1. Summary of Results –Due Fri 11/30 Title Introduction/Background (historical context) Separation of DNA: Explain the process -Why does this technique work? -Why do you need salt, shampoo, & alcohol? Results: Explain what you observed –Describe what you saw –How was the Banana DNA different from your DNA? –How could you verify that what you have is DNA? Conclusion/Evaluation: –How could this lab activity be improved? (Sources of error) –What else could be used? Other techniques?

2. History for the Discovery of DNA Chapter 16 The Molecular Basis of Inheritance

3. Next Unit: **Chapter 16: DNA: History, Structure & Replication **Chapter 17: Genetic Expression (protein synthesis) Chapter 18: Viruses & Bacteria (selected parts) Chapter 19: Regulation (selected parts) **Chapter 20: Genetic Engineering & Biotechnology

4. Overview of Chapter 16: TOPIC Pgs. History & Discovery of DNA 293-296 as Genetic Material Structure of DNA296-298 DNA Replication298-307

5. Key Questions Explored in this Next unit: What are Genes made of? How do Genes work? How can information be stored, retrieved, and modified over time? What keeps this molecule so stable? Why is DNA and not protein responsible for the inheritance of genetic traits?

6. Introductory Questions (#1) 1)How long have we known about the existence of DNA? Who was the first to isolate it? 2)Why are bacteria and viruses so important to our discovery of identifying DNA as our genetic material? 3)What was the significance of Griffith’s Experiment in 1928? 4)What did James Sumner purify in 1926? 5)How was Avery, MacLeod, and McCarty work different from Griffith’s? Why was their work still met with criticism? See pg. 294

7. Key Discoveries Miescher (isolated “nuclein” from soiled bandages) 1869 Garrod (Proteins & inborn errors) 1902 Sutton (Chromosome structure) 1903 Morgan (Gene mapping) 1913 Sumner (Purified Urease, showed it to be an enzyme) 1926 Griffith’s Experiment (Transforming Principle) 1928 Avery, McCarty, and Macleod1944 Chargaff( Base pairing & species specific) 1947 Hershey and Chase1952 Pauling, Wilkins, and Franklin1950’s Watson and Crick1953

8. Discovery of DNA 1868:Miescher first isolated deoxyribonucleic acid, or DNA, from cell nuclei

9. Fredrick Griffith (1928) First suggestion that about what genes are made of. Worked with: 1) Two strains of Pneumococcus bacteria: Smooth strain (S) Virulent (harmful) Rough strain (R) Non-Virulent 2) Mice-were injected with these strains of bacteria and watched to see if the survived. 3) Four separate experiments were done: -injected with rough strain(Lived) -injected with smooth strain(Died) -injected with smooth strain that was heat killed(Lived) -injected with rough strain & heat killed smooth(????)

10. Griffith’s Experiment-1928

11. Conclusion of Griffith’s Experiment Somehow the heat killed smooth bacteria changed the rough cells to a virulent form. These genetically converted strains were called “Transformations” Something (a chemical) must have been transferred from the dead bacteria to the living cells which caused the transformation Griffith called this chemical a “Transformation Principle”

12. Avery, MacLeod, and McCarty (1944) Chemically identified Griffith’s transformation principle as DNA Separated internal contents of the S cells into these fractions: ( lipids, proteins, polysaccharides, and nucleic acids) They tested each fraction to see if it can cause transformation to occur in R cells to become S cells. Only the nucleic acids caused the transformation This was the first concrete evidence that DNA is the genetic material. Some were not completely convinced because they were not sure if this was true for eukaryotes.

13. Next Breakthrough came from the use of Viruses Viruses provided some of the earliest evidence that genes are made of DNA Molecular biology studies how DNA serves as the molecular basis of heredity Only composed of DNA and a protein shell

14. Various Types of Viruses

15. T2 Bacteriophage

16. Phage reproductive cycle Figure 10.1C Phage attaches to bacterial cell. Phage injects DNA. Phage DNA directs host cell to make more phage DNA and protein parts. New phages assemble. Cell lyses and releases new phages.

