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Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation Frederick Griffiths was a bacteriologist studying pneumonia He discovered.

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Presentation on theme: "Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation Frederick Griffiths was a bacteriologist studying pneumonia He discovered."— Presentation transcript:

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2 Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation Frederick Griffiths was a bacteriologist studying pneumonia He discovered two types of bacteria: –Smooth colonies –Rough 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 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. Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation

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 Avery, McCarty, and MacLeod Added the non-deadly Rough Type of Bacteria to the Heat-Killed Smooth TypeCarbohydratesLipidsProteinsRNADNA To the Heat-Killed Smooth Type, added enzymes that destroyed…

7 S-Type Carbohydrates Destroyed S-Type Lipids Destroyed S-Type Proteins Destroyed S-Type RNA Destroyed S-Type DNA Destroyed Conclusion: DNA was the transforming factor!

8 The Hershey-Chase Experiment Alfred Hershey & Martha Chase worked with a bacteriophage: A virus that invades bacteria. It consists of a DNA core and a protein coat DNA Protein coat

9 Protein coats of bacteriophages labeled with Sulfur-35 DNA of bacteriophages labeled with Phosphorus-32 Bacterium Bacterium Phage Phage 1.Hershey and Chase mixed the radioactively-labeled viruses with the bacteria The viruses infect the bacterial cells.

10 Protein coats of bacteriophages labeled with Sulfur-35 DNA of bacteriophages labeled with Phosphorus-32 2.Separated the viruses from the bacteria by agitating the virus- bacteria mixture in a blender

11 Protein coats of bacteriophages labeled with Sulfur-35 DNA of bacteriophages labeled with Phosphorus-32 3.Centrifuged the mixture so that the bacteria would form a pellet at the bottom of the test tube 4.Measured the radioactivity in the pellet and in the liquid

12 How does DNA replicate? Hypotheses: ConservativeSemi-ConservativeDispersive

13 1.Bacteria cultured in medium containing a heavy isotope of Nitrogen ( 15 N) Meselson-Stahl Experiment

14 2.Bacteria transferred to a medium containing elemental Nitrogen ( 14 N) Meselson-Stahl Experiment

15 3.DNA sample centrifuged after First replication

16 Meselson-Stahl Experiment 4.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 end 3. A template strand to which the primer is basepaired Double-stranded DNA that is fully intact and Double-stranded DNA that is fully intact and lacking 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 2Pi Primer 5` n+1 →→ 3` 5` n+2 → 3` Template DNA chain growth is 5’ to 3’

19 Summary of basic mechanism of DNA replication Replication is semiconservative DNA polymerase requires a template-primer complex dNTPs are the substrates for DNA synthesis PPi breakdown to 2 Pi (catalyzed by pyrophosphatase) drives DNA synthesis DNA Polymerase accuracy: 1 mistake every 108 bases


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