Molecular Basis of Inheritance

• Who were the major biologists of the 19th century?

  Mendel

  Darwin

  Miescher
• what did darwin discover?

  Natural selection theory of evolution

  • all species of life have descended from a common ancestor over time
• What did Miescher discover?

  Discoverer of nucleic acid
• what did mendel discover?

  Dominant and recessive trait

  The concept of gene (hertiable factor)

  The formulation of the basic laws of inheritance
• What was Miescher interested in?

  Components of cells

  • need to need to identify a good source of cells with large nuclei, which are easy to purify
• How did miescher purify DNA?

  pus from used bandages -> white blood cells -> purified nuclei -> extracted nuclei and found a precipitate rich in phosphorus and nitrogen (nuclein) -> (N-rich) protein amnd acidic P-rich (nucleic acid)
• What do chromosome contain?

  DNA and protein
• What were the experiments to detemine genetic material?

  Griffith, Avery, Hershey-Chase
• Griffith's experiment

  1. Control groups: Injected mice with live non-virulent strain, mice remained healthy

  2. Injected mice with live virulent strain, mice developed pneumonia and died

  3. Injected mice with heat-killed virulent strain, mice remained healthy

  4. Mixed heat-killed virulent strain with live non-virulent strain and injected, some mice developed pneumonia and died
• Streptococcus pneumoniae strains


  R strain is benign, S strain is virulent
• Griffith concluded that something from the heat-killed virulent strain had transformed the non-virulent strain into a virulent form, which he termed "transformation"
• Griffith did not identify the exact nature of the transforming substance
• What did Avery identify?

  The transforming principle
• How did Avery test the nature of the transforming principle?

  Subjected the heat-killed S-type bacteria to a range of tests
• Results of Avery's tests?

  • Resistant to proteases – so NOT protein
  • Resistant to lipases – so NOT lipid
  • Resistant to ribonucleases – so NOT RNA
  • Ethanol-insoluble (precipitate formed and supernatant was no longer transforming) - so NOT carbohydrate
  • High molecular weight (like DNA)
  • Positive reaction to the Dische test for the deoxyribose of DNA.
  • Conclusion – transforming principle is DNA
• what was the Hershey-Chase experiment?

  1. phage protein labelled with 35S. Results: most radioactivity in supernatant (containing phage ghosts)
  2. phage DNA labelled with 32P. Results: most of the radioactivity in the pellet (containing the intact cells)

  Conclusion - DNA is injected into the cells, not protein
• What is Chargaffs rule?

  Ratio of the four bases is not 1:1:1:1 (suggests it is not simply a structural molecule).

  Ratio is species-specific

  Base composition always obeys a strict rule: A=T and G=C (Chargaff’s Rule)
• How did Franklin, Wilkins, Watson and Crick solve the structure of DNA?

  X-ray diffraction
• What are the 3 components for a nucleotide?

  pentose sugar, Base, Phosphate
• what are the bases in DNA?

  Adenine, Thymine, Guanine, Cytosine
• What are the purine?

  Adenine and Guanine
• what are the pyrimidines?


  cytosine, thymine
• what are the double membered rings in DNA called?

  Purine
• What conclusions can be drawn from X-ray diffraction?

  DNA is a helix

  2nm wide (width of 2 nucleotide chains)

  Length of each turn is 3.4nm

  distance between repeating units is 0.34nm

  therefore 10 nucleotide pairs per turn
• who realised that a purine paired with a pyrimidine?

  Watson and Crick
• How many hydrogen bonds do A and T pair?

  2
• how many hydrogen bonds do G and C form?

  3
• which was does DNA run?

  5' to 3'
• Who suggested that each strand acts as a template for synthesis of a new complementary strand?

  Watson and Crick
• What is the Meselson- Stahl experiment?


  Bacteria was cultured in medium with 15N (heavy isotope) and transferred to mediusm with 14N (lighter isotope)

  DNA sample was centrifuged after first replication, all of DNA of intermediate density showing one had a light strand the other had a heavy strand
• What is used in DNA synthesis?

  DNA polymerase, releasing pyrophosphate which converts into Phosphate
• What does DNA polymerase require?

  single stranded template DNA, all four nucleoside triphosphates (dNTPs), free 3' hydroxyl (primer)
• What does DNA polymerase do?

  synthesises DNA in 5' to 3' direction, inserts complementary nucleotides, uses energy from breaking phosphate bonds, proof-reading ability can remove incorrectly inserted nucleotides
• where does replication begin?

  at an origin of replication which forms a bubble
• Describe a replication fork

  • leading strand
  • lagging strand
• Describe synthesis of the leading strand

  1. helicase unwinds and separates the two strands
  2. single stranded binding proteins prevent DNA strands from re-annelaing
  3. DNA pol III: need a 3' OH group
  4. RNA primer: synthesised and added by primase
• Which way is the leading strand synthesised?

  continuously in 5' to 3' direction
• Describe synthesis of lagging strand

  1. priming
  2. initiation of DNA synthesis
  3. formation of Okazaki fragements
  4. RNA primer romoval
  5. fill-in synthesis
  6. ligating the fragments
  7. completion of replication
• what is the priming step of synthesis of lagging strand?

  Primase (RNA polymerase) synthesis short RNA primers complementary to the template DNA strand
• What occurs in initiation of DNA synthesis?

  DNA polymerase III binds to RNA primer at the replication fork, and synthesised in discontinuous segments