Transcript DNA

DNA:
Prince of the Chemicals
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
Transformation Of Bacteria
Two Strains Of Streptococcus
Rough Strain
(Harmless)
Capsules
Smooth Strain
(Virulent)
©1999 Timothy G. Standish
Transformation Of Bacteria
The Griffith Experiment
OUCH!
+ Control
- Control
- Control
Experimental
©1999 Timothy G. Standish
Avery, MacLeod and McCarty
1944 Avery, MacLeod and McCarty decided to
repeat Griffith’s 1928 experiment and try to
discover the “transforming factor”
They did this by using extracts from the heatkilled cells and digesting specific classes of
molecules with enzymes
Enzyme
Transformation?
Lippase
Yes
Protease
Yes
Saccharase
Yes
Nuclease
No
©1999 Timothy G. Standish
The Hershey-Chase
Experiment
The Hershey-Chase experiment showed
definitively that DNA is the genetic material
Hershey and Chase took advantage of the
fact that T2 phage is made of only two
things: Protein and DNA
OH
H
H2N C C
CH2
CH2
S
CH3
H
O
H2N C C
OH
Methionine
CH2
SH
O
HO P
NH2
O
O
OH
Cysteine
Some amino acids
contain sulfur, thus
proteins contain sulfur,
but not phosphorous.
OH
H
Nucleotides contain phosphorous,
thus DNA contains phosphorous,
but not sulfur.
©1999 Timothy G. Standish
S35
T2 grown in
containing media
incorporate S35
into their proteins
Using S35 Bacteria grown in
T2 attach to bacteria and
inject genetic material
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The supernatant is
radioactive, but the
pellet is not.
Blending causes phage
protein coat to fall off
Did protein enter the bacteria?
Is protein the genetic material?
P32
T2 grown in
containing media
incorporate P32
into their DNA
Using P32 Bacteria grown in
T2 attach to bacteria and
inject genetic material
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The pellet is
radioactive, but the
supernatant is not.
Blending causes phage
protein coat to fall off
Did DNA enter the bacteria?
Is DNA the genetic material?
A Nucleotide
Adenosine Mono Phosphate (AMP)
Phosphate
HO
H+
Nucleotide
OH
P
O
Base
N
H
O
5’CH2
4’
NH2
H
N
O
1’
Sugar
3’
OH
2’
H
OH
N
N
Nucleoside
Purines
NH2
Adenine
N
N
N
O
CH3
(DNA)
N
Guanine
NH
N
Thymine
O
NH2
Uracil
(RNA)
NH
N
O
N
N
Pyrimidines
NH
O
N
O
NH2
Cytosine
N
N
O
Base Pairing
Guanine And Cytosine
-
+
+
+
-
Base Pairing
Adenine And Thymine
+ -
Adenine
-
+
Thymine
Base Pairing
Adenine And Cytosine
+
-
-
Base Pairing
Guanine And Thymine
+
+
P
HO
NH2
O
N
O
CH2
OH
N
N
O
H
N
O
CH2
O
HO
P
O
O
N
O
CH2
OH
H
H2O
NH
N
O
HO
P
O
H
O
NH2
N
O
CH2
O
H
O
H
H2O
N
O
CH2
N
O
O
O
CH2
O
P
HO
H
O
OH
HO
P
NH2
HO
P
O
H
O
HO
O
D
N
A
OH
-
-
-
-
-
-
G
-
3.4 nm
1 nm
-
-
Minor
groove
C
G C
T A
A T
-
The Watson - Crick
Model Of DNA
G C
T A
C G
A T
Major
groove
A T
C G
G C
0.34 nm
T A
-
-
-
-
-
-
-
-
-
-
-
©1999 Timothy G. Standish
-
Large Linear Chromosomes Have
Many Origins Of Replication
Origins of Replication
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
©1999 Timothy G. Standish
Extension - The Replication Fork
5’
3’
3’
5’
3’
5’
5’
3’
5’
Primase
- Makes
RNA
primers
Lagging Strand
Okazaki
fragment
5’
RNA
Primers
3’
5’
Single-strand
binding
proteins Prevent DNA
from reannealing
DNA
Polymerase
5’
3’
Helicase Melts DNA
Leading Strand
5’
3’
©1999 Timothy G. Standish
Extension - Okazaki Fragments
5’
3’
Okazaki Fragment
DNA
Pol.
3’
5’
RNA Primer
DNA Polymerase has 5’ to 3’ exonuclease activity.
When it sees an RNA/DNA hybrid, it chops out the
RNA and some DNA in the 5’ to 3’ direction.
5’
3’
DNA
Pol.
RNA and DNA Fragments
3’
5’
RNA Primer
DNA Polymerase falls off leaving a nick.
5’
3’
Ligase
3’
5’
RNA Primer
Nick
The nick is removed when
DNA ligase joins (ligates) the
DNA fragments.
©1999 Timothy G. Standish
©1999 Timothy G. Standish