Molecular Basis of Inheritance Study Notes

DNA as the Primary Genetic Material

DNA (Deoxyribonucleic acid) is the genetic material in the majority of organisms. In some cases, specifically certain viruses, RNA (Ribonucleic acid) serves as the genetic material.

Key quantitative data on DNA content in various organisms:

$\phi\times174$ Bacteriophage: $5386$ nucleotides.
Bacteriophage Lambda ( $\lambda$ ): $48502$ base pairs (bp).
Escherichia coli (E. coli): $4.6 \times 10^{6}$ bp.
Human DNA (haploid content): $3.3 \times 10^{9}$ bp.

Structure and Roles:

DNA is a long polymer of deoxyribonucleotides.
RNA functions primarily as a messenger, adapter, and in some cases, a catalytic molecule.
The flow of biological information: DNA $\rightarrow$ DNA (Replication) $\rightarrow$ RNA (Transcription) $\rightarrow$ Proteins (Translation).

Structure of the Polynucleotide Chain

A nucleotide has three primary components:

Nitrogenous base: Linked to the pentose sugar via an N-glycosidic linkage.
Pentose sugar: Ribose in RNA, Deoxyribose in DNA.
Phosphate group: Linked to the $5'-OH$ of a nucleoside via a phosphoester linkage.

Nitrogenous Bases are divided into:

Purines: Adenine (A) and Guanine (G).
Pyrimidines: Cytosine (C), Uracil (U) (found only in RNA), and Thymine (T) (found only in DNA). Thymine is also known as 5-methyl uracil.

Chain Formation:

Nucleosides include Adenosine, Guanosine, Cytidine, Uridine, and Deoxythymidine.
Two nucleotides are joined by a $3'-5'$ phosphodiester linkage.
The backbone of the polynucleotide chain is formed by the sugar and phosphate groups, while the nitrogenous bases project inside from the backbone.

Characteristics of RNA and Early DNA Discovery

In RNA, every nucleotide has an additional $-OH$ group present at the $2'$ position of the ribose sugar. Uracil is present in place of Thymine.

History of DNA Identification:

Friedrich Meischer (1869): Identified DNA as an acidic substance in the nucleus and named it 'Nuclein'.
Maurice Wilkins and Rosalind Franklin: Produced X-ray diffraction data.
James Watson and Francis Crick (1953): Proposed the Double Helix model based on the X-ray data.

The Double Helix Model

Erwin Chargaff observed that for double-stranded DNA, the ratios between Adenine and Thymine, and Guanine and Cytosine are constant and equal one: $A=T$ $G=C$ $\frac{A}{T} = \frac{G}{C} = 1$

Key features of the Double Helix:

Two polynucleotide chains with anti-parallel polarity ( $5' \rightarrow 3'$ and $3' \rightarrow 5'$ ).
The backbone is sugar-phosphate; bases face inward.
Bases are paired through hydrogen bonds: $A=T$ (2 hydrogen bonds) and $G \equiv C$ (3 hydrogen bonds).
The chains are coiled in a right-handed fashion.
Pitch of helix: $3.4\,nm$ .
Base pairs per turn: $10\,bp$ .
Distance between base pairs: $0.34\,nm$ .
The plane of one base pair stacks over the other, providing stability to the helical structure via H-bonding.

Central Dogma and DNA Packaging

Francis Crick proposed the Central Dogma, which states the flow of genetic information: DNA (Replication) $\rightarrow$ Transcription $\rightarrow$ mRNA $\rightarrow$ Translation $\rightarrow$ Protein. In some viruses, the flow of information is in the reverse direction (Reverse Transcription).

Packaging of the DNA Helix:

The length of DNA is calculated as: $\text{Total number of bp} \times \text{distance between two bp}$ .
In a typical mammalian cell: $(6.6 \times 10^{9}\,bp) \times (0.34 \times 10^{-9}\,m/bp) = 2.2\,metres$ .
A typical nucleus dimension is only $10^{-6}\,m$ .
In E. coli, the DNA length is approximately $1.36\,mm$ .

DNA Packaging in Organisms

Prokaryotes (e.g., E. coli):

DNA is negatively charged and held by positively charged proteins in a region called the nucleoid.
It is organized in large loops.

