Molecular Basis of Inheritance

2.1 Core concepts

• DNA is a long polymer of deoxyribonucleotides; length is measured in nucleotides/base pairs. phiX174 = 5386 nt, lambda phage = 48,502 bp, E. coli = 4.6 x 10^6 bp, haploid human DNA = 3.3 x 10^9 bp (NCERT §5.1, p. 80).
• A nucleotide = nitrogenous base + pentose sugar (deoxyribose in DNA, ribose in RNA) + phosphate. Purines = Adenine, Guanine; Pyrimidines = Cytosine, Thymine (DNA) / Uracil (RNA). Base + sugar = nucleoside; +phosphate (at 5'-C) = nucleotide. Nucleotides link via 3'-5' phosphodiester bonds (NCERT §5.1.1, p. 80).
• Friedrich Meischer (1869) first isolated DNA from nucleus and called it 'Nuclein'; Watson and Crick (1953) proposed the Double Helix using Wilkins-Franklin X-ray data and Chargaff's rule (A/T = G/C = 1) (NCERT §5.1.1, p. 81).
• Salient features of B-DNA double helix: two anti-parallel chains (5'->3' paired with 3'->5'); A=T (2 H-bonds), G=C (3 H-bonds); right-handed; pitch (per turn) = 3.4 nm; ~10 bp/turn; rise per bp = 0.34 nm; base-pair stacking stabilises the helix (NCERT §5.1.1, pp. 81-82).
• Central Dogma (Francis Crick): genetic information flows DNA -> RNA -> Protein; in some viruses the flow is reversed (RNA -> DNA, reverse transcription) (NCERT §5.1.1, p. 82).
• DNA packaging: prokaryotes hold negatively-charged DNA with positively-charged proteins in a region called the nucleoid as large loops. Eukaryotes use basic histones (rich in lysine + arginine); 8 histones form a histone octamer around which ~200 bp of DNA wraps to form a nucleosome. Nucleosomes are the repeating unit of chromatin ("beads-on-string" under EM) -> chromatin fibres -> metaphase chromosomes; higher-order packaging needs Non-histone Chromosomal (NHC) proteins. Euchromatin = loosely packed, light-staining, transcriptionally active; Heterochromatin = densely packed, dark-staining, inactive (NCERT §5.1.2, pp. 83-84).
• Search for genetic material — Griffith (1928) worked on Streptococcus pneumoniae: live S (smooth, virulent) killed mice; live R (rough) did not; heat-killed S alone did not; heat-killed S + live R -> mice died and live S was recovered -> "transforming principle" transferred genetic material (NCERT §5.2, pp. 84-85).
• Avery, MacLeod & McCarty (1933-44) purified biochemicals from heat-killed S; only DNA caused transformation; proteases and RNases did not block transformation, but DNase did — concluded DNA is the hereditary material (NCERT §5.2, p. 85).
• Hershey-Chase (1952) used T2 bacteriophages grown on 32P (labels DNA) and 35S (labels protein); after infection of E. coli, the blender removed phage coats and centrifugation separated cells from coats. Only 32P (DNA) entered bacteria; 35S (protein) stayed outside -> DNA is the genetic material (NCERT §5.2.1, pp. 85-86).
• Criteria a genetic material must fulfill: (i) replication, (ii) chemical/structural stability, (iii) scope for slow change (mutation), (iv) ability to express as Mendelian characters. DNA wins on stability (no 2'-OH; thymine instead of uracil; double-stranded with repair). RNA, however, evolved first and mutates faster (NCERT §5.2.2, p. 87).
• RNA World — RNA was the first genetic material; it served as both genetic material and catalyst (ribozymes catalyse splicing, peptide-bond formation). DNA evolved from RNA with chemical modifications that made it more stable (NCERT §5.3, p. 88).
• Replication is semi-conservative — proposed by Watson & Crick (1953). Proved by Meselson and Stahl (1958) in E. coli grown in 15NH4Cl, then shifted to 14NH4Cl. After one generation (20 min) DNA was of hybrid density; after two generations (40 min) hybrid + light in equal amounts. Taylor et al. (1958) proved the same in Vicia faba using radioactive thymidine (NCERT §5.4 & §5.4.1, pp. 88-90).
• Replication enzymes — main enzyme is DNA-dependent DNA polymerase; E. coli (4.6 x 10^6 bp) replicates in ~18 min -> average rate ~ 2000 bp/sec. dNTPs serve dual roles (substrate + energy from terminal phosphates). Replication happens at the replication fork; polymerase synthesises only in 5'->3', so the 3'->5' template strand is copied continuously and the 5'->3' template strand is copied discontinuously (Okazaki-style fragments joined by DNA ligase). Replication begins at the origin of replication; in eukaryotes it happens during S-phase (NCERT §5.4.2, pp. 90-91).
• Transcription copies one DNA strand (3'->5' = template strand) into RNA following A-U pairing; the other DNA strand (5'->3') is the coding strand (same sequence as RNA except T for U). A transcription unit = Promoter + Structural gene + Terminator. Promoter is upstream (5'-end of coding strand) and binds RNA polymerase; terminator is downstream (3'-end) (NCERT §5.5 & §5.5.1, pp. 91-92).
• A cistron = DNA segment coding for a polypeptide. Eukaryotic structural genes are monocistronic and split (exons + introns); prokaryotic structural genes are polycistronic and continuous. Exons appear in mature RNA; introns are removed by splicing (NCERT §5.5.2, p. 93).
• Bacteria have a single DNA-dependent RNA polymerase that makes all RNAs; it needs sigma factor for initiation and rho factor for termination. Eukaryotes have three nuclear RNA polymerases: RNA Pol I -> rRNAs (28S, 18S, 5.8S); RNA Pol II -> hnRNA (mRNA precursor); RNA Pol III -> tRNA, 5S rRNA, snRNAs. hnRNA is processed by splicing, 5'-capping (methyl-guanosine triphosphate added) and 3'-tailing (200-300 adenylate residues, template-independent) (NCERT §5.5.3, pp. 93-95).
