Chapter 5: Molecular Basis of Inheritance | Summary

Frederick Griffith's 1928 transformation experiments with Streptococcus pneumoniae demonstrated that a 'transforming principle' transferred hereditary information from heat-killed S-strain to live R-strain bacteria. Avery, MacLeod, and McCarty (1933–44) biochemically characterised this principle, proving that DNA alone caused transformation; DNase inhibited transformation while proteases and RNases did not.

Alfred Hershey and Martha Chase (1952) provided unequivocal proof using bacteriophages labelled with radioactive 32P (in DNA) and 35S (in proteins). Only radioactive 32P entered bacterial cells, confirming that DNA is the genetic material. Both DNA and RNA can serve as genetic material, but DNA is more stable due to the absence of a 2'-OH group, the presence of thymine instead of uracil, and its double-stranded complementary structure that allows repair.

RNA is considered the first genetic material on Earth. Essential life processes such as metabolism, translation, and splicing evolved around RNA. RNA could act both as a genetic material and as a catalyst (ribozyme). However, RNA's reactive 2'-OH group made it unstable, and DNA evolved from RNA with chemical modifications conferring greater stability.

DNA's double-stranded complementary structure also enabled the evolution of DNA repair mechanisms, further enhancing its reliability as a genetic storage molecule. The Central Dogma, proposed by Francis Crick, describes the flow of genetic information as DNA → RNA → Protein, with reverse transcription (RNA → DNA) occurring in some retroviruses.

Watson and Crick proposed semiconservative replication, wherein each strand of the parental DNA serves as a template for a new complementary strand, producing two daughter DNA molecules each with one parental and one new strand. Meselson and Stahl (1958) experimentally confirmed this in E. coli using 15N and 14N isotopes in CsCl density gradient centrifugation. Similar experiments using radioactive thymidine confirmed semiconservative replication in higher organisms (Vicia faba).

Replication occurs at the replication fork, catalysed by DNA-dependent DNA polymerase that synthesises only in the 5'→3' direction. Replication is continuous on the leading strand (3'→5' template) and discontinuous on the lagging strand (5'→3' template), producing Okazaki fragments joined by DNA ligase. Replication requires a specific origin of replication, deoxyribonucleoside triphosphates as energy-rich substrates, and occurs during the S-phase in eukaryotes. E. coli replicates its 4.6 × 10⁶ bp genome in about 18 minutes at ~2000 bp/second.

Transcription is the process of synthesising RNA from a DNA template, following the principle of complementarity (with uracil replacing thymine). Only one strand of DNA (the template strand, 3'→5' polarity) is copied; the other strand is the coding strand. A transcription unit comprises a promoter (upstream, 5'-end of coding strand), the structural gene, and a terminator (downstream, 3'-end). The promoter binds RNA polymerase and determines which strand is the template.

In bacteria, a single RNA polymerase with sigma (σ) and rho (ρ) factors for initiation and termination transcribes all RNA types; transcription and translation can be coupled. In eukaryotes, three RNA polymerases exist (Pol I for rRNA, Pol II for pre-mRNA/hnRNA, Pol III for tRNA and snRNAs). The primary transcript (hnRNA) undergoes processing: 5'-capping (methylguanosine triphosphate), 3'-tailing (200–300 adenylate residues), and splicing (removal of introns, joining of exons) before export as mature mRNA for translation.

The genetic code is a triplet code: 64 codons (4³) encode 20 amino acids and stop signals. George Gamow first theorised the triplet nature. The code is degenerate (multiple codons for one amino acid), non-overlapping, read without punctuation, and nearly universal (e.g., UUU = phenylalanine from bacteria to humans, with exceptions in mitochondria and some protozoans). AUG (methionine) serves as both an amino acid codon and the initiator codon. UAA, UAG, and UGA are stop (terminator) codons.

The tRNA acts as an adapter molecule: its anticodon loop reads the mRNA codon by complementary base pairing, while its amino acid acceptor end carries the specific amino acid. Each amino acid has at least one specific tRNA. The clover-leaf secondary structure of tRNA folds into an inverted-L compact tertiary structure. Point mutations, such as the single base substitution causing sickle cell anaemia (glutamate → valine), illustrate the direct link between genetic code and protein structure. Frameshift mutations (insertion/deletion of 1 or 2 bases) alter the reading frame, while insertion/deletion of 3 bases inserts/deletes one codon without shifting the frame.

Translation is the polymerisation of amino acids into a polypeptide, directed by mRNA sequence. Amino acids are first activated (aminoacylation of tRNA) using ATP energy. The ribosome — consisting of rRNA and ~80 proteins in two subunits — binds mRNA at the start codon (AUG). The small subunit encounters mRNA; the large subunit provides two binding sites (A and P sites) for tRNA-amino acid complexes. The 23S rRNA (in bacteria) acts as a ribozyme catalysing peptide bond formation.

