Molecular Basis of Inheritance (Molecular Biology)

Molecular Basis of Inheritance NEET Notes | High-Yield PYQs & DNA Replication

1. The DNA

Structure of Polynucleotide Chain

Nucleotide components: A nitrogenous base, a pentose sugar (ribose in RNA, deoxyribose in DNA), and a phosphate group.
N-glycosidic linkage: Bonds the nitrogenous base to the 1'-carbon of the pentose sugar to form a nucleoside NEET 2021.
Phosphodiester linkage: Bonds the phosphate group to the 5'-OH of a nucleoside and the 3'-OH of the adjacent nucleotide to form a polynucleotide chain AIPMT 2000 NEET 2017 2021.
Backbone: Formed by sugar and phosphate. The nitrogenous bases project from this backbone.

The Double Helix Model

Proposed by James Watson and Francis Crick (1953) based on X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin AIPMT 1990 1998 NEET 2020.
Erwin Chargaff's Rule: For double-stranded DNA, the ratios between Adenine/Thymine and Guanine/Cytosine are constant and equal to one. Equation: A+G = T+C or (A+G)/(T+C) = 1 AIPMT 1991 2008 NEET 2019 2020.
Exception/Crucial detail: Chargaff's rule applies only to double-stranded DNA, not single-stranded DNA or RNA AIPMT 2008.
Helix Dimensions: Pitch of the helix is 3.4 nm (34 Å). There are roughly 10 bp in each turn. The distance between consecutive base pairs is 0.34 nm (3.4 Å) AIPMT 2008 2015.
Stability: Hydrogen bonds (A=T with 2 H-bonds, G≡C with 3 H-bonds) and the plane of one base pair stacking over the other confer stability to the helical structure.

Packaging of DNA Helix

Histones: Positively charged, basic proteins. They are rich in basic amino acid residues Lysine and Arginine AIPMT 1990 NEET 2020 2021.
Nucleosome: The basic unit of DNA packaging. Formed by DNA (negatively charged) wrapped around a histone octamer. A typical nucleosome contains 200 bp of DNA helix NEET 2020.
Crucial detail: Histone H1 sits outside the octamer core, acting as a linker/seal.
Non-histone Chromosomal (NHC) Proteins: Required for higher-level packaging of chromatin.

Chromatin Type	Structure/Density	Transcriptional Activity	Stain
Euchromatin	Loosely packed	Transcriptionally Active NEET 2017 2019	Stains light
Heterochromatin	Densely packed	Transcriptionally Inactive	Stains dark

2. The Search for Genetic Material

Transforming Principle (Griffith's Experiment)

Conducted by Frederick Griffith (1928) using Streptococcus pneumoniae (causes pneumonia) in mice AIPMT 1999 2002 2014.
Strains used: S strain (smooth, virulent, possesses a polysaccharide capsule) and R strain (rough, non-virulent, no capsule).
Observation: Heat-killed S strain + live R strain injected into mice → Mice died. Live S strain was recovered.
Conclusion: The R strain was "transformed" by a 'Transforming Principle' transferred from the heat-killed S strain, enabling it to synthesize a smooth capsule and become virulent.

Biochemical Characterisation of Transforming Principle

Conducted by Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-44) to determine the biochemical nature of Griffith's transforming principle.
Result: Proteases and RNases did not affect transformation. DNase inhibited transformation, proving that DNA was the transforming chemical NEET 2020 2023.

The Hershey-Chase Experiment (Unequivocal Proof)

Conducted by Alfred Hershey and Martha Chase (1952) using bacteriophage T2 and Escherichia coli. This provided the unequivocal proof that DNA is the genetic material AIPMT 2012 NEET 2017 2019 2022.
Methodology:
- Phages grown in radioactive phosphorus (³²P) had radioactive DNA (phosphorus is in DNA, not protein).
- Phages grown in radioactive sulfur (³⁵S) had radioactive protein (sulfur is in protein, not DNA).
Steps: Infection → Blending (removes viral coats) → Centrifugation (separates virus particles from bacteria).
Conclusion: Bacteria infected with ³²P were radioactive, proving DNA entered the cell. Bacteria infected with ³⁵S were non-radioactive.

3. Properties of Genetic Material (DNA vs RNA)

To act as genetic material, a molecule must:

Generate its replica.
Be chemically and structurally stable.
Provide scope for slow mutation (evolution).
Express itself in the form of Mendelian characters.

Why DNA is more stable than RNA:
- RNA has a 2'-OH group present at every nucleotide, making it labile, reactive, and easily degradable NEET 2017 2018. RNA is also known to be catalytic (ribozyme).
- DNA contains Thymine (5-methyl uracil) instead of Uracil, which confers additional stability NEET 2021.
Crucial detail: RNA is the genetic material in some viruses like Tobacco Mosaic Virus (TMV) and QB bacteriophage NEET 2018. RNA was the first genetic material (RNA World hypothesis).