• Genetic code is triplet (Gamow's proposition; 4^3 = 64 codons). 61 code for amino acids; 3 are stop (UAA, UAG, UGA). Properties: triplet, degenerate (multiple codons per amino acid), contiguous (no punctuation), nearly universal (exceptions in mitochondrial & some protozoan codons), unambiguous (one codon -> one amino acid). AUG has dual function — Methionine + initiator (NCERT §5.6, pp. 95-96).
• Code was deciphered using Khorana's chemically synthesised RNAs (homopolymers/copolymers), Nirenberg's cell-free protein-synthesis system, and Ochoa's polynucleotide phosphorylase (NCERT §5.6, p. 96).
• Point mutation example — sickle-cell anaemia (single bp change -> glutamate replaced by valine in beta-globin). Insertion/deletion of bases not in multiples of 3 = frameshift mutation; insertion/deletion of 3 or multiples leaves reading frame intact (NCERT §5.6.1, pp. 97-98).
• tRNA = adapter molecule (Crick's postulate); has an anticodon loop (reads codon) and an amino-acid acceptor end (binds amino acid). Specific tRNAs for each amino acid; a separate initiator tRNA; no tRNA for stop codons. Secondary structure = clover-leaf; actual 3-D = inverted L (NCERT §5.6.2, p. 98).
• Translation — amino acids polymerise via peptide bonds to make polypeptides. First, amino acids are activated (ATP) and attached to cognate tRNA = charging/aminoacylation of tRNA. Ribosome = rRNA + ~80 proteins; in inactive state = large + small subunits. 23S rRNA in bacteria acts as the peptidyl-transferase ribozyme. mRNA has 5' and 3' UTRs (untranslated regions, flanking start/stop codons) needed for efficient translation. Initiation begins when ribosome binds mRNA at AUG; elongation adds amino acids per codon-anticodon pairing; a release factor binds stop codon to terminate (NCERT §5.7, pp. 98-99).
• Regulation of gene expression — in eukaryotes can occur at (i) transcriptional, (ii) processing/splicing, (iii) mRNA transport, (iv) translational levels. In prokaryotes, transcription-initiation is the dominant control point; regulatory proteins act as activators (+) or repressors (-) via operator sequences (NCERT §5.8, pp. 99-100).
• ***lac* operon (Jacob & Monod) — polycistronic structural genes (z, y, a) regulated by one common promoter (p), one operator (o) and one regulatory gene (i — for "inhibitor"). z -> beta-galactosidase (hydrolyses lactose -> glucose + galactose); y -> permease (increases cell permeability to beta-galactosides); a -> transacetylase. Inducer = lactose / allolactose**. In absence of lactose, the i-gene product (repressor) binds operator and blocks RNA polymerase. In presence of lactose, repressor binds inducer -> inactive -> RNA polymerase transcribes z, y, a. Negative regulation; positive regulation also exists (not detailed). Glucose and galactose are NOT inducers (NCERT §5.8.1, pp. 100-101).
• Human Genome Project (HGP) — launched 1990, completed 2003; 13-year mega-project coordinated by US Dept. of Energy + NIH, with Wellcome Trust (UK), Japan, France, Germany, China. Cost estimate at start: ~US $9 billion (US$3 per bp x 3 x 10^9 bp). Closely linked to growth of Bioinformatics. Goals included identifying ~20,000-25,000 human genes, sequencing ~3 x 10^9 bp, building databases, tool development, technology transfer and addressing ELSI (ethical, legal, social issues) (NCERT §5.9, pp. 102-103).
• HGP methodology — two approaches: Expressed Sequence Tags (ESTs) identified RNA-expressed genes; Sequence Annotation sequenced the entire genome and later assigned functions. Total DNA was fragmented, cloned into BAC (bacterial artificial chromosomes) or YAC (yeast artificial chromosomes), then sequenced on automated sequencers using Frederick Sanger's method. Chromosome 1 was the last sequenced (May 2006); 24 human chromosomes = 22 autosomes + X + Y (NCERT §5.9, p. 103).
• Salient HGP findings — human genome = 3164.7 million bp; average gene ~ 3000 bp; largest gene = dystrophin (2.4 million bp); total genes estimated at ~30,000 (much less than earlier 80,000-1,40,000); 99.9% of bases identical across humans; functions unknown for >50% genes; <2% of genome codes for proteins; repeat sequences make up a large portion. Chromosome 1 has the most genes (2968), Y has the fewest (231). ~1.4 million SNPs (Single Nucleotide Polymorphisms — "snips") identified (NCERT §5.9.1, p. 104).
• DNA Fingerprinting — developed by Alec Jeffreys; identifies polymorphisms in repetitive DNA sequences. Bulk genomic DNA + small density peaks = satellite DNA, classified by base composition, segment length and repeat number into micro-satellites, mini-satellites etc. These don't code for proteins but show high polymorphism. The probe used = Variable Number of Tandem Repeats (VNTR) — a mini-satellite repeated in tandem; VNTR size varies from 0.1 to 20 kb. Steps: (i) isolation of DNA, (ii) restriction digestion, (iii) electrophoresis, (iv) Southern blotting to nitrocellulose/nylon, (v) hybridisation with labelled VNTR probe, (vi) autoradiography. Same in all tissues of one person, but unique between individuals (except identical twins). Sensitivity enhanced by PCR. Applications: forensic science, paternity testing, population/genetic diversity studies (NCERT §5.10, pp. 105-107).

