Chapter 16 - Molecular Inheritence

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Chapter 16 - Molecular Inheritence

  1. 1. DNA The Genetic MaterialAP Biology 2006-2007
  2. 2. Scientific History  The march to understanding that DNA is the genetic material  T.H. Morgan (1908)  Frederick Griffith (1928)  Avery, McCarty & MacLeod (1944)  Erwin Chargaff (1947)  Hershey & Chase (1952)  Watson & Crick (1953)  Meselson & Stahl (1958)AP Biology
  3. 3. 1908 | 1933 Chromosomes related to phenotype  T.H. Morgan  working with Drosophila  fruit flies  associated phenotype with specific chromosome  white-eyed male had specific X chromosomeAP Biology
  4. 4. 1908 | 1933 Genes are on chromosomes  Morgan’s conclusions  genes are on chromosomes  but is it the protein or the DNA of the chromosomes that are the genes?  initially proteins were thought to be genetic material… Why? What’s so impressive about proteins?!AP Biology
  5. 5. 1928 The “Transforming Principle”  Frederick Griffith  Streptococcus pneumonia bacteria  was working to find cure for pneumonia  harmless live bacteria (“rough”) mixed with heat-killed pathogenic bacteria (“smooth”) causes fatal disease in mice  a substance passed from dead bacteria to live bacteria to change their phenotype  “Transforming Principle”AP Biology
  6. 6. The “Transforming Principle” mix heat-killed pathogenic &live pathogenic live non-pathogenic heat-killed non-pathogenicstrain of bacteria strain of bacteria pathogenic bacteria bacteriaA. B. C. D.mice die mice live mice live mice die Transformation = change in phenotype something in heat-killed bacteria could still transmit AP Biology disease-causing properties
  7. 7. 1944 DNA is the “Transforming Principle”  Avery, McCarty & MacLeod  purified both DNA & proteins separately from Streptococcus pneumonia bacteria  which will transform non-pathogenic bacteria?  injected protein into bacteria  no effect  injected DNA into bacteria  transformed harmless bacteria into virulent bacteria mice die What’s theAP Biology conclusion?
  8. 8. 1944 | ??!! Avery, McCarty & MacLeod  Conclusion  first experimental evidence that DNA was the genetic material Oswald AveryAP Biology Maclyn McCarty Colin MacLeod
  9. 9. 1952 | 1969 Confirmation of DNA Hershey  Hershey & Chase  classic “blender” experiment  worked with bacteriophage  viruses that infect bacteria  grew phage viruses in 2 Why use media, radioactively labeled with either Sulfur vs.  35S in their proteinsPhosphorus?  32P in their DNA  infected bacteria with labeled phagesAP Biology
  10. 10. Protein coat labeled DNA labeled with 32P with 35SHershey T2 bacteriophages are labeled with& Chase radioactive isotopes S vs. P bacteriophages infect bacterial cells bacterial cells are agitatedWhich to remove viral protein coatsradioactivemarker is foundinside the cell?Which moleculecarries viral 35S 32P radioactivity radioactivity foundgenetic info? AP Biology found in the medium in the bacterial cells
  11. 11. AP Biology
  12. 12. Blender experiment  Radioactive phage & bacteria in blender  35S phage  radioactive proteins stayed in supernatant  therefore viral protein did NOT enter bacteria  32 P phage  radioactive DNA stayed in pellet  therefore viral DNA did enter bacteria  Confirmed DNA is “transforming factor” Taaa-Daaa!AP Biology
  13. 13. 1952 | 1969 Hershey & Chase HersheyAP Biology Martha Chase Alfred Hershey
  14. 14. 1947 Chargaff  DNA composition: “Chargaff’s rules”  varies from species to species  all 4 bases not in equal quantity  bases present in characteristic ratio  humans: A = 30.9% Rules A = T T = 29.4% C = G G = 19.9% C = 19.8% That’s interesting! What do you notice?AP Biology
  15. 15. 1953 | 1962 Structure of DNA  Watson & Crick  developed double helix model of DNA  other leading scientists working on question:  Rosalind Franklin  Maurice Wilkins  Linus PaulingAP Biology Franklin Wilkins Pauling
  16. 16. 1953 article in Nature Watson and Crick WatsonAP Biology Crick
