Topics to be learn:

  • The Discovery of DNA
  • The Genetic Material is a DNA
  • DNA packaging
  • DNA replication
  • Protein synthesis
  • Regulation of gene expression
  • Operon concept
  • Genomics
  • Human Genome Project
  • DNA Fingerprinting

 The Discovery of DNA

Nuclein:

  • Discovered by Friedrich Miescher in 1869 from the nuclei of pus cells.
  • Acidic substance with high phosphorus content.
  • Isolated using a salt solution to wash pus off bandages, then lysed cells with weak alkaline solution.
  • Initially called nucleic acid due to its isolation from the nucleus.
  • By early 1900s, known that Miescher's nuclein was a mixture of proteins and nucleic acids (DNA and RNA).
Types of Nucleic Acids:  
  1. DNA (deoxyribonucleic acid) 
  2. RNA (ribonucleic acid)
Genetic Material:
  • Initially, proteins were considered as genetic material because they were thought to store information for cell metabolism.
  • DNA was considered small and simple, with little variation among species.
  • Differences in DNA molecules are distinct from variations in proteins' shape, charge, and function.

Important Experiments in DNA Research

1. Griffith's Experiments:

  • In 1928, Frederick Griffith experimented with two strains of bacterium:
  • S-type: Virulent, smooth, pathogenic, encapsulated
  • R-type: Non-virulent, rough, non-pathogenic, non-capsulated
  • Injected a mixture of heat-killed S bacteria and live R bacteria into mice, resulting in death.
  • Live S-strain bacteria obtained from the blood of dead mice.
  • Conclusion: Live R-strain bacteria transformed into S-type, acquiring something from the heat-killed S bacterium (transforming principle).


2. Avery, McCarty, and MacLeod's Experiment: 
  • Purified DNA, RNA, proteins, etc., from heat-killed S-strain cells mixed with R-strain bacteria separately.
  • Only DNA transformed avirulent R-strain into virulent S-strain.
  • No transformation occurred when DNA was digested with DNase.
  • Conclusion (1944): DNA is the genetic material (transforming principle).

3. Hershey-Chase Experiment:
  • Hershey and Chase worked with bacteriophages.
  • Used two types of bacteriophages: one labeled DNA with radioactive phosphorus, and the other labeled protein coat with radioactive sulfur.
  • Steps: infection, blending, centrifugation.
  • Experiment proved that DNA, not protein, enters bacterial cell, confirming DNA as the genetic material.

DNA packaging 
  • DNA Length: In a typical mammalian cell, DNA double helix is about 2.2 meters long.
  • Nucleus Size: Approximate size of a typical nucleus is 10-6 meters.
 
DNA Condensation:
  • DNA molecule is condensed, coiled, and supercoiled to fit inside the small nucleus.
  • Condensation ensures efficient storage of genetic material within the cell.

DNA Packaging in Prokaryotes:

  • Cell Size: E. coli cell size is 2-3 µm.
  • Nucleoid:
  • Small, circular, highly folded, naked DNA (about 1100 µm long in perimeter, contains ~4.6 million base pairs).
  • Circular DNA reduces size to 850 µm in diameter.
  • Folding/looping (40-50 domains) further reduces to 30 µm in diameter.
  • RNA Connectors: Assist in loop formation.
  • Coiling and Supercoiling: Each domain reduced to 2 µm in diameter.
  • Assisting Proteins and Enzymes: Positively charged HU proteins and enzymes like DNA gyrase and DNA topoisomerase-I maintain supercoiled state.
DNA Packaging in Eukaryotic Cells: 
  • DNA (2.2 meters) condensed, coiled, and supercoiled for efficient packaging in nucleus (10-16 µm).
  • Association with Proteins:
  • Histones: Positively charged, basic proteins rich in lysine and arginine.
  • Nucleosome:
  • Nucleosome core (histone octamer) wrapped by negatively charged DNA.
  • H1 protein binds DNA thread.
  • Adjacent nucleosomes linked with linker DNA.
  • Packaging Levels: Beads on string (10 nm), Solenoid fibre (30 nm), Chromatin fibre, Chromosome.
  • Non-Histone Chromosomal Proteins (NHC): Assist in higher-level packaging.

