Chapter4Part1Powerpoint 11.45.23 PM

Chapter4Part1Powerpoint 11.45.23 PM - CHAPTER 4, PART 1 DNA...

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Unformatted text preview: CHAPTER 4, PART 1 DNA and RNA- MOLECULES OF HEREDITY CHAPTER 4, PART 1: LECTURE TOPICS 1. DNA/RNA structures, nomenclature, shorthand conventions 2. DNA and RNA as genetic material 3. General properties of DNA Double Helix 4. Basic mechanism of DNA replication 5. Some important physical and chemical properties of DNA Reminders from Chapter 25 lectures Nucleic acids (DNA and RNA) are polymers consisting of pentose sugars, phosphates and bases DNA and RNA are “Polynucleotides” Levene (1920 book): Tetranucleotide hypothesis of DNA structure G T C A Incorrect!! DNA and RNA are POLYNUCLEOTIDES (chains of nucleotides) 5` A,G,C,T 3` 2’ 2` 2’ 2` A,G,C,U 3` 5` 3` 5` Phosphodiester bond 5` 5` 3` 3` polarity (direction of chain; L to R) RNA/DNA Shorthand Conventions A 3` 5` G C T Base Sugar P P A 3` C P 3` (OH) 5` 5` Phosphodiester bond (A dinucleotide) Nucleic Acids Shorthand Notations Example: pACG 5`-P (or P, etc. if indicated) or: -OH -PP -PPP Assume both ends are OH if not indicated Trinucleotide: [pACG] A (a) 5` P C P G P OH 3` (b) pApCpG b and c are the same as a (c) pACG a=b=c and you can draw full structure from each shorthand Structure of Trinucleotide: [pACG] 5` A C G H 3` CHAPTER 4, PART 1: LECTURE TOPICS 1. DNA/RNA structures, nomenclature, shorthand conventions 2. DNA and RNA as genetic material 3. General properties of DNA Double Helix 4. Basic mechanism of DNA replication 5. Some important physical and chemical properties of DNA Nucleic acids discovered (1870’s) – Crimean War bandages DNA and RNA as genetic material: Proofs • Transformation with pure DNA (Pneumococcus; 1928,1944) • Bacteriophage T2 transmits DNA, not protein (1952) • Tobacco mosaic virus transmits RNA, not protein (1953) • Retroviruses (RNA to DNA to RNA) • Prions (PROTEINS - not DNA or RNA) as transmissible disease agents Even in early 1950’s, still asking: Is a gene protein or DNA?? Hershey-Chase (1952): show that DNA (not protein) transmits genetic information Hershey-Chase Experiment: Biological system [To ask: Are genes protein or DNA??] T2 bacteriophage (from Paris sewers) E. coli cell DNA BACTERIOPHAGE VIRUS LIFE CYCLE Scenario: attach to cell inject DNA reproduce (new DNA and proteins) kill cells release progeny (new) virus infect more cells new Hershey-Chase experiment (1952): DNA not protein transmits genetic information (32P) (35S) - protein - DNA Infect Cell DNA (32P) Shake off virus (35S) New virus has: 30% of 32P (DNA) <1% of 35S (protein) Tobacco Mosaic Virus (TMV) An RNA virus RNA as Genetic Material (RNA RNA) Ex: Tobacco Mosaic Virus Type 1 RNA and protein (1970) Retroviruses: RNA in virus transmits genetic information via a DNA intermediate in infected cells RNA RNA RNA (makes a cDNA strand) DNA DNA RNA DNA DNA (cuts the RNA strand) Example: HIV (makes 2nd cDNA strand) PRIONS – Proteins that are transmissible agents [PrPc] - Promiscuous host range and cause CNS diseases SLOW BRAIN INFECTIONS Scrapie (Czeckoslovakia) Bovine spongiform encephalopathy (BSE) Mad Cow disease (England) Sheep, Goats Cattle Transmissible mink encephalopathy Mink Creutzfeldt-Jakob disease * Kuru, Gerstmann-Strauss syndrome Chronic wasting disease with spongiform encephalopathy Humans * Spongiform encephalopathy Nyala gemsbok Domestic cat * Choreographer Balanchine died from this. Captive