Eukaryotes:

Packaging is more complex. It utilizes positively charged basic proteins called histones.
Histones are rich in basic amino acid residues: Lysines and Arginines.
Eight histone molecules form a histone octamer.
The Nucleosome consists of negatively charged DNA wrapped around the histone octamer, containing roughly $200\,bp$ of DNA helix.
Chromatin: Repeating units of nucleosomes in the nucleus, appearing as "beads on a string".
Chromatin fibers coil and condense during the metaphase stage of cell division to form Chromosomes.
Non-histone chromosomal (NHC) proteins are required for packaging at higher levels.

Chromatin Types:

Euchromatin: Loosely packed, stains light, transcriptionally active.
Heterochromatin: Densely packed, stains dark, transcriptionally inactive.

The Search for Genetic Material

Frederick Griffith (1928) - Transforming Principle:

Conducted experiments with Streptococcus pneumoniae (Pneumococcus).
S-strain (Smooth): Has a mucous (polysaccharide) coat; virulent. Mice die when infected.
R-strain (Rough): Lacks coat; non-virulent. Mice live.
Heat-killed S-strain: Mice live.
Heat-killed S-strain + Live R-strain: Mice die. Griffith concluded a "transforming principle" transferred from heat-killed S to live R, making them virulent.

Biochemical Characterization (1933-44):

Oswald Avery, Colin Macleod, and Maclyn McCarty purified biochemicals (proteins, DNA, RNA) from heat-killed S-cells.
Proteases and RNases did not affect transformation.
DNases inhibited transformation, proving DNA is the hereditary material.

The Hershey-Chase Experiment (1952):

Performed by Alfred Hershey and Martha Chase using bacteriophages.
Phages grown in radioactive Phosphorus ( $^{32}P$ ) had radioactive DNA.
Phages grown in radioactive Sulphur ( $^{35}S$ ) had radioactive protein.
After infection of E. coli, blending (to remove viral coats), and centrifugation, radioactivity was found in the cells (pellet) only for the $^{32}P$ batch, proving DNA enters the cell, not protein.

Properties of Genetic Material and Replication

Essential properties of genetic material:

Ability to replicate.
Chemical and structural stability.
Ability to allow slow mutations (evolution).
Ability to express itself in the form of Mendelian characters.

Comparing DNA and RNA:

RNA is less stable due to the $2'-OH$ group and is often catalytic.
DNA contains Thymine, providing more stability.
RNA can mutate faster (advantageous for some viruses).
DNA is better for storage; RNA is better for transmission.

Semiconservative DNA Replication:

Proposed by Watson and Crick: the two strands separate and each acts as a template for a new complementary strand.
Experimental Proof (1958): Matthew Meselson and Franklin Stahl.
E. coli grown in $^{15}NH_{4}Cl$ (heavy nitrogen) until DNA was heavy.
Transferred to $^{14}NH_{4}Cl$ (light nitrogen). After 20 mins (one generation), DNA was hybrid/intermediate density. After 40 mins, DNA was equal parts hybrid and light. Measured via $CsCl$ density gradient centrifugation.
Taylor and colleagues (1958): Used radioactive thymidine in Vicia faba to prove semi-conservative replication in chromosomes.

Machinery and Mechanism of Replication

Main enzyme: DNA dependent DNA polymerase.

Highly efficient: E. coli replicates $4.6 \times 10^{6}$ bp in 18 minutes (~2000 bp/sec).
Energetically expensive; Uses Deoxyribonucleoside triphosphates (dNTPs) as both substrate and energy source.

Replication Process:

Occurs within a replication fork.
Polymerises only in the $5' \rightarrow 3'$ direction.
Leading Strand: Continuous synthesis ( $3' \rightarrow 5'$ template).
Lagging Strand: Discontinuous synthesis ( $5' \rightarrow 3'$ template); creating Okazaki fragments joined by DNA ligase.
Origin of Replication: Definite region where replication starts.
In eukaryotes, replication occurs during the S-phase. Failure in cell division after DNA replication results in polyploidy.

