Molecular Basis of Inheritance Notes

CHAPTER 5: MOLECULAR BASIS OF INHERITANCE

5.1 THE DNA

• Definition: DNA (deoxyribonucleic acid) is a long polymer of deoxyribonucleotides.

• Length of DNA: Defined by the number of nucleotides (or base pairs) it contains.

  • Examples of DNA lengths in different organisms:

  • Bacteriophage $ ext{φ} imes 174$: 5386 nucleotides

  • Bacteriophage lambda: 48502 base pairs (bp)

  • Escherichia coli: $4.6 imes 10^{6}$ bp

  • Human DNA (haploid): $3.3 imes 10^{9}$ bp.

5.1.1 Structure of Polynucleotide Chain

• Components of a Nucleotide:

  • Nitrogenous base

  • Pentose sugar (ribose for RNA, deoxyribose for DNA)

  • Phosphate group

• Types of Nitrogenous Bases:

  • Purines: Adenine (A), Guanine (G)

  • Pyrimidines: Cytosine (C), Uracil (U, in RNA), Thymine (T, in DNA)

• Nucleotide Formation:

  • Nucleoside formation: Nitrogenous base linked to the 1' carbon of sugar via N-glycosidic linkage.

  • Nucleotide formation: Phosphate group linked to the 5' carbon of nucleoside via phosphoester linkage.

• Linkage:

  • Dinucleotide formation: Two nucleotides linked through a 3'-5' phosphodiester linkage.

• Structure Characteristics:

  • Free phosphate on the 5' end: referred to as the 5'-end of the polynucleotide chain.

  • Free OH on the 3' carbon: referred to as the 3'-end.

  • Backbone: Composed of sugar and phosphate, with bases projecting from it.

5.1.2 Packaging of DNA Helix

• Length Calculation:

  • Length in mammalian cells (where DNA is approximately $6.6 imes 10^{9}$ bp) would be around 2.2 meters (calculated as $6.6 imes 10^9 ext{ bp} imes 0.34 imes 10^{-9} ext{ m/bp}$).

• Organization in Prokaryotes:

  • DNA organized in nucleoid, held by positively charged proteins, forming large loops.

• Organization in Eukaryotes:

  • Basic proteins called histones are involved, organized into histone octamers (8 molecules).

  • DNA wraps around histones to form nucleosomes (200 bp of DNA per nucleosome).

  • Nucleosomes form chromatin, visible as a beads-on-a-string structure under electron microscopy.

• Types of Chromatin:

  • Euchromatin: Loosely packed, transcriptionally active.

  • Heterochromatin: Densely packed, transcriptionally inactive.

5.2 THE SEARCH FOR GENETIC MATERIAL

• Despite early discoveries by Meischer and Mendel, it took time to identify DNA as the genetic material.

5.2.1 Transforming Principle

• Frederick Griffith's Experiment (1928): Studied Streptococcus pneumoniae, observed transformation in bacteria.

  • Observations:

  • S strain (virulent) produces smooth colonies; R strain (non-virulent) produces rough colonies.

  • Mice infected with S strain die; R strain does not cause disease.

  • Heat-Killed S Strain: Injected into mice, did not cause death.

  • Mixture of Heat-Killed S and Live R: Mice died, recovered living S bacteria. Suggested a "transforming principle" was present.

5.2.2 Biochemical Characterisation of Transforming Principle

• Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-44) identified DNA as the transforming principle.

• They could purify various biochemicals and found that only DNA could transform R strain into S strain.

• Confirmed that proteins and RNA did not affect transformation., while DNase inhibited transformation, suggesting that DNA was the genetic material.

5.2.3 The Genetic Material is DNA

• Hershey-Chase Experiment (1952): Used bacteriophages to demonstrate DNA is the genetic material.

  • Radioactive phosphorus was used to label DNA and sulfur for proteins.

  • Radioactive DNA entered bacterial cells, while radioactive protein did not.

