Presentation on theme: "Griffith (Frederick) Hershey and Chase (Alfred and Martha)"— Presentation transcript:
1 We will focus on these 3 classic experiments highlighted in this chapter…… Griffith (Frederick)Hershey and Chase (Alfred and Martha)Meselsen and Stahl (Matt and Frank)
2 There are unknown heritable substances… GriffithThere are unknown heritable substances…Turned to a bacterial pathogen…Streptococcus pneumoniaeIs it gram positive or negative?
3 Work by Avery identified the transforming substance as DNA Figure 13.2ExperimentMixture ofheat-killedS cells andliving R cellsLivingS cells(control)LivingR cells(control)Heat-killedS cells(control)Figure 13.2 Inquiry: Can a genetic trait be transferred between different bacterial strains?ResultsMouse diesMouse healthyMouse healthyMouse diesWork by Avery identified the transforming substance as DNALiving S cells
4 Transformation-did not really understand mechanism Can we do this-pick up DNA from our environment?
5 Hershey and Chase (1952)Their work pointed to DNA rather than proteins…Bacteriophages what are they???? (worked with one called T2)
6 Some phages grown in media for a couple hrs with radioactive Sulphur…(which should be incorporated into some proteins Methionine, Cysteine)Other phages grown in media for a couple hrs with radioactive Phosphorus….(which should be incorporated into DNA)
7 Batch 1: Radioactive sulfur (35S) in phage protein Figure 13.4ExperimentBatch 1: Radioactive sulfur (35S) in phage protein1Labeled phagesinfect cells.2Agitation frees outsidephage parts from cells.3Centrifuged cellsform a pellet.Radioactiveprotein4Radioactivity(phage protein)found in liquidCentrifugePelletBatch 2: Radioactive phosphorus (32P) in phage DNARadioactiveDNAFigure 13.4 Inquiry: Is protein or DNA the genetic material of phage T2?Centrifuge4Radioactivity (phageDNA) found in pelletPellet7
8 Watson-Crick Model predicted…. Each of two daughter molecules would have one parental strand and one newly made!Meselson and Stahl-clever experiment…What did they do??
9 Experiment Bacteria cultured in medium with 15N (heavy isotope) Figure 13.11Experiment1Bacteriacultured inmediumwith 15N(heavyisotope)2Bacteriatransferredto mediumwith 14N(lighterisotope)Results3DNA samplecentrifugedafter firstreplication4DNA samplecentrifugedafter secondreplicationLessdenseMoredenseConclusionPredictions:First replicationSecond replicationConservativemodelFigure Inquiry: Does DNA replication follow the conservative, semiconservative, or dispersive model?SemiconservativemodelDispersivemodel9
10 Figure 13.1Watson and CrickFigure 13.1 How was the structure of DNA determined?
11 What do these terms refer to… How does this replication thing work??origin of replicationhelicasetopoisomerasereplication forkprimase and the RNA primersingle stranded binding proteinsDNA polymeraseSearch online for stronger and weaker video clips-which one is the very best and the very worst?me the links to your very best and worst (with your group members names)Jot down on the board enough of the web address that we can distinguish which ones are the same
12 1 2 5 end 3 end 3 end 5 end T A G C C G A T Figure 13.7b Figure 13.7b The double helix (part 2: chemical structure)AT3 end5 end12
14 Overall directions of replication Figure 13.15OverviewLeadingstrandOrigin of replicationLaggingstrandPrimerLeadingstrandLagging strandOveralldirectionsof replicationOrigin of replication355RNA primer33Sliding clampDNA pol IIIParental DNAFigure Synthesis of the leading strand during DNA replication5355Continuous elongationin the 5 to 3 direction33514
15 Overall directions of replication Figure 13.16aOverviewLaggingstrandOrigin of replicationLeadingstrandLaggingstrandLeadingstrandOverall directionsof replicationFigure 13.16a Synthesis of the lagging strand (part 1)What is going to latch on at #1?15
16 Primase makes RNA primer. Template strand 1 3 5 3 5 Figure 13.16b-11Primase makesRNA primer.353Templatestrand5Figure 13.16b-1 Synthesis of the lagging strand (part 2, step 1)16
17 Primase makes RNA primer. Template strand RNA primer DNA pol III Figure 13.16b-21Primase makesRNA primer.353Templatestrand5RNA primerfor fragment 12DNA pol IIImakes Okazakifragment 1.3535Figure 13.16b-2 Synthesis of the lagging strand (part 2, step 2)17