4. DNA Replication

Semiconservative Nature

Proposed by Watson and Crick simultaneously with their double-helix model AIPMT 2001 2008.
Proved experimentally by Matthew Meselson and Franklin Stahl (1958) in E. coli AIPMT 1995 2012 NEET 2018 2023.
Experiment:
- Grew E. coli in a medium containing ¹⁵N (¹⁵NH₄Cl, heavy isotope) for many generations.
- Transferred cells to normal ¹⁴N medium.
- Separated DNA based on density using Cesium Chloride (CsCl) density gradient centrifugation.
- After 20 mins (one generation), DNA had intermediate/hybrid density. After 40 mins, equal amounts of hybrid and light DNA were found.
Exception/Crucial detail: Taylor and colleagues (1958) proved semiconservative replication in chromosomes using radioactive thymidine on Vicia faba (fava beans) AIPMT 2016.

The Machinery and Enzymes

DNA-dependent DNA polymerase: The main enzyme. It is highly efficient and polymerizes deoxynucleotides exclusively in the 5' → 3' direction NEET 2022.
Leading vs Lagging Strand:
- On the template strand with 3' → 5' polarity, replication is continuous (leading strand).
- On the template strand with 5' → 3' polarity, replication is discontinuous (lagging strand).
Okazaki fragments: The discontinuously synthesized short DNA fragments. They are later joined by DNA ligase AIPMT 2015 NEET 2023.
Origin of Replication (ori): The definite region in E. coli DNA where replication originates.

5. Transcription

The Transcription Unit

Promoter: Located at the 5'-end (upstream) of the structural gene with respect to the coding strand. It provides the binding site for RNA polymerase AIPMT 2015 NEET 2019.
Structural Gene: The DNA sequence to be transcribed.
Terminator: Located at the 3'-end (downstream) of the coding strand. Defines the end of transcription.
Template vs Coding Strand:
- Template Strand: Has 3' → 5' polarity. Acts as the template for RNA synthesis. Also called the antisense/non-coding strand AIPMT 1998 2015 NEET 2022.
- Coding Strand: Has 5' → 3' polarity. Its sequence is identical to the synthesized RNA (except Thymine is replaced by Uracil). Also called the sense strand NEET 2022.

Transcription in Prokaryotes

Bacteria have a single DNA-dependent RNA polymerase that catalyzes transcription of all three types of RNA (mRNA, tRNA, rRNA).
Initiation: RNA polymerase binds to the promoter and uses the Sigma (σ) factor to initiate transcription AIPMT 2004 NEET 2021.
Elongation: RNA polymerase alone elongates the RNA chain.
Termination: Polymerase reaches the terminator region and falls off, facilitated by the Rho (ρ) factor NEET 2018.

Transcription in Eukaryotes

Eukaryotes have a clear division of labor with three different RNA polymerases in the nucleus NEET 2020 2021 2023.

Enzyme	RNA Transcribed	Function
RNA Polymerase I	28S, 18S, 5.8S rRNA	Ribosome components
RNA Polymerase II	hnRNA (Heterogeneous nuclear RNA)	Precursor to mature mRNA
RNA Polymerase III	tRNA, 5S rRNA, snRNA	Adapter molecule, splicing machinery

Post-Transcriptional Modifications (Processing of hnRNA)

NEET 2017 2020 2023

Capping: An unusual nucleotide (methyl guanosine triphosphate) is added to the 5'-end of hnRNA.
Tailing (Polyadenylation): Poly-A tail (200-300 adenylate residues) is added at the 3'-end in a template-independent manner.
Splicing: Introns (intervening, non-coding sequences) are removed by spliceosomes (snRNPs), and exons (coding, expressed sequences) are joined together NEET 2018.

6. Genetic Code & Mutations

Deciphering the Code

George Gamow: Argued that to code for 20 amino acids with 4 bases, the code must be a triplet (4³ = 64 codons).
Marshall Nirenberg and Har Gobind Khorana: Developed chemical methods to synthesize RNA molecules with defined combinations.
Severo Ochoa enzyme (Polynucleotide phosphorylase): Allowed synthesis of RNA with defined sequences in a template-independent manner (enzymatic synthesis of RNA) AIPMT 1997 NEET 2023.

Salient Features of the Genetic Code

Triplet: 61 codons code for amino acids, while 3 codons (UAA, UAG, UGA) do not code for any amino acids and function as stop codons AIPMT 1993 NEET 2019 2021.
Unambiguous and Specific: One codon codes for only one specific amino acid.
Degenerate: Some amino acids are coded by more than one codon NEET 2017 2022.
Universal: UUU codes for Phenylalanine from bacteria to humans (with minor exceptions in mitochondria and some protozoans).
Commaless: Read in a contiguous fashion with no punctuations.
Dual Function of AUG: Acts as the Start Codon (initiator) AND codes for the amino acid Methionine (met) AIPMT 1991 2006 NEET 2019.