17. A Typical Bacteriophage

18. Alfred Hershey & Martha Chase (1952) Worked with T-2 Bacteriophages Infected Escherchia coli (E. coli) = Host cell Used Radioactive Isotopes: (S 35 ) Sulfur-35 (P 32 ) Phosphorus-32 Why did they use these particular isotopes? *Sulfur is found in proteins and not in DNA *Phosphorus is found in DNA but not in protein

19. Labeling of Virus Structures

20. Details of the Hershey & Chase Experiment

21. The Hershey-Chase Experiment Figure 10.1B Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells. Phage Bacterium Radioactive protein DNA Empty protein shell 12 Agitate in a blender to separate phages outside the bacteria from the cells and their contents. 3 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube. 4 Measure the radioactivity in the pellet and liquid. Batch 1 Radioactive protein Batch 2 Radioactive DNA Radioactive DNA Phage DNA Centrifuge Pellet Radioactivity in liquid Radioactivity in pellet Pellet Centrifuge

22. Video clip of Hershey Chase Experiment http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html# Key findings: the phage DNA entered in the host cell and when these cells were returned to the culture medium the infection ran its course producing E.coli and other bacteriophages with the radioactive phosphorus.(pg. 298)

23. James Watson and Francis Crick worked out the three-dimensional structure of DNA, based on work by Rosalind Franklin DNA is a Double-Stranded Helix Figure 10.3A, B

24. Rosalind Franklin’s Image (pg. 297) and Media

25. Video #1 DNA: The Blueprint of Life 1.Name the technology used in the movie Jurassic Park. 2.Where did Meissner extract the “nuclein” material that later was identified as DNA? 3. How did Hershey & Chase separate the virus from its bacterial host? How did they trace (track) the DNA and protein? 4. What did x-ray crystallography reveal about DNA? 5. What purpose do enzymes serve in the replication process? Segment #2: **Need Five key Statements for the segment

26. Introductory Questions (#1) 1)How long have we known about the existence of DNA? Who was the first to isolate it? 2)Why are bacteria and viruses so important to our discovery of identifying DNA as our genetic material? 3)What was the significance of Griffith’s Experiment in 1928? 4)What did James Sumner purify in 1926? 5)How was Avery, MacLeod, and McCarty work different from Griffith’s? Why was their work still met with criticism? See pg. 294

27. DNA is a nucleic acid, made of long chains of nucleotides DNA and RNA are polymers of Nucleotides Figure 10.2A Nucleotide Phosphate group Nitrogenous base Sugar PolynucleotideSugar-phosphate backbone DNA nucleotide Phosphate group Nitrogenous base (A, G, C, or T) Thymine (T) Sugar (deoxyribose)

28. DNA has four kinds of bases, A, T, C, and G Figure 10.2B Pyrimidines Thymine (T)Cytosine (C) Purines Adenine (A)Guanine (G)

29. DNA Maintains a Uniform Diameter See pg. 298

30. DNA Bonding Purines: ‘A’ & ‘G’ Pyrimidines: ‘C’ & ‘T’ (Chargaff rules) ‘A’ H+ bonds (2) with ‘T’ and ‘C’ H+ bonds (3) with ‘G’ Van der Waals attractions between the stacked pairs

31. RNA is also a nucleic acid –RNA has a slightly different sugar –RNA has U instead of T Figure 10.2C, D Phosphate group Nitrogenous base (A, G, C, or U) Uracil (U) Sugar (ribose)

32. Hydrogen bonds between bases hold the strands together –Each base pairs with a complementary partner –A pairs with T –G pairs with C

33. DNA Structure Chargaff ratio of nucleotide bases (A=T; C=G) Watson & Crick (Wilkins, Franklin) The Double Helix √ nucleotides: nitrogenous base (thymine, adenine, cytosine, guanine); sugar deoxyribose; phosphate group

34. Three representations of DNA Figure 10.3D Ribbon modelPartial chemical structureComputer model Hydrogen bond

35. Each strand of the double helix is oriented in the opposite direction Figure 10.5B 5 end3 end 5 end P P P P P P P P

36. DNA Replication: History & Discovery First model suggested by Watson & Crick Three models were proposed: -Semiconservative (half old & half new) -Conservative (old strands remain together) -Dispersive (random mixture) Heavy isotopic nitrogen (N-15) was used to label the nitrogenous bases in the DNA Density gradient centrifugation was used DNA was mixed with Cesium chloride (CsCl)

37. Video #1 DNA: The Blueprint of Life Name the technology used in the movie Jurassic Park. Where did Meissner extract the “nuclein” material that later was identified as DNA? How did Hershey & Chase separate the virus from its bacterial host? How did they trace (track) the DNA and protein? What did x-ray crystallography reveal about DNA? What purpose do enzymes serve in the replication process? Segment #2: Name the disorder that Andrew and his sister inherited. What were the major symptoms of this disorder? How can this genetic defect be treated? Name the gene that is defective. How can a gene be transported and carried to a cell? What is a vector? Give an example. What purpose do restriction enzymes serve? What about ligase? What does PCR stand for? Segment #3: What is the first step of gene therapy? How long would all of the DNA contained in all of the chromosomes in a human cell be if they were connected end to end? Which chromosome consists of 5% of all the genes in the human genome?