Rocky mountain elk and mule deer PRIONS [NOT DNA or RNA] – Mode of Action Twisted abnormal prions induce shape change in normal ones and they accumulate around neurons. Elk brain tissue: Light spots show spongy areas where tissue has been destroyed. Incidence of Chronic Wasting Disease among elk and deer (2002) Arizona (www.gf.state.az.us/) SUMMARY 2. DNA and RNA are genetic material Transformation with DNA T2 DNA not Protein TMV RNA PRIONS (Proteins are transmissible agent) Prions are not DNA or RNA – not Genes! CHAPTER 4, PART 1: LECTURE TOPICS 1. DNA/RNA structures, nomenclature, shorthand conventions 2. DNA and RNA as genetic material 3. General properties of DNA Double Helix 4. Basic mechanism of DNA replication 5. Some important physical and chemical properties of DNA Lysed E. coli cell reveals chromosomal DNA DNA E. Coli chromosome size: 4.6x106 base pairs DNA molecules are usually double helices Double helix X-ray diffraction from a hydrated DNA-B fiber. 3.4 Å Sugar Base The central cross is diagnostic of a helical structure. The strong arcs on the meridian arise from the stack of base pairs DNA Base Composition: A=T G=C =1 =1 % GC Low High (genes) A=T G=C DNA: usually double-stranded with antiparallel strands 5` C T 5’ G 3` A G=C A=T 3’ 5’ 3’ 3` 5` Watson-Crick model B-DNA A space-filling model of B-DNA from x-ray data [Fig. 4.11a] Bases Minor groove Major groove Right handed helix 10 bp/turn; 3.4 A/bp Top view, looking down the helical axis [Figs 4.11b and 4.13] Deoxyribose/ phosphate backbone Bases Hydrogen bonds between paired bases C G A T Compare with a chapter 25 slide showing groups involved in base pairing C=O NH2 A-T base pair (A-U) in RNA T A Ring N atom 2 H-bonds NH2 G-C base pair 3 H-bonds C C=O G 3 Base pair stack in DNA Sugars are perpendicular to the bases Sugar 3` Phosphate Sugar 3` Bases are parallel SUMMARY 3. General properties of Watson-Crick DNA Double helix Antiparallel strands Right-handed helix 10 bp / helix turn, 3.4A/bp Bases on inside and parallel Bases perpendicular to the deoxyribose-P chain A=T and G=C base pairs CHAPTER 4, PART 1: LECTURE TOPICS 4. Basic mechanism of DNA replication Buoyant density of DNA Distance from axix of rotation 1.68 1.7 Density (g/cc) 1.72 Use gradient of CsCl of same density range as DNA Buoyant density of DNA N14 DNA N15 DNA % GC 1.68 1.7 Density (g/cc) 1.72 Use gradient of CsCl of same density range as DNA E. Coli cells grown in media that contains 14N- or 15N-NH4+ 14N 15N UV photo of DNA Centrifugal force or DNA buoyant density (g/cc) or CsCl concentration Photo density DNA replication is semiconservative (1958) [Meselson and Stahl] CsCl 15N Start 15N 15N/14N Transfer to 14N medium 1 cell division 15N/14N 14N 15N/14N 2 cell divisions Half 15N/14N and Mix half 14N/14N 14N 15N (2) 14N DNA replication E.Coli DNA polymerase I requires: 1. All four dNTPs (dATP, dGTP, dCTP and dTTP) 2. A primer chain with a free 3`-OH end 3. A template strand to which the primer is basepaired • Double-stranded DNA that is fully intact and lacking a free 3`-OH end will not be replicated (Ex: Intact circular DNA) 4. Mg2+ Draw a template-primer complex to learn how it works. DNA synthesis: DNA Polymerase Reaction (DNA)n + dNTP Primer 2Pi (DNA)n+1 + PPi 5` n+1 3` 2Pi 2Pi Template DNA chain growth is 5’ to 3’ 5` n+2 3` DNA Polymerase Reaction Mechanism: A nucleophilic attack of the 3`-OH on the α–P of dNTP 3` γ 3` α β 5` Nucleophilic attack 5` New 3` 5` phosphodiester bond DNA REPLICATION DNA