  17. 17. Rosalind Franklin (1920-1958)AP Biology
  18. 18. But how is DNA copied?  Replication of DNA  base pairing suggests that it will allow each side to serve as a template for a new strand“It has not escaped our notice that the specific pairing we have postulatedimmediately suggests a possible copying mechanism for the genetic AP Biologymaterial.” — Watson & Crick
  19. 19. Can you design a nifty experiment to verify? Models of DNA Replication  Alternative models  become experimental predictions conservative semiconservative dispersiveP12AP Biology
  20. 20. 1958 Semiconservative replication  Meselson & Stahl  label “parent” nucleotides in DNA strands with heavy nitrogen = 15N  label new nucleotides with lighter isotope = 14N “The Most Beautiful Experiment in Biology” parent replicationMake predictions… 15N/15N 15N parent strandsAP Biology
  21. 21.  Predictions 14N/14N 15N/14N 15N/14N 1st round of 15N/15N replication semi- conservative dispersive conservative 2nd round of replication    14N/14N 14N/14NP 15N/14N 15N/14N 15N/15N1 15N/15N semi-2 15N parent conservative dispersive AP Biology conservative strands
  22. 22. Meselson & StahlMatthew Meselson Franklin Stahl Franklin Stahl Matthew MeselsonAP Biology
  23. 23. Scientific History  March to understanding that DNA is the genetic material  T.H. Morgan (1908)  genes are on chromosomes  Frederick Griffith (1928)  a transforming factor can change phenotype  Avery, McCarty & MacLeod (1944)  transforming factor is DNA  Erwin Chargaff (1947)  Chargaff rules: A = T, C = G  Hershey & Chase (1952)  confirmation that DNA is genetic material  Watson & Crick (1953)  determined double helix structure of DNA  Meselson & Stahl (1958)AP Biology  semi-conservative replication
  24. 24. The “Central Dogma”  Flow of genetic information in a cell transcription translation DNA RNA protein replicationAP Biology
  25. 25. DNA Replication 2007-2008AP Biology
  26. 26. 1953 article in Nature Watson and CrickAP Biology
  27. 27. Double helix structure of DNA“It has not escaped our notice that the specific pairing we have postulatedimmediately suggests a possible copying mechanism for the genetic AP Biologymaterial.” Watson & Crick
  28. 28. Directionality of DNA  You need to PO4 nucleotide number the carbons!  it matters! N base 5 CH2 This will be O IMPORTANT!! 4 ribose 1 3 2AP Biology OH
  29. 29. 5 The DNA backbone PO4  Putting the DNA backbone together base 5 CH2  refer to the 3 and 5 O 4 1 ends of the DNA C 3 2  the last trailing carbon O –O P O Sounds trivial, but… O base this will be 5 CH2 IMPORTANT!! O 4 1 3 2 OHAP Biology 3
  30. 30. Anti-parallel strands  Nucleotides in DNA backbone are bonded from phosphate to sugar 5 3 between 3 & 5 carbons  DNA molecule has “direction”  complementary strand runs in opposite directionAP Biology 3 5
  31. 31. Bonding in DNA hydrogen bonds 5 3 covalent phosphodiester bonds 3 5….strong or weak bonds?AP Biology the bonds fit the mechanism for copying DNA?How do
  32. 32. Base pairing in DNA  Purines  adenine (A)  guanine (G)  Pyrimidines  thymine (T)  cytosine (C)  Pairing  A:T  2 bonds  C:G  3 bondsAP Biology
  33. 33. Copying DNA  Replication of DNA  base pairing allows each strand to serve as a template for a new strand  new strand is 1/2 parent template & 1/2 new DNA  semi-conservative copy processAP Biology
  34. 34. Let’s meet the team… DNA Replication  Large team of enzymes coordinates replicationAP Biology
  35. 35. I’d love to be helicase & unzip Replication: 1st step your genes…  Unwind DNA  helicase enzyme  unwinds part of DNA helix  stabilized by single-stranded binding proteins helicase single-stranded binding proteinsAP Biology replication fork
  36. 36. Replication: 2nd step  Build daughter DNA strand  add new complementary bases  DNA polymerase III But… Where’s the We’re missing ENERGY DNA something! for the bonding! Polymerase III What?AP Biology