38. Introductory Questions (#1) 1)What was the significance of Griffith’s Experiment in 1928? 2)Give three reasons why Neurospora was in genetic studies to discover the “one gene, one enzyme” principle? (See Chapter 17 also) 3)What did James Sumner purify in 1926? 4)How was Avery, MacLoed, and McCarty work different from Griffith’s? 5)Matching: Garrod (ch. 17) A. Urease GriffithB. T 2 Bacteriophage Beadle & Tatum (ch. 17)C. Alkaptonuria SumnerD. Neurospora Hershey & ChaseE. Transformation Principle

39. Introductory Questions #2 1)Briefly explain what density gradient centrifugation is and what it is used for. 2)Name the organism used by Meselson & Stahl to label the DNA. 3)Name all of the enzymes required for DNA replication to occur and what purpose they serve. 4)In what direction is the newly synthesized strand made? What end of the old strand do the nucleotides add to? 5)What direction is the new strand growing? (towards or away from the replication fork) 6)How long (# nucleotides) are the Okasaki fragments? How long are the RNA primers?

40. Three Proposed Models of DNA Replication

41. Meselson & Stahl’s Experiment

42. Meselson-Stahl Experiment

43. Meselson & Stahl Experiment (Pg. 300) Grew E. coli on a medium containing isotopic Nitrogen ( 15 N) in the form of NH 4 Cl Nitrogenous bases incorporated the isotopic nitrogen DNA was extracted from the cells Density gradient centrifugation was used on the DNA to determine the banding region of the heavy isotopic nitrogen. The rest of the bacteria was then grown on a medium containing normal nitrogen and allowed to grow.

44. Meselson & Stahl Experiment cont’d. The newly synthesized strands of DNA were expected to have the lighter normal nitrogen in their bases. The older original strands were labeled with the heavier isotopic nitrogen. Two generations were grown in order to rule out the conservative and dispersion models.

45. The structure of DNA consists of two polynucleotide strands wrapped around each other in a double helix Figure 10.3C Twist 1 chocolate coat, Blind (PRA)

46. In DNA replication, the strands separate –Enzymes use each strand as a template to assemble the new strands DNA replication depends on specific base pairing Parental molecule of DNA Figure 10.4A Both parental strands serve as templates Two identical daughter molecules of DNA Nucleotides A A

47. Untwisting and replication of DNA Figure 10.4B

48. Anti-parallel Structure of DNA

49. Antiparallel nature 5’ end corresponds to the Phosphate end 3’ end corresponds to the –OH sugar Replication runs in BOTH directions One strand runs 5’ to 3’ while the other runs 3’ to 5’ Nucleotides are added on the 3’ end of the newly synthesized strand The new DNA strand forms and grows in the 5’  3’ direction only

50. How a Nucleotides adds to the old Strand 5’ end 3’ end 5’ end

51. Building New Strands of DNA Each nucleotide it a triphosphate: (GTP, TTP, CTP, and ATP) Nucleotides only add to the 3’ end of the growing strand (never on the 5’ end) Two phosphates are released (exergonic) and the energy released drives the polymerization process.

52. Origin of replication (“bubbles”): beginning of replication (pg. 301)

53. Key Enzymes Required for DNA Replication (pg. 303-304) Helicase - catalyzes the untwisting of the DNA at the replication fork DNA Polymerase - catalyzes the elongation of new DNA and adds new nucleotides on the 3’ end the growing strand. SSBP’s - single stranded binding proteins, prevents the double helix from reforming Topoisomerase – Breaks the DNA strands and prevents excessive coiling RNA primase – synthesizes the RNA primers and starts the replication first by laying down a few nucleotides initially. **DNA primase will get replaced by DNA polymerase

54. RNA Primers Initiates the Replication process and begins the building of the newly formed strands. Laid down by RNA primase Consists of 5 to 14 nucleotides Synthesized at the point where replication begins Will be laid down on both template strands of the DNA

55. How DNA daughter strands are synthesized 5 end P P Parental DNA Figure 10.5C DNA polymerase molecule 5 3 3 5 3 5 Daughter strand synthesized continuously Daughter strand synthesized in pieces DNA ligase Overall direction of replication 5 3 The daughter strands are identical to the parent molecule

56. Laying Down RNA Primers

57. DNA Replication-New strand Development Leading strand: synthesis is toward the replication fork (only in a 5’ to 3’ direction from the 3’ to 5’ master strand) -Continuous Lagging strand: synthesis is away from the replication fork -Only short pieces are made called “Okazaki fragments” - Okazaki fragments are 100 to 2000 nucleotides long -Each piece requires a separate RNA primer -DNA ligase joins the small segments together (must wait for 3’ end to open; again in a 5’ to 3’ direction) View video clip: http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

