8/13/2016 Chapter 16 DNA molecule responsible for all cell activities DNA and contains the genetic code The Molecular Basis of Genetic ic Code method cells use to store the program that is passed from one Inheritance generation to another 1928: Frederick Griffith DISCOVERY OF THE GENETIC CODE 1. Grew 2 strains of bacteria on Experiment 1 plates - smooth colonies- caused disease (virule lent) 1928: Frederick Griffith - rough edge colonies-did not cause disease (aviru rule lent) Transformation 2. Injected into mice Results: - smooth colonies: died - rought colonies: lived Conclusion: bacteria didn’t produce a toxin to kill mice After Experiment 1. Injected mice with heat Experiment 2 killed virulent strain Cultured bacteria from dead mice and they grew 2. Injected mice with virulent strain. non -virulent strain + heat killed virulent strain Results: Griffith hypothesized that a factor was - heat killed: lived transferred from heat killed cells - mixed strains: mice developed pneumonia to live cells . Conclusion: heat killed virulent strain TRANS NSFOR FORMA MATIO ION passed disease causing abilities to non virulent strain 1
8/13/2016 1952: Hershey / Chase 1944: Avery (et al) - studied how viruses (bacteriophage) affect bacteria. 1. Repeated Griffith’s experiment with same results. Bacterio riophage ge - result: transformation occurred Virus composed of DNA core and protein coat that infect bacteria 2. Did a second experiment using enzymes that would destroy RNA. - result: transformation occurred 3. Did third experiment using enzymes that would destroy DNA. - result: no transformation CONCLUSION DNA was transforming factor Hershey Chase Experiment DISCOV OVERY RY OF STRUCTU TURE OF DNA 1. They labeled virus protein coat with radioactive sulfur 2. They labeled virus DNA with radioactive phosphorous Result lt observed that bacteria had phosphorous *** virus injected bacterial cells with its phosphorous labeled DNA*** Conclu clusion DNA carried genetic code since bacteria made new DNA. animation Early 1950’s: Rosalind Franklin (English) Same time period: x ray cryst stallogra graphy hy eviden dence ce: Chargaff (American biochemist) X pattern showed that fibers of DNA twisted and Chargaff’s Rule: molecules are spaced at regular intevals on length fiber. Maurice Wilkins: x ray diffraction, worked with Franklin 2
8/13/2016 DNA 1953 Watson (American) & Crick (English) - double strand of nucleotides **double helix model** won Nobel prize in 1962 - may have 1000’s of nucleotides in 1 strand (very long molecule) Discovered the double - bases join up in specific (complementary) pairs: helix by building models • complementary pairs (base pairing rules) to conform to Franklin’s X- ray data and Chargaff’s 1 purine bonds with 1 pyrimid idin ine on one rung of Rules the ladder connected by a weak H bond C - G A – T Order r of nucleotid ides not import rtant, proper r comple lementary ry bases must be paired. Nucleotide Structure STRUCT CTURE URE OF DNA Backbone Purines Pyrimidines Phosphate + Deoxyribose sugar (5 C) Rungs 4 Nitrogenous bases - Purines Adenine A Guanine G Sugar - Pyrimidines Base Thymine T Cytosine C Phosphate D bases attached to sugar E. bases attached to each other by weak H bond DNA Comparison DNA makes up Chromosomes (chromatin packing) Prokaryotic DNA Eukaryotic DNA • Double-stranded • Double-stranded • Circular • Linear • One chromosome • Usually 1+ chromosomes • In cytoplasm • In nucleus • No histones • DNA wrapped around histones (proteins) • Supercoiled DNA • Forms chromatin 3
8/13/2016 REPLIC ICATION ION OF DNA DNA REPLICATION Process of duplication of DNA - Before cell can divide a new copy of DNA must be made for the new cell - Semiconser ervative ive replication ion: each strand acts as a template (pattern) for new strand to be made End Result lt: one old strand, one new daughter strand Discovery of Replication Model Models of DNA Replication Meselson and Stahl • Cultured E coli with heavy isotope 15 N for many generations. • Transferred to light isotope 14 N • Sampled after first and second replication • Removed bacteria and extracted DNA • Centrifuged to separate different density DNA Steps of Replication Meselson and Stahl 1. Enzyme