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Summary of Results –Due Fri 11/30 PowerPoint Presentation
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Summary of Results –Due Fri 11/30

Summary of Results –Due Fri 11/30

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Summary of Results –Due Fri 11/30

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  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: TOPICPgs. History & Discovery of DNA 293-296 as Genetic Material Structure of DNA 296-298 DNA Replication 298-307

  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) • How long have we known about the existence of DNA? Who was the first to isolate it? • Why are bacteria and viruses so important to our discovery of identifying DNA as our genetic material? • What was the significance of Griffith’s Experiment in 1928? • What did James Sumner purify in 1926? • 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 Macleod 1944 • Chargaff (Base pairing & species specific) 1947 • Hershey and Chase 1952 • Pauling, Wilkins, and Franklin 1950’s • Watson and Crick 1953

  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 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. Figure 10.1C

  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: (S35) Sulfur-35 (P32) 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. Agitate in a blender to separate phages outside the bacteria from the cells and their contents. Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube. Measure the radioactivity in the pellet and liquid. Mix radioactivelylabeled phages with bacteria. The phages infect the bacterial cells. 1 2 3 4 • The Hershey-Chase Experiment Radioactiveprotein Emptyprotein shell Radioactivityin liquid Phage Bacterium PhageDNA DNA Batch 1Radioactiveprotein Centrifuge Pellet RadioactiveDNA Batch 2RadioactiveDNA Centrifuge Radioactivityin pellet Pellet Figure 10.1B

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

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

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

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

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

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

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

  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 Hydrogen bond Ribbon model Partial chemical structure Computer model Figure 10.3D

  35. 5 end 3 end P • Each strand of the double helix is oriented in the opposite direction P P P P P P P 3 end 5 end Figure 10.5B

  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) • What was the significance of Griffith’s Experiment in 1928? • Give three reasons why Neurospora was in genetic studies to discover the “one gene, one enzyme” principle? (See Chapter 17 also) • What did James Sumner purify in 1926? • How was Avery, MacLoed, and McCarty work different from Griffith’s? • Matching: Garrod (ch. 17) A. Urease Griffith B. T2 Bacteriophage Beadle & Tatum (ch. 17) C. Alkaptonuria Sumner D. Neurospora Hershey & Chase E. Transformation Principle

  39. Introductory Questions #2 • Briefly explain what density gradient centrifugation is and what it is used for. • Name the organism used by Meselson & Stahl to label the DNA. • Name all of the enzymes required for DNA replication to occur and what purpose they serve. • In what direction is the newly synthesized strand made? What end of the old strand do the nucleotides add to? • What direction is the new strand growing? (towards or away from the replication fork) • 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 (15N) in the form of NH4Cl • 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 1 chocolate coat, Blind (PRA) Figure 10.3C Twist

  46. DNA replication depends on specific base pairing • In DNA replication, the strands separate • Enzymes use each strand as a template to assemble the new strands A A Nucleotides Parental moleculeof DNA Both parental strands serveas templates Two identical daughtermolecules of DNA Figure 10.4A

  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