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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? History for the Discovery of DNA • Chapter 16 The Molecular Basis of Inheritance 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 Overview of Chapter 16: TOPIC History & Discovery of DNA as Genetic Material Pgs. 293-296 Structure of DNA DNA Replication 296-298 298-307 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? 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 Key Discoveries • • • • • • • • • • • Miescher (isolated “nuclein” from soiled bandages) Garrod (Proteins & inborn errors) Sutton (Chromosome structure) Morgan (Gene mapping) Sumner (Purified Urease, showed it to be an enzyme) Griffith’s Experiment (Transforming Principle) Avery, McCarty, and Macleod Chargaff (Base pairing & species specific) Hershey and Chase Pauling, Wilkins, and Franklin Watson and Crick 1869 1902 1903 1913 1926 1928 1944 1947 1952 1950’s 1953 Discovery of DNA • 1868: Miescher first isolated deoxyribonucleic acid, or DNA, from cell nuclei 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 -injected with smooth strain -injected with smooth strain that was heat killed -injected with rough strain & heat killed smooth (Lived) (Died) (Lived) (????) Griffith’s Experiment-1928 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” 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. 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 Various Types of Viruses T2 Bacteriophage • 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 A Typical Bacteriophage 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 Labeling of Virus Structures Details of the Hershey & Chase Experiment • The Hershey-Chase Experiment 1 Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells. Phage 2 Agitate in a blender to separate phages outside the bacteria from the cells and their contents. Radioactive protein Bacterium 3 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube. 4 Empty protein shell Measure the radioactivity in the pellet and liquid. Radioactivity in liquid Phage DNA DNA Batch 1 Radioactive protein Centrifuge Pellet Batch 2 Radioactive DNA Figure 10.1B Radioactive DNA Centrifuge Pellet Radioactivity in pellet 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) 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 Rosalind Franklin’s Image (pg. 297) • and Media 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 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 DNA and RNA are polymers of Nucleotides • DNA is a nucleic acid, made of long chains of nucleotides Phosphate group Nitrogenous base Sugar Phosphate group Nitrogenous base (A, G, C, or T) Nucleotide Thymine (T) Sugar (deoxyribose) DNA nucleotide Polynucleotide Sugar-phosphate backbone Figure 10.2A • DNA has four kinds of bases, A, T, C, and G Thymine (T) Cytosine (C) Pyrimidines Adenine (A) Guanine (G) Purines Figure 10.2B DNA Maintains a Uniform Diameter See pg. 298 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 • RNA is also a nucleic acid – RNA has a slightly different sugar – RNA has U instead of T Nitrogenous base (A, G, C, or U) Phosphate group Uracil (U) Sugar (ribose) Figure 10.2C, D • Hydrogen bonds between bases hold the strands together – Each base pairs with a complementary partner – A pairs with T – G pairs with C 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 • Three representations of DNA Hydrogen bond Ribbon model Partial chemical structure Computer model Figure 10.3D 3 end P P P P P P P • Each strand of the double helix is oriented in the opposite direction 5 end P Figure 10.5B 3 end 5 end 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) 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? Introductory Questions (#1) 1) 2) 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) 3) 4) 5) 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 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? Three Proposed Models of DNA Replication Meselson & Stahl’s Experiment Meselson-Stahl Experiment 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. 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. • The structure of DNA consists of two polynucleotide strands wrapped around each other in a double helix 1 chocolate coat, Blind (PRA) Twist Figure 10.3C 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 Nucleotides Parental molecule of DNA Both parental strands serve as templates Two identical daughter molecules of DNA Figure 10.4A • Untwisting and replication of DNA Figure 10.4B Anti-parallel Structure of DNA 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 How a Nucleotides adds to the old Strand 5’ end 3’ end 5’ end 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. Origin of replication (“bubbles”): beginning of replication (pg. 301) 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 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 • How DNA daughter strands are synthesized • The daughter strands are identical to the parent molecule DNA polymerase molecule 5 end Daughter strand synthesized continuously Parental DNA 5 3 Daughter strand synthesized in pieces 3 5 P 5 3 3 5 P DNA ligase Overall direction of replication Figure 10.5C Laying Down RNA Primers 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# DNA Replication Fork Video Clip of DNA Replication • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html# Prokaryotic vs Eukaryotic Replication • Prokaryotes – Circular DNA (no free ends) – Contains 4 x 106 base pairs (1.35 mm) – Only one origination point • Eukaryotes -Have free ends -Contains 3 x 109 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 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) 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 Telomeres 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? Chapter 17 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. 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” Metabolic Pathway for the breakdown of Tyrosine Tyrosine ↓ Hydroxyphenylpyruvate ↓ Homogentisic acid Alkaptonuria (Inactive enzyme) Maleyacetoacetate (active ↓ enzyme) CO2 & H2O 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) 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 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. Beadle & Tatum’s Conclusion “One Gene affects One Enzyme” Later Revised “One Gene affects One Protein” Later Revised “One Gene affects One Polypeptide Chain” 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