2.2 Definitions to memorise

2.3 Diagrams / processes to remember

• Figure 5.1 / 5.2 (pp. 80, 82) — Polynucleotide chain & double-stranded DNA with 5' phosphate, 3' hydroxyl, sugar-phosphate backbone, A-T (2 H-bonds), G-C (3 H-bonds).
• Figure 5.3 (p. 82) — DNA double helix (right-handed, base pairs stacked, sugar-phosphate backbone outside).
• Figure 5.4a/b (p. 83) — Nucleosome (DNA around histone octamer + H1) and EM "beads-on-string" chromatin.
• Figure 5.5 (p. 86) — Hershey-Chase experiment (32P enters bacteria; 35S stays in supernatant after blending + centrifugation).
• Figure 5.6 (p. 88) — Watson-Crick semi-conservative replication model.
• Figure 5.7 (p. 89) — Meselson-Stahl CsCl gradient showing heavy / hybrid / light DNA at 0 / 20 / 40 min.
• Figure 5.8 (p. 91) — Replication fork: continuous (5'->3' on 3'->5' template) vs discontinuous (Okazaki-style) synthesis.
• Figure 5.9 (p. 92) — Transcription unit: Promoter – Structural gene – Terminator (with coding/template strands).
• Figure 5.10 (p. 93) — Bacterial transcription: Initiation (sigma), Elongation, Termination (rho).
• Figure 5.11 (p. 94) — Eukaryotic transcription with capping (m-G-ppp), splicing of introns, polyadenylation (poly-A tail).
• Table 5.1 (p. 96) — Codon checker-board for all 64 codons.
• Figure 5.12 (p. 98) — tRNA clover-leaf with anticodon loop and amino-acid acceptor end.
• Figure 5.13 (p. 99) — Translation on ribosome (mRNA, tRNAs, growing polypeptide).
• Figure 5.14 (p. 101) — lac operon in absence vs presence of inducer.
• Figure 5.15 (p. 103) — HGP workflow (cell -> chromosome -> DNA -> automated sequencer -> computer alignment).
• Figure 5.16 (p. 107) — DNA fingerprinting via VNTR copy number, restriction digestion, electrophoresis, autoradiography.

2.4 Common confusions / NTA trap points

• A-T has 2 hydrogen bonds, G-C has 3 — NTA loves to flip these.
• Pitch (per full turn) = 3.4 nm; rise per bp = 0.34 nm; bp/turn = 10. Don't mix up "pitch" with "distance between adjacent bp".
• Coding strand = the one NOT transcribed (5'->3', same as mRNA except T->U). Template strand = the one read by RNA pol (3'->5'). Most students reverse these.
• i gene of lac operon means "inhibitor", NOT "inducer". The repressor itself is constitutively expressed.
• Inducer for lac operon = lactose / allolactose only; glucose and galactose CANNOT induce it.
• RNA Pol I -> rRNA (28S, 18S, 5.8S); RNA Pol II -> hnRNA/mRNA; RNA Pol III -> tRNA, 5S rRNA, snRNA. The "II makes mRNA" mapping is the most trapped fact.
• Hershey-Chase used 32P (DNA) and 35S (protein) — NOT 14N or 15N (that was Meselson-Stahl).
• Human genome = ~3 x 10^9 bp total / 3.3 x 10^9 haploid / 6.6 x 10^9 diploid / 3164.7 million bp per HGP findings — pay attention to which figure the stem asks for.
• Chr 1 has most genes (2968), Y has fewest (231) — students often guess "X" for fewest.