template-primer complex for DNA polymerase 3` Template DNA 5` Primer DNA Direction of chain growth 5` 3` 5` GA CT 3`-OH dCTP dTTP…..etc 1 2 PPi 2Pi Accuracy: 1 “permanent” mistake every 108 bases Summary of basic mechanism of DNA replication Replication is semiconservative DNA polymerase requires a template-primer complex dNTPs are the substrates for DNA synthesis PPi breakdown to 2 Pi (catalyzed by pyrophosphatase) drives DNA synthesis DNA Polymerase accuracy: 1 mistake every 108 bases CHAPTER 4, PART 1: LECTURE TOPICS 5) Some important physical and chemical properties of DNA Some important physical-chemical properties of DNA Buoyant density of DNA is proportional to %G-C bp Reversible separation-reassociation of DNA strands Denature/Renature/melting temperature (Tm) proportional to %G-C bp) Enormous range of lengths [:m to cm lengths] Conformation of DNA can be: Linear circle (open/supercoiled) single (SS) or double- stranded (DS) Buoyant density of DNA Why would a G-C base pair have a higher density than an A-T base pair? N14 DNA % GC 1.68 1.7 Density (g/cc) 1.72 Use gradient of CsCl of same density range as DNA C=O NH2 A-T base pair (A-U) in RNA T A Ring N atom 2 H-bonds NH2 G-C base pair 3 H-bonds C C=O G DNA reversible strand separation Melting (denature; break H-bonds) Heat ordered structure Cool (<A260) + disordered structure (>A260) Reannealing: renature; reform H-bonds (zipper analogy) (Denatured) disordered” U.V. light absorbance “Ordered” (Native d.s.) Melting of DNA is reversible 50% of base pairs “broken” (denatured) A-T rich A-T pairs melt first G-C rich G-C pairs more stable Tm (50% base paired) DNA melting temperature DNA Size Definitions: 1 kb (kilobase) of DNA or RNA = 1000 bases singlestranded (1 kb) or double-stranded (1 kbp). [p=pair] 1 kb double-stranded DNA = 0.34 :m long 1 kb double-stranded DNA = 660 kd [660 d/base pair] ENORMOUS SIZE RANGES OF DNA GENOMES 1.7 micron (circle) 1.36 mm (circle) 99 cm (linear) CIRCULAR DNAs Mitochondria Other circular DNA’s: Plasmids Bacterial Chromosomes (E. coli) Chloroplasts Open Supercoiled SUMMARY 1. DNA/RNA structures Know nomenclature + shorthand conventions SUMMARY 2. DNA and RNA as genetic material Transformation with DNA T2 DNA not Protein TMV RNA PRIONS (Proteins as transmissible agent) SUMMARY 3. General properties of Watson-Crick DNA Double helix Antiparallel strands Right-handed helix 10 bp / helix turn, 3.4A/bp Bases on inside and parallel Bases perpendicular to the deoxyribose-P chain A=T and G=C base pairs SUMMARY 4. Basic mechanism of DNA replication Semiconservative DNA polymerase I (template-primer complex) PPi 2Pi drives DNA synthesis SUMMARY 5. Some important physical-chemical properties of DNA Buoyant density (proportional to %G-C) Denature-renature-melting (Tm proportional to %G-C) :m to cm lengths Linear, circles (open/supercoiled), SS, DS If lined up end to end, How long a line would All the DNA in your body make?? To: Phoenix Los Angeles Moon and Back Pluto and Back Sun and Back 6 times Around the world One way to Mars If lined up end to end, How long a line would All the DNA in your body make?? To: Phoenix Los Angeles Moon and Back Pluto and Back Sun and Back 6 times Around the world One way to Mars ...
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This note was uploaded on 12/16/2011 for the course BIOLOGY 101 taught by Professor Mr.wallace during the Fall '11 term at Montgomery College.

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