Transformation-Griffith’s Expt

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Transformation-Griffith’s Expt. 1928. DNA Mediates Transformation. Convert IIR to IIIS By DNA?. Avery MacLeod and McCarty Experiment. Circa 1943. Transforming Principle. DNAse activity. + means that activity is present. All RNA gets degraded during enzyme preparation.
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Transformation-Griffith’s Expt1928DNA Mediates TransformationConvert IIR to IIIS By DNA? Avery MacLeod and McCarty ExperimentCirca 1943Transforming Principle DNAse activity+ means that activity is presentAll RNA gets degraded during enzyme preparationA-DNA, B-DNA and Z-DNAThe Z-DNA helix is left-handed and has a structure that repeats every 2 base pairs. The major and minor grooves, unlike A- and B-DNA, show little difference in widthNon-B DNA in diseaseChapter 10Replication of DNA and ChromosomesDNA Replication is Semiconservative
  • Each strand serves as a template
  • Complementary base pairing determines the sequence of the new strand
  • Each strand of the parental helix is conserved
  • Possible Modes ofDNA ReplicationThe Meselson-Stahl Experiment:DNA Replication in E. coli is SemiconservativeVisualization of Replication in E. coliReplication in E. coliNote:OriC is 245bpThe Origin of Replication in E. coliThe Core Origin of Replication in SV 40Prepriming at oriC in E. coliDNA Polymerases and DNA Synthesis In VitroRequirements of DNA Polymerases
  • Primer DNA with free 3'-OH
  • Template DNA to specify the sequence of the new strand
  • Substrates: dNTPs
  • Mg2+ (where?)
  • Nucleophilic attack of alpha phosphate whichreleases pyrophosphateMg2+ (where?)bgDNA Polymerase I:5'3' Polymerase ActivityOften called: Kornberg PolymeraseDNA Polymerase I:5'3' Exonuclease ActivityCleaves ahead of itselfDNA Polymerase I:3'5' Exonuclease ActivityProofreadingKlenow fragment…..is?DNA Polymerases
  • Polymerases in E. coli
  • DNA Replication: DNA Polymerases III and I
  • DNA Repair: DNA Polymerases II, IV, and V
  • Polymerases in Eukaryotes
  • Replication of Nuclear DNA: Polymerase  and/or 
  • Replication of Mitochondrial DNA: Polymerase 
  • DNA Repair: Polymerases and
  • All of these enzymes synthesize DNA 5' to 3' and require a free 3'-OH at the end of a primer
  • DNA Polymerase III is the True DNA Replicase of E. coliDNA replication is a complex process, requiring the concerted action of a large number of proteins.E. coli DNA Polymerase III HoloenzymeReplication in E. coliNote:OriC is 245bpThe Origin of Replication in E. coliPrepriming at oriC in E. coliDNA Replication
  • Synthesis of the leading strand is continuous.
  • Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together.
  • What’s wrong with this picture?RNA Primers are Used to Initiate DNA SynthesisDNA Helicase Unwinds the Parental Double HelixDNA Ligase Covalently Closes Nicks in DNADNA ligase forms a high energy intermediate that Aside:Calf Intestinal Phosphotase?Cut with EcoR1GAATTCCTTAAGG-OHp-AATTCCTTAA-pHO-GCalf Intestinal Phosphotase?Cut with EcoR1G-OHp-AATTCCTTAA-pHO-GG-OHHO-AATTCCTTAA-OHHO-GCalf Intestinal Phosphotase?Cut with EcoR1p-AATTCgatacagagagactcatgacgG-OHHO-GctatgtctctctgagtactgcCTTAA-pG-OHHO-AATTCCTTAA-OHHO-GVector won’t religate,But will take in insertSingle-Strand DNA Binding (SSB) ProteinSupercoiling of Unwound DNADNA Topoisomerase I Produces Single-Strand Breaks in DNADNA Topoisomerase II Produces Double-Strand Breaks in DNAThe Replication Apparatus in E. coliThe E. coli ReplisomeDNA Replication in Eukaryotes
  • Shorter RNA primers and Okazaki fragments
  • DNA replication only during S phase
  • Multiple origins of replication
  • Telomeres
  • Bidirectional Replication from Multiple Origins in EukaryotesThe Eukaryotic ReplisomeDNA polymerase -DNA primase—initiation; priming of Okazaki fragmentsDNA polymerase —processive DNA synthesisDNA polymerase —DNA replication and repair in vivoPCNA (proliferating cell nuclear antigen)—sliding clampReplication factor-C Rf-C)—loading of PCNARibonuclease H1 and Ribonuclease FEN-1—removal of RNA primersEukaryotic Replication ProteinsThe E. coli ReplisomeThe Telomere ProblemTelomeraseTelomere Length and Aging
  • Most human somatic cells lack telomerase activity.
  • Shorter telomeres are associated with cellular senescence and death.
  • Diseases causing premature aging are associated with short telomeres.
  • BACsGeometric Doubling Progression12481632641282565121024=103=210….10 more doublings is another 210So 20 doublings is 220=103+3=106So 30 doublings is 230=103+3+3=109So 40 doublings is 240=103+3+3+3=1012Molecular Weight of NucleosidessBase plus riboseSingle phosphate330 Da= 330g/mol/nt (nucleotide)660 Da= 660g/mol/bp (base pair)Molecular Weight of Plasmid DNA330 Da= 330g/mol/nt (nucleotide)660 Da= 660g/mol/bp (base pair)For 3000bp of DNA (a starting plasmid vector)3000 bp x 660 g/mol/bp= 1000 x 3 x 660 = 1x 103 x 2 x 103 = 2 x 106 g/mol for a 3kb plasmid2 x 106 g/mol is how many grams per molecule6 x 1023 molecules/molThus 2 x 106 / 6 x 1023 = g/molecules1g/ 3 x 1017 molecules for a given 3kb plasmid2 x 106 g/mol is how many grams per molecule6 x 1023 molecules/molThus 2 x 106 / 6 x 1023 = g/molecules1g/ 3 x 1017 molecules for a given 3kb plasmid1g/ 3 x 1017 molecules is the same as1mg/ 3 x 1014 molecules1ug/ 3 x 1011 molecules1ng/ 3 x 108 molecules1pg/ 3x 105 molecules1fg/ 3 x 102 (300) moleculesIf each bacterium can hold 3000 molecules, then eachBacterium makes 10fg of plasmid DNAIf one makes 1mg of plasmid DNA, then this is 1012 fg as wellIf each bacterium can hold 3000 molecules, then eachBacterium makes 10fg of plasmid DNAIf one makes 1mg of plasmid DNA, then this is 1012 fg as wellSince each bacterium has 10fg DNA, then only 1011Are needed to produce 1mg DNA.…..So 40 doublings is 240=103+3+3+3=1012236=~101136 doublings for 1011 bacteria3 cell divisions per hour or about 12 hoursWhat are the factors that affect DNA replication?Geometric Doubling Progression12481632641282565121024=103=210….10 more doublings is another 210So 20 doublings is 220=103+3=106So 30 doublings is 230=103+3+3=109So 40 doublings is 240=103+3+3+3=1012
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