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Chapter 11 DNA: The Carrier of Genetic Information

Chapter 11 DNA: The Carrier of Genetic Information. Experiments in DNA. ???Protein as the genetic material 20 AA – many different combinations = unique properties Genes control protein synthesis DNA and RNA – only 4 nucleotides = dull. Experiments in DNA. Frederick Griffith – 1928

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Chapter 11 DNA: The Carrier of Genetic Information

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  1. Chapter 11DNA: The Carrier of Genetic Information

  2. Experiments in DNA • ???Protein as the genetic material • 20 AA – many different combinations = unique properties • Genes control protein synthesis • DNA and RNA – only 4 nucleotides = dull

  3. Experiments in DNA • Frederick Griffith – 1928 • Bacteria – pneumococcus – 2 strains • (S) smooth strain – virulent (lethal) • Mice – pneumonia - death • (R) rough strain – avirulent • Mice survive • Heat killed (S) strain • Mice survive • Heat killed (S) + live (R) • Mice died • Found living (S) in dead mice

  4. Griffith continued •  transformation - type of permanent genetic change where the properties of 1 strain of dead cells are conferred on a different strain of living cells • “transforming principle” was transferred from dead to living cells

  5. Fig. 16-2 Mixture of heat-killed S cells and living R cells EXPERIMENT Living R cells (control) Living S cells (control) Heat-killed S cells (control) RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells

  6. Avery, MacLeod, McCarty - 1944 • Identified Griffith’s transforming principle as DNA • Live (R) + purified DNA from (S)  R cells transformed • R + (S) DNA  die • R = (S) protein  live • DNA responsible for transformation • Really?

  7. Hershey and Chase – 1952 • Bacteriophages • Radioactive labels • Viral protein – sulfur • Viral DNA - phosphorus • infect bacteria, agitate in blender, centrifuge • Found • Sulfur sample – all radioactivity in supernatant (not cells) • Phosphorus sample – radioactivity in pellet (inside cells) • SO – bacteriophages inject DNA into bacteria, leaving protein on outside • DNA = hereditary material

  8. Fig. 16-3 Phage head Tail sheath Tail fiber DNA 100 nm Bacterial cell

  9. Fig. 16-4-3 EXPERIMENT Empty protein shell Radioactivity (phage protein) in liquid Radioactive protein Phage Bacterial cell DNA Batch 1: radioactive sulfur (35S) Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: radioactive phosphorus (32P) Centrifuge Radioactivity (phage DNA) in pellet Pellet

  10. Rosalind Franklin (in lab of Wilkins) • X-ray diffraction on crystals of purified DNA • (X-ray crystallography) • Determine distance between atoms of molecules arranged in a regular, repeating crystalline structure • Helix structure • Nucleotide bases like rungs on ladder

  11. Fig. 16-6 (b) Franklin’s X-ray diffraction photograph of DNA (a) Rosalind Franklin

  12. James Watson and Francis Crick – 1953 • Model for DNA structure = double helix • DNA now widely accepted as genetic material • Took all available info on DNA and put together • Showed – • DNA can carry info for proteins • Serve as own template for replication

  13. Structure of DNA • Nucleotides • Deoxyribose • Phosphate • Nitrogenous base (ATCG) • Purines – adenine, guanine – 2 rings • Pyrimidines – thymine, cytosine – 1 ring • covalent bonds link = sugar-phosphate backbone • 3’ C of sugar bonded to 5’ phosphate = phophodiester linkage • 5’ end – 5’ C attached to phosphate • 3’ end – 3’ C attached to hydroxyl

  14. Chargaff - 1950 • # purines = # pyrimidines • #A = #T • #C = # G • Each cross rung of ladder • 1 purine + 1 pyrimidine

  15. Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

  16. Hydrogen bonding between N bases • A-T = 2 H bonds • G-C = 3 H bonds • Complementary base pairs • # possible sequences virtually unlimited •  many genes, much info

  17. Fig. 16-8 Adenine (A) Thymine (T) Cytosine (C) Guanine (G)

  18. Fig. 16-5 Nitrogenous bases Sugar–phosphate backbone 5 end Thymine (T) Adenine (A) Cytosine (C) DNA nucleotide Phosphate Sugar (deoxyribose) 3 end Guanine (G)

  19. Fig. 16-7a 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end (a) Key features of DNA structure (b) Partial chemical structure

  20. DNA Replication • Semiconservative – each strand of DNA is template to make opposite new strand • Meselson and Stahl • E. coli and isotopes of N • 15N – heavy/dense; 14N “normal” • Bacteria with 15N in DNA replicated with medium having 14N • Centrifuge  • Supports semiconservative model • Explains how mutagens can be passed on

  21. Fig. 16-11a EXPERIMENT Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N 2 1 RESULTS DNA sample centrifuged after 20 min (after first application) DNA sample centrifuged after 20 min (after second replication) Less dense 3 4 More dense

  22. Fig. 16-9-3 A T A T A T A T C G C G C G C G A T A T A A T T T A T A T T A A C C G C G C G G (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands (a) Parent molecule

  23. Fig. 16-10 First replication Second replication Parent cell (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model

  24. Steps of DNA Replication • 1. DNA helicase – • 2. Helix-destabilizing proteins – • 3. Topoisomerases – • 4. RNA primer – • 5. DNA polymerase – • 6. Origin of replication – • Leading strand • Lagging strand • 7. DNA Ligase

  25. Leading Strand

  26. Fig. 16-12b Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0.25 µm Replication fork Bubble Two daughter DNA molecules (b) Origins of replication in eukaryotes

  27. Fig. 16-14 New strand 5 end Template strand 3 end 5 end 3 end Sugar T A A T Base Phosphate C G C G G C G C DNA polymerase 3 end A A T T 3 end Pyrophosphate C C Nucleoside triphosphate 5 end 5 end

  28. Fig. 16-15a Overview Origin of replication Leading strand Lagging strand Primer Leading strand Lagging strand Overall directions of replication

  29. Fig. 16-17 Overview Origin of replication Lagging strand Leading strand Leading strand Lagging strand Single-strand binding protein Overall directions of replication Helicase Leading strand 5 DNA pol III 3 3 Primer Primase 5 3 Parental DNA Lagging strand DNA pol III 5 DNA pol I DNA ligase 4 3 5 3 2 1 3 5

  30. Telomeres • Telomeres – caps end of chromosome; short non-coding sequences repeated many times • Cell can divide many times before losing crucial info • Lagging strand is discontinuous, so DNA polymerase unable to complete replication , leaving small part unreplicated  small part lost with each cycle

  31. Fig. 16-19 5 Ends of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 New leading strand 3 5 New lagging strand 3 Further rounds of replication Shorter and shorter daughter molecules

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