58. DNA Replication Fork

59. Video Clip of DNA Replication http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

60. Prokaryotic vs Eukaryotic Replication Prokaryotes –Circular DNA (no free ends) –Contains 4 x 10 6 base pairs (1.35 mm) –Only one origination point Eukaryotes -Have free ends -Contains 3 x 10 9 base pairs (haploid cells) = 1 meter -Lagging strand is not completely replicated -Small pieces of DNA are lost with every cell cycle -End caps (Telomeres) protect and help to retain the genetic information

61. Issues with Replication Prokaryotes: (ex. E. coli) –Have one singular loop of DNA –E. coli has approx. 4.6 million Nucleotide base pairs –Rate for replication: 500 nucleotides per second Eukaryotes w/Chromosomes: –Each chromosome is one DNA molecule –Humans (46) has approx. billion base pairs –Rate for replication: 50 per second (humans) Errors: –Rate is one every 10 billion nucleotides copied –Proofreading is achieved by DNA polymerase (pg. 305)

62. Telomeres Short, non-coding pieces of DNA Contains repeated sequences (ie. TTGGGG 20 times) Can lengthen with an enzyme called Telomerase Lengthening telomeres will allow more replications to occur. Telomerase is found in cells that have an unlimited number of cell cycles (commonly observed in cancer cells) Artificially giving cells telemerase can induce cells to become cancerous Shortening of these telomeres may contribute to cell aging and Apotosis (programmed cell death) Ex. A 70 yr old person’s cells divide approx. 20-30X vs an infant which will divide 80-90X

63. Telomeres

64. Introductory Questions #2 1)Briefly explain what density gradient centrifugation is and what it is used for. 2)Name the organism used by Meselson & Stahl to label the DNA. 3)Name all of the enzymes required for DNA replication to occur and what purpose they serve. 4)In what direction is the newly synthesized strand made? What end of the old strand do the nucleotides add to? 5)What direction is the new strand growing? (towards or away from the replication fork) 6)How long (# nucleotides) are the Okasaki fragments? How long are the RNA primers?

65. Chapter 17

66. James Sumner (1926) Isolated the enzyme “Urease” First to identify an enzyme as a protein First to crystallize an enzyme Awarded the Nobel prize in 1946 in chemistry for his crystallization of an enzyme.

67. Archibald Garrod (1902-1908) Studied a rare genetic disorder: Alkaptonuria Thought to be a recessive disorder Tyrosine is not broken down properly into carbon dioxide and water. An Intermediate substance: “Homogentisic acid” accumulates in the urine turning it BLACK when exposed to air. An enzyme was thought to be lacking A genetic mutation was thought to be the cause “An Inborn Error of Metabolism”

68. Metabolic Pathway for the breakdown of Tyrosine Tyrosine ↓ Hydroxyphenylpyruvate ↓ Homogentisic acid Alkaptonuria Maleyacetoacetate (Inactive enzyme) (active ↓ enzyme) CO 2 & H 2 O

69. Garrod’s Conclusion A mutation in a specific gene is associated with the absence of a specific enzyme. Led to the idea of: “One gene, One Enzyme” Not validated until Beadle & Tatum’s work in the 1940’s with Neurospora (breadmold)

70. George Beadle & EdwardTatum (1940’s) Discovered the “One Gene, One Enzyme” Principle Analyzed mutations that interfered with a known metabolic pathway Organism they chose to work with: Neurospora (breadmold) -Grows easily -Grows as a haploid: (no homologs) -Mutants are easily identified: Dominant allele won’t be expressed Neurospora can grow easily in only: salt, sugar, & Biotin

71. George Beadle & EdwardTatum (1940’s) cont’d Mutants-are unable to make certain organic molecules: amino acids, lipids, etc. These substances are added to the media which will allow mutants to grow successfully Exposed the haploid spores to x rays & UV to induce mutations Haploid spores were crossed, grown in a variety of media to determine what kind of mutation was occurring **They examined the effect of the mutation instead of identifying the enzyme.

72. Beadle & Tatum’s Conclusion “One Gene affects One Enzyme” Later  Revised “One Gene affects One Protein” Later  Revised “One Gene affects One Polypeptide Chain”

73. Suggestions on how to Review Make a List of all Bold Terms (See summaries) Make a list of key people & generate a timeline Answer all MC questions at end of each chapter Review all your Quizzes from textbook website Review all the MC Questions from your study guides Look at all the key figures & diagrams discussed Review all Tables from the four chapters Re-Look at the Powerpoint Pres. From my website. Think back to what was emphasized Anticipate questions to be asked Make an outline of all chapters & connect the concepts discussed