Elongation proceeds codon by codon as charged tRNAs enter the A-site, form a peptide bond, and translocate to the P-site. At a stop codon (UAA/UAG/UGA), a release factor binds, terminating translation and releasing the polypeptide. Untranslated regions (UTRs) at both 5'- and 3'-ends of mRNA are required for efficient translation. The evolution of translation around RNA supports the RNA World hypothesis.

Gene expression can be regulated at transcriptional, processing, transport, and translational levels. In prokaryotes, transcriptional regulation predominates. Regulatory proteins called repressors (negative regulation) and activators (positive regulation) interact with operator sequences adjacent to promoters to control RNA polymerase access. In bacteria, functionally related genes are grouped in operons regulated by a common promoter and operator.

The lac operon (Jacob and Monod) in E. coli consists of the regulatory i gene (encoding the lac repressor) and three structural genes: z (β-galactosidase), y (permease), and a (transacetylase). In the absence of lactose, the repressor binds the operator and blocks transcription. When lactose (or allolactose) is present, it acts as an inducer, inactivating the repressor and allowing transcription. When lactose is exhausted, the repressor again binds the operator, shutting off the operon. This is an example of substrate-mediated regulation of enzyme synthesis and is the prototype of negative regulation.

The Human Genome Project (HGP), launched in 1990 and completed in 2003, was a 13-year mega project coordinated by the U.S. Department of Energy and NIH, with contributions from the Wellcome Trust (UK), Japan, France, Germany, China, and others. Its goals included identifying all ~20,000–25,000 human genes, sequencing 3 billion base pairs, storing data in databases, improving analytical tools, and addressing ethical, legal, and social issues (ELSI). Methodologies included EST (Expressed Sequence Tags) sequencing and whole-genome shotgun sequencing, using BAC and YAC vectors, automated Sanger sequencing, and bioinformatics.

Key findings: the human genome contains 3164.7 million bp; average gene size is 3000 bases (largest: dystrophin at 2.4 Mb); total genes ~30,000; 99.9% of bases are identical across humans; less than 2% of the genome codes for proteins; repeated sequences form a large portion; chromosome 1 has most genes (2968) and Y chromosome the fewest (231); ~1.4 million SNP locations identified. The project led to the emergence of Bioinformatics and opened new avenues for diagnosis, treatment, and understanding of human biology.

DNA fingerprinting, developed by Alec Jeffreys, exploits polymorphism in repetitive DNA sequences — particularly Variable Number of Tandem Repeats (VNTR), a type of mini-satellite DNA. VNTR copy numbers vary highly between chromosomes and individuals (polymorphism), but are the same in all tissues of an individual. The technique involves DNA isolation, restriction endonuclease digestion, gel electrophoresis, Southern blot hybridisation with a radiolabelled VNTR probe, and autoradiography to generate a unique banding pattern for each individual.

VNTR sizes range from 0.1 to 20 kb, and the resulting autoradiogram bands differ between individuals except in monozygotic (identical) twins. Sensitivity is enhanced by PCR, allowing fingerprinting from a single cell. Applications include forensic identification of suspects, paternity testing, population genetics, and studying genetic diversity and evolutionary biology. Polymorphism arises through mutations in germ cells; if a mutation spreads in a population at frequency >0.01, it is termed DNA polymorphism.

Chapter 5 establishes DNA as the principal genetic material through a combination of biochemical evidence (Griffith, Avery et al., Hershey-Chase) and structural understanding (Watson-Crick model). The chapter demonstrates how genetic information encoded in the sequence of bases flows from DNA to RNA to protein — the Central Dogma — through the processes of replication (semiconservative), transcription, and translation. The genetic code, nearly universal and triplet in nature, links nucleic acid sequence to amino acid sequence. Gene expression is tightly regulated (e.g., lac operon) to conserve cellular energy. The Human Genome Project and DNA fingerprinting represent powerful applications of molecular biology with far-reaching implications for medicine, forensics, and evolutionary biology.

For CBSE board examinations, this chapter is high-yield as it covers several core topics: the experimental proofs for DNA as genetic material, the Double Helix model and base pairing rules, semiconservative replication (Meselson-Stahl experiment), transcription and translation mechanisms, the features of genetic code, lac operon regulation, HGP goals and findings, and DNA fingerprinting techniques. Students must be comfortable with diagrams of nucleosome packaging, the transcription unit, tRNA cloverleaf, ribosome function, and Southern blot hybridisation, as these are commonly tested in both short-answer and long-answer questions.