  37. 37. Energy of Replication Where does energy for bonding usually come from? We come with our own energy! You energy remember energy ATP! Are there other waysother energyto get energynucleotides? out of it? You bet! And we leave behind a ATP TTP GTP CTP nucleotide! CMP TMP GMP AMP ADPAP Biology modified nucleotide
  38. 38. Energy of Replication  The nucleotides arrive as nucleosides  DNA bases with P–P–P  P-P-P = energy for bonding  DNA bases arrive with their own energy source for bonding  bonded by enzyme: DNA polymerase III ATP GTP TTP CTPAP Biology
  39. 39. 5 3 Replication energy DNA Adding bases Polymerase III  can only add energy nucleotides to DNA 3 end of a growing Polymerase III DNA strand energy DNA  need a “starter” Polymerase III nucleotide to bond to DNA energy  strand only grows Polymerase III 53 B.Y.O. ENERGY! The energy rules 3 5 the processAP Biology
  40. 40. 5 3 5 need “primer” bases to add on to 3 energy no energy  to bond energy energy energy energy ligase energy energy AP Biology3 5 3 5
  41. 41. Okazaki Leading & Lagging strandsLimits of DNA polymerase III can only build onto 3 end of 5 an existing DNA strand 3 5 3 5 3 5 5 3 Lagging strand ligase growing 3 replication fork 5 Leading strandLagging strand 3 5 3 DNA polymerase III Okazaki fragments joined by ligase Leading strandAP Biology  “spot welder” enzyme  continuous synthesis
  42. 42. Replication fork / Replication bubble 3 5 5 3 DNA polymerase III leading strand 5 3 3 5 5 5 5 3 3 lagging strand 3 5 53 lagging strand leading strand 5 growing 3 replication fork 55 growing replication fork 5 leading strand 3 lagging strand 3 5 5 5 AP Biology
  43. 43. Starting DNA synthesis: RNA primersLimits of DNA polymerase III can only build onto 3 end of 5 an existing DNA strand 3 5 3 5 3 3 5 growing 3 primase replication fork DNA polymerase III 5 RNA 5RNA primer 3 built by primase serves as starter sequenceAP for DNA polymerase III Biology
  44. 44. Replacing RNA primers with DNADNA polymerase I removes sections of RNA DNA polymerase I primer and replaces with 5 DNA nucleotides 3 3 5 ligase growing 3 replication fork 5 RNA 5 3But DNA polymerase I stillcan only build onto 3 end ofan BiologyAP existing DNA strand
  45. 45. Houston, we have a problem! Chromosome erosionAll DNA polymerases canonly add to 3 end of an DNA polymerase Iexisting DNA strand 5 3 3 5 growing 3 replication fork DNA polymerase III 5 RNA 5Loss of bases at 5 ends 3in every replication chromosomes get shorter with each replicationAP limit to number of cell divisions? Biology
  46. 46. TelomeresRepeating, non-coding sequences at the endof chromosomes = protective cap 5 limit to ~50 cell divisions 3 3 5 growing 3 telomerase replication fork 5 5Telomerase TTAAGGG TTAAGGG 3 enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cellsAP Biology  high in stem cells & cancers -- Why?
  47. 47. Replication fork DNA polymerase III lagging strand DNA polymerase I 3’ Okazaki primase fragments 5’ 5’ ligase 3’ 5’ SSB 3’ helicase DNA polymerase III 5’ leading strand 3’ direction of replicationAP Biology SSB = single-stranded binding proteins
  48. 48. DNA polymerases  DNA polymerase III  1000 bases/second! Thomas Kornberg ??  main DNA builder  DNA polymerase I  20 bases/second  editing, repair & primer removalDNA polymerase III Arthur Kornberg enzyme 1959 AP Biology
  49. 49. Editing & proofreading DNA  1000 bases/second = lots of typos!  DNA polymerase I  proofreads & corrects typos  repairs mismatched bases  removes abnormal bases  repairs damage throughout life  reduces error rate from 1 in 10,000 to 1 in 100 million basesAP Biology
  50. 50. Fast & accurate!  It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome  divide to form 2 identical daughter cells  Human cell copies its 6 billion bases & divide into daughter cells in only few hours  remarkably accurate  only ~1 error per 100 million bases  ~30 errors per cell cycleAP Biology
  51. 51. What does it really look like? 1 2 3 4AP Biology
  52. 52. Any Questions?? 2007-2008AP Biology

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