DNA helica case attaches to DNA molecule at origins of replication and breaks H bonds so strand unwinds • Compared results to - single strand binding proteins bind to unpaired bases to keep models of replication them from re-binding - topoisomerase relieves additional strain on forward DNA by breaking and rejoining DNA strands • 1 st replication: Hybrid DNA (14 and 15) - replica icatio ion forks: two areas on either end of the DNA where -Eliminated double helix separates conservative model - forms replica catio ion bubble: “bubble” under electron microscope • 2 nd replication: Light and hybrid DNA - Eliminated dispersive model - Supported semi-conservative model of replicaiton. 4
8/13/2016 DNA Directionality 2. enzyme RNA Primase lays down an RNA primer on DNA strand antiparallel strands - RNA primer segment signals beginning of replication 3’ 5’ 3. Enzyme DNA polymera rase III moves along each of DNA strand and adds complementary bases of nucleotides floating freely in nucleus A. DNA polymerase III begins synthesis at RNA primer segment B. DNA polymerase I replaces 5’ RNA primers with DNA 3’ nucleotides - Directionality: DNA polymerase III reads the template in the 3’ to 5’ direction But if there exist no DNA polymerases capable of polymerizing DNA in the Daughter DNA strand 3' to 5' direction, how could this be? (since it is complementary) must be synthesized in the 5’ to 3’ direction Strands are antiparallel. Discontinuous synthesis - synthesis only occurs when a large amount of single strand D. DNA ligase stitches DNA is present together Okazaki - daughter DNA is then synthesized fragments into a in 5’ to 3’ direction single, unfragmented daughter molecule - leading and lagging strands: E. enzyme chops off - leading strand – continuously synthesized DNA strand RNA primer and replaces it with DNA - lagging strand - delayed, fragmented, daughter DNA - Okazaki fragments- discontinuous fragmented DNA segments 5
8/13/2016 3. DNA polymerase III catalyzes formation of H bonds between nucleotides of template and newly arriving nucleotides which will form daughter DNA 4. Once all DNA is copied, daughter DNA detaches Animation DNA Replication Fork Okazaki fragment animation End Replication Problem Solution to End Replication Problem telomere res: repeated units of non-coding short nucleotide sequences -On one end, RNA primer (TTAGGG) at ends of DNA cannot be replaced with - become shorter with repeated cell divisions DNA because it is a 5’ - once telomeres are gone, coding sections of chrom. are lost and cell does not have enough DNA to (DNA polymerase III function can only read from 3’ to 5’) ***telomere theory of aging*** - Causes daughter DNA’s - telomera rase: special enzyme that contains an RNA template to be shorter with each molecule so that telomeres can be added back on to DNA replication (cell division) (rebuilds telomeres) ** found in: Cancer cells - immortal in culture Stem cells ** not found in most differentiated cells Telomeres and Telomerase Speed of Replication • Multiple replication forks- replication occurs simultaneously on many points of the DNA molecule • Would take 16 days to replicate 1 strand from one end to the other on a fruit fly DNA without multiple forks • Actually takes ~ 3 minutes / 6000 sites replicate at one time • Human chromosome replicated in about 8 hours with multiple replication forks working together 6
8/13/2016 Nucleotide Excision Repair Accuracy and Repair • DNA polymerases proofread as bases are added - can remove damaged nucleotides and replace Errors: with new ones for accurate replication Pairing errors: 1 in 100,000 nucleotides • Mismatch repair: special enzymes fix incorrect pairings Complete DNA: 1 in 10 • Nucleotide excision repair: billion nucleotides • Nucl clea eases ses cut damaged DNA • DNA polymerase and ligase fill in gaps RNA does not have this ability- reason RNA viruses mutate so much Importance of DNA 1. Controls formation of all substances in the cell by the genetic code 2. Directs the synthesis of specific strands of m RNA to make proteins RNA A (Ribonucleic acid) Another nucleic acid takes orders from DNA Used in protein synthesis 7
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