Unit 2: Genetics and Evolution - Chapter 2: Molecular Basis of Inheritance

2.2 Molecular Basis of Inheritance

Search for Genetic Material

1. Griffith's Experiment (Transforming Principle - 1928):

   • Frederick Griffith worked with Streptococcus pneumoniae (pneumococcus) bacteria and mice.
   • R strain (rough): Non-virulent, no capsule.
   • S strain (smooth): Virulent, with capsule.
   • Observations:
     • Live R strain → Mouse lives.
     • Live S strain → Mouse dies.
     • Heat-killed S strain → Mouse lives.
     • Heat-killed S strain + Live R strain → Mouse dies. Live S strain bacteria were recovered from the dead mouse.
   • Conclusion: Griffith concluded that the R strain bacteria had been transformed by the heat-killed S strain. He called this the "transforming principle," but the biochemical nature of the genetic material was not defined.

2. Avery, MacLeod, and McCarty Experiment (1933-1944):

   • Oswald Avery, Colin MacLeod, and Maclyn McCarty purified biochemicals (proteins, DNA, RNA) from heat-killed S cells to determine which one caused the transformation.
   • Experiment: They treated heat-killed S strain extract with proteases (to digest proteins), RNases (to digest RNA), and DNases (to digest DNA) and then mixed each with live R strain.
   • Observations:
     • Transformation occurred when proteins and RNA were digested.
     • Transformation was inhibited when DNA was digested (i.e., no S strain appeared when DNase was added).
   • Conclusion: They concluded that DNA is the genetic material, but not all biologists were convinced.

3. Hershey-Chase Experiment (1952):

   • Alfred Hershey and Martha Chase provided unequivocal proof that DNA is the genetic material, using bacteriophages (viruses that infect bacteria).
   • Experiment:
     • They grew some bacteriophages on a medium containing radioactive phosphorus (³²P) to label DNA (DNA contains P, protein does not).
     • They grew others on a medium containing radioactive sulfur (³⁵S) to label proteins (proteins contain S, DNA does not).
     • These labeled phages were allowed to infect E. coli bacteria.
     • Blending: The viral coats were agitated off the bacteria in a blender.
     • Centrifugation: The mixture was centrifuged to separate the viral particles from the bacterial cells.
   • Observations:
     • Bacteria infected with ³²P-labeled phages were radioactive, and ³²P was found in the progeny phages.
     • Bacteria infected with ³⁵S-labeled phages were not radioactive, and ³⁵S remained in the supernatant (viral coats).
   • Conclusion: DNA, not protein, is the genetic material that is passed from phage to bacteria.

DNA Structure: Watson & Crick Model, Chargaff’s Rule

• DNA (Deoxyribonucleic Acid): A polymer of deoxyribonucleotides.
• Watson and Crick Model (1953): Based on X-ray diffraction data (Rosalind Franklin and Maurice Wilkins) and Chargaff's rules, James Watson and Francis Crick proposed the double helix model of DNA.
  • Key Features:
    • DNA is made of two polynucleotide chains.
    • The backbones are sugar-phosphate, and the bases project inwards.
    • The two chains have anti-parallel polarity (one runs 5'→3', the other 3'→5').
    • The bases in the two strands are paired through hydrogen bonds (A=T, G≡C).
    • Adenine (A) always pairs with Thymine (T) with two hydrogen bonds.
    • Guanine (G) always pairs with Cytosine (C) with three hydrogen bonds.
    • The two chains are coiled in a right-handed helix.
    • Pitch of the helix is 3.4 nm, with approximately 10 base pairs per turn.
    • The distance between two base pairs is 0.34 nm.

• Chargaff’s Rule:
  • Erwin Chargaff (1950) observed that in DNA:
    • The amount of Adenine (A) is always equal to the amount of Thymine (T) (A=T).
    • The amount of Guanine (G) is always equal to the amount of Cytosine (C) (G=C).
    • The ratio of (A+G) to (T+C) is always equal to one.
    • The ratio of (A+T) to (G+C) varies from species to species.

RNA Structure: tRNA, mRNA, rRNA, hnRNA, snRNA

• RNA (Ribonucleic Acid): A polymer of ribonucleotides. Generally single-stranded, but can fold back on itself to form complex 3D structures.
• Types of RNA:
  • mRNA (messenger RNA): Carries genetic information from DNA in the nucleus to the ribosomes in the cytoplasm for protein synthesis. It is linear.
  • tRNA (transfer RNA): Smallest RNA, acts as an adaptor molecule. It has an anticodon loop (binds to mRNA codon) and an amino acid acceptor end (binds to specific amino acid). It has a clover-leaf like 2D structure and an L-shaped 3D structure.
  • rRNA (ribosomal RNA): Most abundant RNA, a structural and catalytic component of ribosomes. It provides the site for protein synthesis.
  • hnRNA (heterogeneous nuclear RNA): The primary transcript in eukaryotes, contains both introns and exons. It undergoes processing to form mRNA.
  • snRNA (small nuclear RNA): Involved in splicing of hnRNA.

