  6. Autoradiography → band pattern unique to individual
• PCR-STR modern upgrades: single cell or degraded samples suffice
• Applications
  • Forensic ID, paternity/maternity disputes
  • Population genetics, biodiversity assessments, evolutionary studies

Integrative Connections & Ethical Implications

• Central dogma links DNA structure, replication, expression & regulation; each level exploited in biotech & medicine.
• HGP & fingerprinting raise ELSI debates: privacy, discrimination, consent.
• RNA-world, ribozymes, split genes & introns highlight evolutionary layers in modern cells.