2Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation Frederick Griffiths was a bacteriologist studying pneumoniaHe discovered two types of bacteria:Smooth coloniesRough colonies:
3Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation When heat was applied to the deadly smooth type…And injected into a mouse…The mouse lived!
4Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation Griffith injected the heat-killed type and the non-deadly rough type of bacteria.The bacteria “transformed” itself from the heated non-deadly type to the deadly type.
5Griffith’s Experiment did not prove that DNA was responsible for transformation How would you design an experiment to prove that DNA was responsible for transformation?
6To the Heat-Killed Smooth Type, added enzymes that destroyed… Avery, McCarty, and MacLeod Added the non-deadly Rough Type of Bacteria to the Heat-Killed Smooth TypeTo the Heat-Killed Smooth Type, added enzymes that destroyed…CarbohydratesLipidsProteinsRNADNA
7DNA was the transforming factor! S-Type Carbohydrates DestroyedS-Type Lipids DestroyedS-Type Proteins DestroyedS-Type RNA DestroyedS-Type DNA DestroyedConclusion:DNA was the transforming factor!
8The Hershey-Chase Experiment Protein coatAlfred Hershey & Martha Chase worked with a bacteriophage:A virus that invades bacteria. It consists of a DNA core and a protein coatDNA
9Protein coats of bacteriophages labeled with Sulfur-35 Hershey and Chase mixed the radioactively-labeled viruses with the bacteriaBacteriumPhageThe viruses infect the bacterial cells.BacteriumDNA of bacteriophages labeled with Phosphorus-32
10Protein coats of bacteriophages labeled with Sulfur-35 Separated the viruses from the bacteria by agitating the virus-bacteria mixture in a blenderDNA of bacteriophages labeled with Phosphorus-32
11Protein coats of bacteriophages labeled with Sulfur-35 Centrifuged the mixture so that the bacteria would form a pellet at the bottom of the test tubeMeasured the radioactivity in the pellet and in the liquidDNA of bacteriophages labeled with Phosphorus-32
12How does DNA replicate? Hypotheses: Conservative Semi-Conservative Dispersive
13Meselson-Stahl Experiment Bacteria cultured in medium containing a heavy isotope of Nitrogen (15N)
14Meselson-Stahl Experiment Bacteria transferred to a medium containing elemental Nitrogen (14N)
15Meselson-Stahl Experiment DNA sample centrifuged after First replication
16Meselson-Stahl Experiment DNA sample centrifuged after Second replication
17DNA 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 end3. A template strand to which the primer is basepaired• Double-stranded DNA that is fully intact andlacking a free 3`-OH end will not be replicated(Ex: Intact circular DNA)4. Mg2+
18DNA synthesis: DNA Polymerase Reaction (DNA)n + dNTP (DNA)n+1 + PPi 2PiPrimer5` n+1→→ 3`5` n+2 →3`TemplateDNA chain growth is 5’ to 3’
19DNA polymerase requires a template-primer complex Summary of basic mechanism of DNA replicationReplication is semiconservativeDNA polymerase requires a template-primer complexdNTPs are the substrates for DNA synthesisPPi breakdown to 2 Pi (catalyzed by pyrophosphatase) drives DNA synthesisDNA Polymerase accuracy: 1 mistake every 108 bases