Transcription in Prokaryotes

Transcription involves copying genetic information from DNA to RNA. Only one strand is copied because:

If both strands acted as templates, they would code for different amino acid sequences, complicating genetic information.
Two complementary RNA molecules would form double-stranded RNA, preventing translation.

Transcription Unit:

Promoter: Binding site for RNA polymerase (located at $5'$ end of coding strand).
Structural gene: The DNA segment to be transcribed.
Terminator: Defines the end of transcription (located at $3'$ end of coding strand).

Reference points are based on the Coding Strand ( $5' \rightarrow 3'$ polarity). The Template Strand has $3' \rightarrow 5'$ polarity.

In Bacteria:

A single DNA dependent RNA polymerase catalyzes transcription of all RNAs (mRNA, tRNA, rRNA).
Initiation: RNA polymerase binds to the promoter with the help of the Initiation factor ( $\sigma$ - sigma).
Elongation: Facilitated by the opening of the DNA helix.
Termination: When the terminator region is reached, nascent RNA falls off with the help of the Termination factor ( $\rho$ - rho).
In bacteria, mRNA does not require processing, allowing transcription and translation to be coupled.

Transcription in Eukaryotes

Eukaryotes have two additional complexities:

Three RNA Polymerases:
- RNA Pol I: Transcribes rRNAs ( $28S$ , $18S$ , $5.8S$ ).
- RNA Pol II: Transcribes mRNA precursor (hnRNA - heterogeneous nuclear RNA).
- RNA Pol III: Transcribes tRNA, $5S\,rRNA$ , and snRNAs (small nuclear RNAs).
Post-transcriptional Processing:
- The primary transcript (hnRNA) contains both exons (coding/expressed sequences) and introns (non-coding/unexpressed sequences).
- Splicing: Introns are removed and exons are joined.
- Capping: Methyl guanosine triphosphate is added to the $5'$ end.
- Tailing: Adenylate residues ( $200-300$ ) are added to the $3'$ end (Poly-A tail).
- Fully processed hnRNA is now called mRNA and is transported out of the nucleus.

The Genetic Code

The genetic code directs amino acid sequences during protein synthesis.

Key Contributors:

George Gamow: Argued the code must be a triplet ( $4^{3} = 64$ codons) to account for 20 amino acids.
Hargobind Khurana: Developed chemical methods to synthesize RNA with defined base combinations.
Marshall Nirenberg: Created a cell-free system for protein synthesis to decipher the code.
Severo Ochoa enzyme (polynucleotide phosphorylase): Allowed template-independent RNA polymerization.

Salient Features of the Genetic Code:

Triplet: 61 codons for amino acids, 3 stop codons (UAA, UAG, UGA).
Unambiguous and Specific: One codon codes for only one amino acid.
Degenerate: Some amino acids are coded by more than one codon.
Commaless: Read in a continuous fashion without punctuation.
Universal: UUU codes for Phenylalanine from bacteria to humans.
Dual function (AUG): Codes for Methionine (met) and acts as the initiator codon.

Mutations, tRNA, and Translation

Mutations:

Point mutation: e.g., Sickle cell anemia, where a single base pair change in the $\beta$ -globin chain converts Glutamate to Valine.
Frameshift mutation: Insertion or deletion of 1 or 2 bases shifts the reading frame. Insertion/deletion of 3 (or multiples of 3) bases inserts/deletes amino acids without altering the rest of the frame.

tRNA (The Adapter Molecule):

Postulated by Francis Crick; previously known as sRNA (soluble RNA).
Anticodon loop: Complementary to the mRNA code.
Amino acid acceptor end: Binds specific amino acids.
Initiator tRNA: Specific for initiation; no tRNA for stop codons.
Structure: $2^{\circ}$ structure looks like a clover leaf; actual $3^{\circ}$ structure is an inverted L.

Translation Process:

Polymerization of amino acids into a polypeptide via peptide bonds.
Phase 1: Charging (Aminoacylation): Amino acids are activated (using ATP) and linked to their cognate tRNA.
Ribosome: Consists of structural RNAs and ~80 proteins. Has a large subunit and a small subunit.
23S rRNA in bacteria: Acts as a ribozyme (catalyst) for peptide bond formation.
UTRs (Untranslated Regions): Sequences at both $5'$ and $3'$ ends of mRNA required for efficient translation.
Termination: A release factor binds to the stop codon.