Heterochromatin and Euchromatin:Heterochromatin:
  • Proposed by Heitz.
  • Genetically almost inactive, condensed parts of chromonema/chromosomes.
  • Located near centromere, telomeres.
  • Richer in DNA than euchromatin.
Euchromatin:
  • Non-condensed regions of chromonema.
  • Genetically very active and fast replicating.

DNA Replication

Definition: Process where DNA duplicates itself, forming two identical copies. Occurs once in eukaryotes, specifically during the S-phase of interphase.

Functions of DNA:

  • Regulates cellular activities.
  • Ensures equal distribution of genetic material during cell division.
  • Carrier of genetic information.
  • Heterocatalytic Function: Directs synthesis of other molecules like RNA (transcription) and proteins (translation).
  • Autocatalytic Function: Directs synthesis of DNA itself (replication).
  • Master molecule guiding and regulating protein synthesis.

 

Steps in DNA Replication:

1. Activation of Nucleotides: Nucleotides activated by ATP to form deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP).

2. Point of Origin (Initiation): Replication starts at specific origin (O) and ends at termination point (T). Endonuclease breaks one DNA strand at O temporarily.
3. Unwinding of DNA Molecule:DNA helicase breaks weak hydrogen bonds, separating and unwinding strands bidirectionally.Single-strand binding proteins (SSBP) prevent strands from recoiling.
4. Replicating Fork: Y-shaped replication fork formed due to unwinding. Super-helix relaxing enzyme releases strain.
5. Synthesis of New Strands:
  • Each strand acts as template for complementary strand synthesis.
  • RNA primer attaches to 3' end, attracting complementary nucleotides.
  • Nucleotides bind via hydrogen bonds, forming polynucleotide strand.
  • DNA polymerase catalyzes synthesis in 5'->3' direction.
6. Leading and Lagging Strand:
  • Leading: Continuous synthesis towards replicating fork.
  • Lagging: Discontinuous synthesis away from fork in Okazaki fragments.
  • Maturation of fragments by DNA ligase.
7. Formation of Daughter DNA Molecules: Semiconservative replication: Each daughter molecule has one parental and one newly synthesized strand.
Experimental Confirmation of Semiconservative DNA Replication
  • Researchers: Matthew Meselson and Franklin Stahl (1958)
  • Technique Used: Equilibrium Density Gradient Centrifugation

Experimental Procedure:

  • Cultured bacteria E. coli in medium containing 14N (light nitrogen).
  • Obtained equilibrium density gradient band using 6M CsCl2, recorded its position.
  • Transferred E. coli cells to 15N medium (heavy isotopic nitrogen), allowed replication for several generations.
  • Obtained equilibrium density gradient band using 6M CsCl2, recorded its position.
  • Heavy DNA (15N) distinguished from normal DNA by centrifugation in CsCl2 density gradient, forming a band.
  • Transferred cells to 14N medium after replication.
  • After first generation, obtained density gradient band for 14N-15N and recorded its position.
  • After second generation, obtained two bands: one at 14N-15N and other at 14N position.

Conclusion: Position of bands after two generations proved DNA replication is semiconservative.

Protein Synthesis

Importance of Proteins: Structural components, enzymes, and hormones.
Process: Protein synthesis involves transcription and translation.

Central Dogma:

  • Proposed by F.H.C. Crick (1958).
  • DNA → Transcription → mRNA → Translation → Polypeptide.
  • Unidirectional flow of genetic information.

Transcription:

  • Copying genetic information from one DNA strand to complementary RNA transcript.
  • Catalyzed by RNA polymerase.

Transcription Unit:

  • Promoter: Binding site for RNA polymerase.
  • Structural Genes: Template (antisense) strand and sense strand.
  • Terminator: Defines end of transcription.

Three Stages of Transcription:

  1. Initiation: RNA polymerase binds to promoter, unwinds DNA, and starts transcription.
  2. Elongation: RNA nucleotides attach to exposed DNA bases, forming mRNA.
  3. Termination: RNA polymerase reaches terminator, releasing mRNA.

Transcription Unit and Gene:

  • Gene: DNA sequence coding for mRNA, tRNA, or rRNA.
  • Cistron: DNA segment coding for a polypeptide.
  • Monocistronic Gene: Single structural gene.
  • Polycistronic Gene: Transcription unit with multiple structural genes.
  • Interrupted Genes: Contain exons (coding sequences) and introns (non-coding sequences).