5.2.4 Properties of Genetic Material

• Requirements for a molecule to serve as genetic material:

  1. Capable of replication.

  2. Chemically and structurally stable.

  3. Allow for mutation (evolution).

  4. Able to express Mendelian characters.

• RNA functions dynamically but is chemically less stable than DNA. DNA is preferred for storage of genetic information due to its stability.

5.3 RNA WORLD

• First Genetic Material: Evidence suggests RNA was the first genetic material.

• Functions of RNA include acting as genetic material and catalyst (ribozyme). DNA evolved from RNA, with modifications for stability.

5.4 REPLICATION

• Watson and Crick's Proposal: Suggested a copying mechanism based on their double helix model.

• Semiconservative Replication: Each strand acts as a template for a new complementary strand.

5.4.1 Experimental Proof

• Meselson and Stahl (1958):

  • Experiment with E. coli to demonstrate semiconservative replication using isotopes to mark DNA strands.

  • Result showed hybrid densities of DNA corresponding to the origins of replication.

5.4.2 Machinery and Enzymes

• Key Enzyme: DNA-dependent DNA polymerase.

• Process:

  • Replication occurs in a replication fork; leading strand synthesised continuously, lagging strand discontinuously.

  • Uses ATP as energy sources.

• Importance of Accuracy: DNA polymerases must minimize errors to avoid mutations.

5.5 TRANSCRIPTION

• Definition: Process of copying genetic information from DNA to RNA.

• Works on complementarity except uracil replaces thymine.

• Existence of promoter and terminator sequences define the area of transcription.

5.5.1 Transcription Unit

• Components:

  1. Promoter

  2. Structural gene

  3. Terminator

• Coding and Template Strands:

  • Template strand acts as a 3'->5' during transcription, coding strand has 5'->3'.

5.5.2 Transcription Unit and the Gene

• Gene Definition: Functional unit of inheritance.

• Monocistronic vs. Polycistronic:

  • Monocistronic (eukaryotic genes) versus polycistronic (bacterial genes) organization.

5.5.3 Types of RNA and Transcription Process

• Types of RNA: mRNA, tRNA, rRNA; all essential for protein synthesis.

• RNA Polymerase: One enzyme in prokaryotes catalyses transcription.

• Eukaryotic Complexity: Involves multiple RNA polymerases and extensive processing of transcripts.

5.6 GENETIC CODE

• Definition and Importance: Directs the sequence of amino acids in proteins.

• Codon Features:

  • Triplet coding system, 61 codons for amino acids.

  • Code is universal with some exceptions in mitochondria.

5.6.1 Mutations and Genetic Code

• Point Mutations: Single base changes affect genes, exemplified by sickle cell anemia.

• Frameshift Mutations: Insertion or deletion changes reading frame, significantly affecting protein synthesis.

5.6.2 tRNA and Adapter Molecule

• Structure: tRNA has acceptor ends for amino acids and anticodons for mRNA translation.

5.7 TRANSLATION

• Process: Involves polymerisation of amino acids to form polypeptides.

• Roles of Ribosomes: Site for mRNA binding and peptide bond formation.

• Phases: Initiation, elongation, termination characterized by interactions between tRNA and mRNA.

5.8 REGULATION OF GENE EXPRESSION

• Gene expression can be regulated at transcriptional, processing, transport, and translational levels.

• Lac Operon: Regulates beta-galactosidase synthesis in E. coli, responding to lactose availability.

• Mechanism: Inducer and repressor proteins control expression based on metabolite presence.

5.9 HUMAN GENOME PROJECT

• Overview: Aimed to sequence the entire human genome including approximately 3 billion base pairs.

• Importance: Identified genes, studied variations in DNA sequences leading to medical advancements.

• Goals and Methodologies: Involves high-throughput technologies and bioinformatics.

5.9.1 Salient Features of Human Genome

• Contains approximately 3164.7 million bp; total gene count significantly lower than expected.

• Less than 2% of genome codes for proteins; repetitive sequences form a major part of DNA.

5.10 DNA FINGERPRINTING

• Definition: Technique to identify genetic differences through variations in repetitive DNA sequences.

• Applications: Forensics, paternity testing, and population studies; uses Unique Variably Number of Tandem Repeats (VNTR).