Mutations and Genetic Code

Point Mutation: Change in a single base pair.
- Example: Sickle cell anemia, where a single base substitution (GAG → GUG) changes Glutamic acid to Valine at the 6th position of the beta-globin chain AIPMT 2009.
Frameshift Mutation: Insertion or deletion of one or two bases changes the reading frame from the point of insertion/deletion onwards.

7. tRNA and Translation

tRNA - The Adapter Molecule

Also known as soluble RNA (sRNA).
Structure: Looks like a clover-leaf in 2D secondary structure, but is a compact inverted L-shape in actual 3D structure AIPMT 1990 2004.
Key regions:
- Anticodon loop: Has bases complementary to the mRNA codon.
- Amino acid acceptor end (3'-end): Binds to the specific amino acid AIPMT 2001.
Crucial detail: There are NO tRNAs for stop codons.

Translation Mechanism

The process of polymerising amino acids to form a polypeptide, dictated by mRNA.
Charging of tRNA (Aminoacylation): Amino acids are activated in the presence of ATP and linked to their cognate tRNA.
Ribosome function: Contains a large and small subunit. The large subunit has two sites for subsequent amino acids to bind and form a peptide bond.
Ribozyme: In bacteria, the 23S rRNA of the large ribosomal subunit acts as the enzyme (peptidyl transferase) for peptide bond formation AIPMT 2016 NEET 2020.
UTRs (Untranslated Regions): Present on mRNA at both the 5'-end (before start codon) and 3'-end (after stop codon). They are not translated but are required for efficient translation.

8. Regulation of Gene Expression (The Lac Operon)

Operon concept: Proposed by geneticist Francois Jacob and biochemist Jacques Monod in E. coli NEET 2021.
Operon structure: A polycistronic structural gene regulated by a common promoter and regulatory genes.

Gene	Codes For	Function / Role
i gene (Regulatory)	Repressor protein	Constitutively active. Binds to the operator to turn operon OFF AIPMT 1999 NEET 2018 2024.
z gene (Structural)	β-galactosidase (β-gal)	Cleaves lactose into galactose and glucose AIPMT 2008 NEET 2019-22.
y gene (Structural)	Permease	Increases cellular permeability to β-galactosides (lactose).
a gene (Structural)	Transacetylase	Assists in the removal of toxic by-products of lactose digestion.

Mechanism

Absence of Inducer (Lactose OFF): The repressor protein (transcribed continuously by i gene) binds to the operator region. This prevents RNA polymerase from transcribing the operon NEET 2024.
Presence of Inducer (Lactose ON): Lactose (or allolactose) acts as an inducer. It binds to the repressor, inactivating it. The operator is now free, allowing RNA polymerase to transcribe the z, y, a genes AIPMT 2002 NEET 2019.
Crucial detail: The regulation of the lac operon by the repressor is termed Negative Regulation because the regulatory protein shuts down transcription.

9. Human Genome Project (HGP)

A 13-year mega project coordinated by the US Department of Energy and the National Institutes of Health (1990-2003).

Methodologies

Expressed Sequence Tags (ESTs): Identifying all the genes that are expressed as RNA NEET 2019 2022.
Sequence Annotation: Blindly sequencing the whole set of genome (coding + non-coding sequences) and later assigning different regions with functions.
Cloning Vectors used: BAC (Bacterial Artificial Chromosome) and YAC (Yeast Artificial Chromosome) AIPMT 2004.
Sequencing principle: Based on automated DNA sequencers developed by Frederick Sanger NEET 2021.

Salient Features of the Human Genome

The human genome contains 3164.7 million nucleotide bases.
Average gene consists of 3000 bases.
Largest known human gene is Dystrophin at 2.4 million bases AIPMT 2014.
Total number of estimated genes is ~30,000.
Less than 2% of the genome codes for proteins NEET 2020.
Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (231) NEET 2019.
Scientists have identified about 1.4 million locations where Single Nucleotide Polymorphisms (SNPs) occur.

10. DNA Fingerprinting

Developed by Alec Jeffreys to identify individuals based on DNA variations AIPMT 2004 NEET 2020.
Principle: Relies on identifying differences in specific regions in DNA called Repetitive DNA.
Satellite DNA: Repetitive DNA sequences that form minor peaks during density gradient centrifugation. They show a high degree of polymorphism and form the basis of DNA fingerprinting.
VNTR (Variable Number of Tandem Repeats): A type of mini-satellite used as a radiolabelled probe by Alec Jeffreys. It shows very high polymorphism NEET 2022 2023.

Steps of DNA Fingerprinting

AIPMT 2006 NEET 2020 2021

Isolation of DNA from the biological sample (blood, hair follicle, semen).
Digestion of DNA by Restriction Endonucleases.
Separation of DNA fragments by Gel Electrophoresis.
Blotting (Southern Blotting): Transferring the separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon NEET 2021.
Hybridisation: Using a radiolabelled VNTR probe to bind complementary DNA strands.
Autoradiography: Exposing the hybridized membrane to X-ray film to detect the specific bands, which appear as a barcode unique to an individual.

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