Regulation of Gene Expression

In eukaryotes, regulation occurs at four levels:

Transcriptional level.
Processing level (splicing).
Transport of mRNA from nucleus to cytoplasm.
Translational level.

Metabolic, physiological, or environmental conditions regulate gene expression. Example: $\beta$ -Galactosidase in E. coli breaks Lactose into Glucose and Galactose. Expressed only when lactose is present.

The Lac Operon (Jacob and Monod):

A polycistronic structural gene regulated by a common promoter and regulatory genes.
Regulatory gene (i): Codes for the repressor protein (synthesized constitutively).
Structural genes:
- z: $\beta$ -galactosidase.
- y: Permease.
- a: Transacetylase.
Inducer (Lactose/Allolactose): Binds to the repressor, inactivating it, allowing RNA polymerase access to the promoter.
Regulatory system is primarily under negative regulation.

Human Genome Project (HGP)

Launched in 1990; completed in 2003. A "Mega project" to determine the DNA sequence of the human genome ( $3 \times 10^{9}$ bp).

Project Scale:

Cost: US $\$3$ per bp; Total approx. US $\$9$ billion.
Data storage: Required 3300 books, each 1000 pages, each page 1000 letters.
Closely associated with the rise of Bioinformatics.

Goals:

Identify 20,000–25,000 human genes.
Determine sequence of 3 billion chemical base pairs.
Improve tools for data analysis.
Address ethical, legal, and social issues (ELSI).

Project Contributors:

US Department of Energy and National Institute of Health.
Wellcome Trust (UK) was a major partner; other contributors included Japan, France, Germany, and China.
Non-human models sequenced: Bacteria, Yeast, Caenorhabditis elegans (nematode), Drosophila (fruit fly), plants (rice and Arabidopsis).

Methodologies:

Expressed Sequence Tags (EST): Identifying all sequences expressed as RNA.
Sequence Annotation: Sequencing the whole genome then assigning functions.
Tools: DNA fragments cloned in BAC (Bacterial Artificial Chromosomes) and YAC (Yeast Artificial Chromosomes); automated sequencers based on Frederick Sanger’s principle.

Findings of the Human Genome Project

Salient Features:

Total bases: $3164.7$ Million nucleotide bases.
Average gene size: $3000$ bases. Dystrophin is the largest known human gene at $2.4$ Million bases.
Total genes: ~30,000 (lower than previous estimates of 80,000–140,000).
Similarity: $99.9\%$ of nucleotide bases are identical in all people.
Unknown function: About $50\%$ of discovered genes have unknown functions.
Coding DNA: Less than $2\%$ of the genome codes for proteins.
Repeated sequences: Make up a large portion; offer insight into chromosome structure and evolution.
Gene density: Chromosome 1 has the most genes ( $2968$ ); the Y chromosome has the fewest ( $231$ ).
SNPs (Single Nucleotide Polymorphisms): 1.4 Million locations where single base DNA differences occur, useful for disease tracking and history.

DNA Fingerprinting

Developed by Alec Jeffreys, this technique involves identifying differences in repetitive DNA sequences.

Mechanism:

Satellite DNA: Repetitive DNA separated from bulk DNA as smaller peaks during density gradient centrifugation.
Categorized by base composition, segment length, and number of repetitive units (Mini-satellites and Micro-satellites).
Polymorphism: High degree of variation; specifically defined as allele frequency > 0.01 at a locus. Inheritable and forms the basis of paternity testing and evolution studies.
VNTR (Variable Number Tandem Repeats): A class of mini-satellite used as a radiolabelled probe in DNA fingerprinting.

Steps of DNA Fingerprinting:

DNA isolation.
Digestion by restriction endonucleases.
Separation of fragments by Electrophoresis.
Transfer (blotting) to synthetic membranes (nitrocellulose or nylon).
Hybridization using labelled VNTR probe.
Detection of hybridized fragments via autoradiography.