Processing of hnRNA:

  • Eukaryotes have split genes.
  • hnRNA undergoes capping, tailing, and splicing.
  • Capping: Methylated guanosine triphosphate added to 5' end.
  • Tailing: Polyadenylation at 3' end.
  • Splicing: Removal of introns, joining of exons.
  • Processed hnRNA becomes functional mRNA for translation.

Genetic Code
  • About: 20 amino acids synthesized from DNA's 4 nitrogen bases.
  • Discovery:
    • Yanofski and Sarabhai (1964): DNA contains info for protein synthesis.
    • F.H.C. Crick: Genetic code as a coded language, triplet nature.
    • G. Gamow (1954): Proposed codon as a sequence of 3 nucleotides.

Cracking of Genetic Code:

  • M. Nirenberg and Matthaei:
  • Synthesized poly-U mRNA → Phenylalanine.
  • Dr. Har Gobind Khorana:
  • Synthesized artificial mRNA with known sequences.
  • E.g., CUC, UCU, resulting in leucine and serine.
  • Severo Ochoa:
  • Enzymatic synthesis of RNA with defined sequences.
  • All 64 codons deciphered.

Characteristics of Genetic Code:

  • Triplet Code: 3 bases (codon) for 1 amino acid.
  • Distinct Polarity: Read 5' → 3'.
  • Non-overlapping, Commaless: Each nucleotide part of 1 codon.
  • Degeneracy: Some amino acids coded by multiple codons.
  • Universal: Same codon specifies same amino acid in all organisms.
  • Non-ambiguous: Each codon specifies one amino acid.
  • Initiation and Termination Codons: AUG for methionine, UAA, UAG, UGA for termination.
  • Codon, Anticodon: Triplet on DNA, complementary triplet on tRNA.
  • Wobble Hypothesis: Third base in codon-anticodon pairing may not be complementary.
Mutations and Genetic Code:

  • Mutation:

    • Sudden change in DNA sequence.
    • Results in genotype change.

  • Mutation and Recombination:

    • Raw material for evolution.
    • Generates variations.

Types of Mutations:

  • Chromosomal Mutations:Loss (deletion) or gain (insertion/duplication) of DNA segment.Alters chromosome structure.
  • Point Mutations: Single base pair change in DNA. E.g., Sickle-cell anemia mutation.
  • Deletion or Insertion of Base Pairs:
  • Causes frame-shift mutations.
  • Alters reading frame.
  • Insertion/deletion of:
  • One or two bases → Change in reading frame.
  • Three or multiples of three bases → Insertion/deletion of amino acids, reading frame unchanged.
Transfer-RNA (t-RNA):
  • 1. Adapter Molecule: t-RNA reads the codon. Binds with amino acid.

2. Clover Leaf Structure (2D) of t-RNA:

  • Four Arms:
DHU Arm: Amino acyl binding loop.
Middle Arm: Anticodon loop.
T & C Arm: Ribosome binding loop.
Variable Arm.
  • G nucleotide at 5’ end.
  • Amino Acid Acceptor End (3’ end):
Unpaired CCA bases.
Amino acid binding site.
  • Specific t-RNA for each amino acid.
  • Initiator t-RNA: Specific for methionine.
  • No t-RNAs for stop codons.

3. Actual Structure: t-RNA resembles an inverted L (3D structure).

Translation — Protein Synthesis:
  • Definition: Process of decoding mRNA codons to add amino acids in sequence to form a polypeptide on ribosomes.
  • Components Required: 20 amino acids, mRNA, tRNA, Ribosomes, ATP, Mg++ ions, Enzymes, Elongation, translocation, and release factors.

Three Steps of Translation:

  1. 1. Initiation of Polypeptide Chain:

    • Ribosome binds to mRNA at 5’ end.
    • Start codon (AUG) positioned at P-site.
    • Initiator tRNA (carrying methionine) binds to initiation codon (AUG) on mRNA.
    • Large ribosomal subunit joins.
    • P-site occupied by initiator tRNA.

  2. 2. Elongation of Polypeptide Chain:

    • Codon Recognition:
    • Anticodon of second tRNA binds with codon at A-site.
    • Peptide Bond Formation:
    • Ribozyme catalyzes peptide bond formation between amino acids.
    • Translocation:
    Ribosome moves along mRNA, exposing new codons.
    Previous tRNA released.

