2 Griffith’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:
3 Griffith’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!
4 Griffith’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.
5 Griffith’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?
6 To 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
7 DNA 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!
8 The 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
9 Protein 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
10 Protein 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
11 Protein 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
12 How does DNA replicate? Hypotheses: Conservative Semi-Conservative Dispersive
13 Meselson-Stahl Experiment Bacteria cultured in medium containing a heavy isotope of Nitrogen (15N)
14 Meselson-Stahl Experiment Bacteria transferred to a medium containing elemental Nitrogen (14N)
15 Meselson-Stahl Experiment DNA sample centrifuged after First replication
16 Meselson-Stahl Experiment DNA sample centrifuged after Second replication
17 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 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+
18 DNA 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’
19 DNA 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