Differences between DNA and RNA

DNA Packaging: Nucleosome

• In prokaryotes, DNA is organized in a nucleoid region.
• In eukaryotes, DNA is highly coiled and packaged into chromosomes.
• Nucleosome: The basic unit of DNA packaging in eukaryotes.
  • It consists of a segment of DNA wound around a core of eight histone proteins (histone octamer).
  • The histone octamer is composed of two molecules each of H2A, H2B, H3, and H4 histones.
  • H1 histone acts as a linker histone, binding the DNA where it enters and leaves the nucleosome.
  • The nucleosomes are repeated units that form a structure called chromatin, which looks like "beads-on-string" under an electron microscope.
  • Chromatin further coils and condenses to form chromosomes.

Central Dogma, Reverse Transcription

• Central Dogma of Molecular Biology: Proposed by Francis Crick.
  • States that genetic information flows from DNA → RNA → Protein.
  • DNA (Replication) → DNA (Transcription) → RNA (Translation) → Protein

• Reverse Transcription:
  • In some viruses (retroviruses, e.g., HIV), the flow of genetic information is reversed.
  • RNA acts as a template to synthesize DNA.
  • RNA → DNA (Reverse Transcription) → RNA → Protein
  • This process is catalyzed by the enzyme reverse transcriptase.

DNA Replication: Semi-conservative, Enzymes Involved

• DNA Replication: The process by which a DNA molecule makes an exact copy of itself.
• Semi-conservative Replication: Proposed by Watson and Crick, and experimentally proven by Meselson and Stahl.
  • Each new DNA molecule consists of one original (parental) strand and one newly synthesized strand.

• Meselson and Stahl Experiment (1958):

  • They grew E. coli in a medium containing ¹⁵N (heavy isotope of nitrogen) for several generations, so that ¹⁵N was incorporated into the bacterial DNA.
  • Then, they transferred the bacteria to a medium containing ¹⁴N (light isotope of nitrogen).
  • After one generation, the DNA was extracted and centrifuged in a CsCl density gradient. They found an intermediate hybrid band (¹⁵N-¹⁴N).
  • After two generations, they found two bands: one hybrid (¹⁵N-¹⁴N) and one light (¹⁴N-¹⁴N).
  • This confirmed the semi-conservative nature of DNA replication.

• Enzymes Involved in DNA Replication:

  • DNA Helicase: Unwinds the DNA double helix.
  • DNA Polymerase: Synthesizes new DNA strands by adding nucleotides complementary to the template strand. It works in a 5'→3' direction.
  • DNA Ligase: Joins the Okazaki fragments on the lagging strand.
  • Primase: Synthesizes RNA primers.
  • Topoisomerase: Relieves supercoiling ahead of the replication fork.

Transcription: Post-transcriptional Processing

• Transcription: The process of synthesizing RNA from a DNA template.
• In Prokaryotes: Transcription and translation occur in the cytoplasm and can be coupled.
• In Eukaryotes:
  • Occurs in the nucleus.
  • Involves RNA polymerase (RNA Pol I for rRNA, RNA Pol II for mRNA precursors/hnRNA, RNA Pol III for tRNA and 5s rRNA).
  • The primary transcript is hnRNA (heterogeneous nuclear RNA), which contains both coding sequences (exons) and non-coding sequences (introns).
• Post-transcriptional Processing (hnRNA to mRNA):
  1. Splicing: Introns (non-coding regions) are removed, and exons (coding regions) are ligated together by a process called splicing, carried out by spliceosomes (snRNPs).
  2. Capping: An unusual nucleotide, methyl guanosine triphosphate, is added to the 5'-end of hnRNA.
  3. Tailing: Adenylate residues (200-300) are added to the 3'-end of hnRNA in a template-independent manner.
  • The fully processed hnRNA (now mRNA) is transported out of the nucleus for translation.

Genetic Code: Features, Codons

• Genetic Code: The sequence of nucleotide bases in mRNA that specifies the amino acid sequence of a protein.
• Codon: A sequence of three consecutive nucleotides in mRNA that codes for a specific amino acid or a stop signal.

• Features of the Genetic Code:
  • Triplet Code: Each codon consists of three nucleotides.
  • Degenerate: Most amino acids are coded by more than one codon.
  • Unambiguous and Specific: One codon codes for only one specific amino acid.
  • Universal: The same codon codes for the same amino acid in almost all organisms (with a few exceptions).
  • Non-overlapping: No base is shared between adjacent codons.
  • Comma-less: No punctuation between codons.
  • Start Codon: AUG (codes for Methionine, also acts as initiator codon).
  • Stop Codons (Nonsense Codons): UAA, UAG, UGA (do not code for any amino acid, signal termination of translation).