  3. 3. Termination and Release of Polypeptide:

    • Stop codon exposed at A-site.
    • Release factor binds to stop codon.
    • Polypeptide released.
    • Ribosome subunits dissociate.

Regulation of Gene Expression:

Definition: Multistep process regulating gene synthesis.
  • Levels of Regulation (in Eukaryotes):
    • Transcriptional level.
    • Processing level.
    • mRNA transport.
    • Translational level.
Operon Concept:
  • Definition: Transcriptional control mechanism of gene regulation.
  • Proposed by: Francois Jacob and Jacques Monod (1961).
  • Explanation: Metabolic pathways regulated as a unit.

Lac Operon of E. coli:

  • Type: Inducible operon.
  • Activation: Switched on by chemical inducer, lactose.

Components of Lac Operon:

  • Regulator Gene: Precedes promoter gene, Codes for repressor protein, Represses operator gene action.
  • Promoter Gene: Adjacent to operator gene, RNA Polymerase binds here.
  • Operator Gene: Precedes structural genes, Controls transcription.
  • Structural Genes: lac-Z, lac-Y, lac-A. Produce enzymes: β-galactosidase, β-galactoside permease, transacetylase.

Inducer: Allolactose acts as an inducer. Inactivates repressor by binding with it.

  • Genomics:

    • Introduced by H. Winkler in 1920.
    • Total genetic constitution of an organism or one complete set of chromosomes.
    • Term coined by T. H. Roderick in 1986.
    • Study of genomes through analysis, sequencing, and mapping of genes along with their functions.
Types of Genomics:
  • Structural Genomics: Involves mapping, sequencing, and analysis of genomes.
  • Functional Genomics: Study of functions of all gene sequences and their expressions in organisms.

Applications of Genomics:

  • Improvement of: Crop plants, Human health, Livestock
  • Used in: Medicine,Biotechnology,Social sciences.
  • Helps in: Treatment of genetic disorders, Developing transgenic crops, Forensic analysis, Producing enzymes, therapeutic proteins, and biofuels
Human Genome Project (HGP)1990:
 
Aims:
  • Mapping the entire human genome
  • Storing information in databases
  • Developing analysis tools
  • Addressing legal, ethical, and social issues
  • Providing accurate sequence of 3 billion DNA base pairs
  • Estimating number of human genes (about 33,000)
  • Sequencing genomes of other organisms

Significance of HGP:

  • Increased knowledge about gene and protein functions.
  • Major impact in medicine, biotechnology, and life sciences.
  • Enhanced understanding of gene structure and function in other species, aiding in human evolution studies.

 Comparative genome sizes of humans and other models organisms.


DNA Fingerprinting: Unique genetic makeup of an individual, akin to a fingerprint.
Reasons for Uniqueness:
  • Recombination of paternal and maternal genes.
  • Infrequent mutations during gamete formation.
DNA Profiling Technique:
  • Developed by Dr. Alec Jeffreys in 1984.
  • Identifies individuals through DNA restriction analysis.
  • Based on identification of nucleotide sequences.
Key Components: 99.9% similarity in nucleotide sequence.
Variable Number of Tandem Repeats (VNTRs): Unusual sequences of 20-100 base pairs repeated multiple times.

Steps Involved:

  • Isolation of DNA: From tissues like blood, hair roots, skin, etc.
  • Restriction Digestion: DNA treated with restriction enzymes to form variable-length fragments.
  • Gel Electrophoresis: DNA fragments separated based on length.
  • Southern Blotting: Fragments transferred to a membrane.
  • Selection of DNA Probe: Known sequence of single-stranded DNA, labeled with radioactive isotopes.
  • Hybridization: Probe pairs with complementary DNA sequences on the membrane.
  •  Photography: DNA bands captured on X-ray film.

Applications:

  • Forensic sciences (e.g., solving rape and murder cases).
  • Determining disputed parentage.
  • Pedigree analysis in various species.
  • Father of DNA Fingerprinting in India:Scientist - Dr. Lalji Singh (1947-2017):  Developed a unique segment from the Y chromosome of the banded krait snake (BKM-DNA) for DNA fingerprinting.
  • Contributions:
    • Established research laboratories.
    • Founded the Centre for DNA Fingerprinting and Diagnostics (CDFD) and Laboratory for Conservation of Endangered Species (LaCONES).
    • Applied DNA fingerprinting technology in wildlife conservation, forensics, and evolutionary research.