Download History_of_DNA

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
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!
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.
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?
Avery, McCarty, and MacLeod
Added the non-deadly Rough Type of
Bacteria to the Heat-Killed Smooth Type
To the Heat-Killed Smooth Type,
added enzymes that destroyed…
Carbohydrates
Lipids
Proteins
RNA
DNA
S-Type
S-Type
S-Type
S-Type
S-Type
Carbohydrates Lipids
Proteins
RNA
DNA
Destroyed Destroyed Destroyed Destroyed Destroyed
Conclusion:
DNA was the
transforming factor!
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
Protein coat
DNA
Protein coats of bacteriophages labeled with Sulfur-35
Phage
1. Hershey and Chase
Bacterium
mixed the
radioactively-labeled
viruses with the
bacteria
Phage
Bacterium
The viruses infect the
bacterial cells.
DNA of bacteriophages labeled with Phosphorus-32
Protein coats of bacteriophages labeled with Sulfur-35
2. Separated the viruses
from the bacteria by
agitating the virusbacteria mixture in a
blender
DNA of bacteriophages labeled with Phosphorus-32
Protein coats of bacteriophages labeled with Sulfur-35
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
DNA of bacteriophages labeled with Phosphorus-32
How does DNA replicate?
Hypotheses:
Conservative
Semi-Conservative
Dispersive
Meselson-Stahl Experiment
1. Bacteria cultured in medium containing
a heavy isotope of Nitrogen (15N)
Meselson-Stahl Experiment
2. Bacteria transferred to a medium
containing elemental Nitrogen (14N)
Meselson-Stahl Experiment
3. DNA sample centrifuged after First
replication
Meselson-Stahl Experiment
4. DNA sample centrifuged after Second
replication
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
lacking a free 3`-OH end will not be replicated
(Ex: Intact circular DNA)
4. Mg2+
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’
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