Translation: Process in Prokaryotes

• Translation: The process of synthesizing protein from an mRNA template.
• In Prokaryotes:
  1. Initiation: The small ribosomal subunit binds to the mRNA near the start codon (AUG). The initiator tRNA (carrying formylmethionine) binds to the start codon. The large ribosomal subunit then joins, forming the initiation complex.
  2. Elongation: Amino acids are added one by one to the polypeptide chain.
     • tRNA molecules, carrying specific amino acids, bind to the codons in the A-site of the ribosome.
     • A peptide bond is formed between the amino acid in the A-site and the growing polypeptide chain in the P-site (peptidyl transferase activity of rRNA).
     • The ribosome moves along the mRNA (translocation), shifting the tRNA from the A-site to the P-site, and the uncharged tRNA from the P-site to the E-site (exit site).
  3. Termination: When a stop codon (UAA, UAG, or UGA) arrives at the A-site, no tRNA binds. Release factors bind to the stop codon, causing the release of the polypeptide chain and dissociation of the ribosomal subunits.

Gene Expression: Lac Operon

• Gene Expression: The process by which information from a gene is used in the synthesis of a functional gene product (protein or RNA).
• Regulation of Gene Expression: In prokaryotes, gene expression is often regulated at the transcriptional level.
• Lac Operon (in E. coli): An example of an inducible operon that regulates the metabolism of lactose.
  • Components:
    • Promoter (P): Binding site for RNA polymerase.
    • Operator (O): Binding site for the repressor protein.
    • Structural Genes:
      • lacZ: Codes for β-galactosidase (hydrolyzes lactose into glucose and galactose).
      • lacY: Codes for permease (increases permeability of the cell to β-galactosides).
      • lacA: Codes for transacetylase.
    • Regulator Gene (i gene): Codes for the repressor protein.
  • When Lactose is Absent: The repressor protein (synthesized by the i gene) binds to the operator region, preventing RNA polymerase from transcribing the structural genes. The operon is switched OFF.
  • When Lactose is Present: Lactose (or allolactose, an isomer of lactose) acts as an inducer. It binds to the repressor protein, causing a conformational change that prevents the repressor from binding to the operator. RNA polymerase can then bind to the promoter and transcribe the structural genes. The operon is switched ON.

Human Genome Project: Goals, Methods, Applications

• Human Genome Project (HGP): An international scientific research project with the primary goal of determining the sequence of chemical base pairs that make up human DNA, and of identifying and mapping all of the genes of the human genome.
• Goals:
  • Identify all the approximately 20,000-25,000 genes in human DNA.
  • Determine the sequences of the 3 billion chemical base pairs that make up human DNA.
  • Store this information in databases.
  • Improve tools for data analysis.
  • Transfer related technologies to other sectors.
  • Address the ethical, legal, and social issues (ELSI) that may arise from the project.
• Methods:
  • Expressed Sequence Tags (ESTs): Focuses on identifying all the genes that are expressed as RNA.
  • Sequence Annotation: Sequencing the entire genome (coding and non-coding regions) and then assigning functions to different regions.
• Applications:
  • Understanding human biology, health, and disease.
  • Diagnosis, treatment, and prevention of genetic disorders.
  • Gene therapy.
  • Pharmacogenomics (personalized medicine).
  • Forensic science.

DNA Fingerprinting: Technique, Applications, Ethics

• DNA Fingerprinting (DNA Profiling): A technique used to identify individuals based on their unique DNA patterns.
• Principle: Based on the presence of Variable Number Tandem Repeats (VNTRs), which are short, repetitive DNA sequences that vary in number from person to person.
• Technique (Steps):
  1. Isolation of DNA: DNA is extracted from cells (blood, hair, saliva, semen).
  2. Digestion of DNA: DNA is cut into fragments using restriction enzymes.
  3. Separation of DNA Fragments: Fragments are separated by size using gel electrophoresis.
  4. Southern Blotting: Separated DNA fragments are transferred from the gel to a nylon membrane.
  5. Hybridization: The membrane is exposed to a labeled VNTR probe (a single-stranded DNA sequence complementary to the VNTRs).
  6. Autoradiography: The membrane is exposed to X-ray film, which reveals the unique banding pattern (DNA fingerprint).
• Applications:
  • Forensic Science: Identification of criminals, paternity disputes, identification of victims in mass disasters.
  • Paternity Testing: To determine biological parentage.
  • Conservation Biology: To identify endangered species, track animal populations.
  • Medical Diagnosis: To detect genetic diseases.
• Ethics:
  • Privacy concerns (storage and use of DNA data).
  • Potential for discrimination (e.g., in employment or insurance).
  • Misinterpretation of results.
  • Consent issues.

Interactive Practice