18 Where is DNA pol III going to go next?? Figure 13.16b-31Primase makesRNA primer.353Templatestrand5RNA primerfor fragment 12DNA pol IIImakes Okazakifragment 1.3535Figure 13.16b-3 Synthesis of the lagging strand (part 2, step 3)3DNA pol IIIdetaches.3Okazakifragment 1535Where is DNA pol III going to go next??18
19 Figure 13.16c-1RNA primer for fragment 2Okazakifragment 254DNA pol IIImakes Okazakifragment 2.335Now you have all these bits what has to happen next? And who does that?Figure 13.16c-1 Synthesis of the lagging strand (part 3, step 1)19
20 RNA primer for fragment 2 Figure 13.16c-2RNA primer for fragment 2Okazakifragment 254DNA pol IIImakes Okazakifragment 2.33555DNA pol Ireplaces RNAwith DNA.335Figure 13.16c-2 Synthesis of the lagging strand (part 3, step 2)20
21 RNA primer for fragment 2 Figure 13.16c-3RNA primer for fragment 2Okazakifragment 254DNA pol IIImakes Okazakifragment 2.33555DNA pol Ireplaces RNAwith DNA.3356DNA ligase formsbonds betweenDNA fragments.Figure 13.16c-3 Synthesis of the lagging strand (part 3, step 3)5335Overall direction of replication21
22 Overall directions of replication Figure 13.16OverviewLaggingstrandOrigin of replicationLeadingstrandLaggingstrandLeadingstrandRNA primerfor fragment 2Overall directionsof replicationOkazakifragment 254DNA pol IIImakes Okazakifragment 2.31Primase makesRNA primer.3533Templatestrand5555DNA pol Ireplaces RNAwith DNA.3RNA primerfor fragment 12DNA pol IIImakes Okazakifragment 1.3Figure Synthesis of the lagging strand53356DNA ligase formsbonds betweenDNA fragments.5533DNA pol IIIdetaches.3Okazakifragment 153535Overall direction of replication22
23 Leading strand template Single-strand binding proteins Leading strand Figure 13.17OverviewOrigin of replicationLeading strandtemplateLeading strandLaggingstrandSingle-strandbinding proteinsLagging strandLeading strandOverall directionsof replicationLeading strandHelicaseDNA pol III53Primer53Primase3Parental DNALagging strandDNA pol IIIFigure A summary of bacterial DNA replication5Lagging strandtemplate3DNA pol IDNA ligase53523
24 Overall directions of replication Figure 13.17aOverviewOrigin of replicationLeading strandLaggingstrandLagging strandLeading strandFigure 13.17a A summary of bacterial DNA replication (part 1)Overall directionsof replication24
25 Leading strand template Single-strand binding proteins Leading strand Figure 13.17bLeading strandtemplateSingle-strandbinding proteinsLeading strandHelicaseDNA pol III5Primer35Primase33Figure 13.17b A summary of bacterial DNA replication (part 2)Parental DNALagging strandtemplate25
26 Lagging strand DNA pol III 5 3 DNA pol I DNA ligase 5 3 5 Figure 13.17cLagging strandDNA pol III53DNA pol IDNA ligase535Figure 13.17c A summary of bacterial DNA replication (part 3)26
27 DNA poly- merase Pyro- phosphate Figure 13.14New strandTemplate strand5353SugarATATBasePhosphateCGCGDNApoly-meraseGCGC3ATATFigure Addition of a nucleotide to a DNA strandPPPPiPC3CPyro-phosphateNucleotide552Pi27
28 Question 1.True of Leading strand, Lagging strand, or Both????Daughter strand elongates away from replication forkSynthesizes 5’ to 3’Multiple primers neededMade in segmentsMade continuouslyDaughter strand elongates toward replication fork
29 True of Leading strand, Lagging strand, or Both???? Daughter strand elongates away from replication fork LagSynthesizes 5’ to 3’ BothMultiple primers needed LaggMade in segments LagMade continuously LeadDaughter strand elongates toward replication fork from Lead
30 Question 2. The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands.Rank the primers in the order they were produced. If two primers were produced at the same time, overlap them.
31 Question 3. The lagging strand is synthesized as a series of segments called Okazaki fragments Fragment A is the most recently synthesized and Fragment B will be synthesized next in the space between primers A and B.-----Start DNA polymerase III binds to 3’ end of primer BA. DNA polymerase I replaces primer with DNAB. DNA polymerase I binds to 5’ end of primer AC. DNA polymerase III moves 5’ to 3’ adding DNA nucleotides to primer BD. DNA ligase links fragments A and B
32 In an analysis of the nucleotide composition of DNA, which of the following will be found? A = G and C = TG + C = T + AA = CA + C = G + T
33 Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine?31%42%8%16%It cannot be determined from the information provided.