Fig. 7-0a - St. Olaf Pages

Report
We will focus on these 3 classic experiments highlighted in this
chapter……
•Griffith (Frederick)
•Hershey and Chase (Alfred and Martha)
•Meselsen and Stahl (Matt and Frank)
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Griffith
There are unknown heritable substances…
Turned to a bacterial pathogen…Streptococcus pneumoniae
Is it gram positive or negative?
http://en.wikipedia.org/wiki/File:Fred_Griffith_and_%22Bobby%2
2_1936.jpg
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Figure 13.2
Experiment
Living
S cells
(control)
Living
R cells
(control)
Heat-killed
S cells
(control)
Mixture of
heat-killed
S cells and
living R cells
Results
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Work by Avery identified the transforming substance as
DNA
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Living S cells
Transformation-did not really understand mechanism
• Can we do this-pick up DNA from our environment?
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Hershey and Chase (1952)
Their work pointed to DNA rather than proteins…
Bacteriophages what are they???? (worked with one called T2)
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Some phages grown in media for a couple hrs with radioactive
Sulphur…(which should be incorporated into some proteins
Methionine, Cysteine)
Other phages grown in media for a couple hrs with radioactive
Phosphorus….(which should be incorporated into DNA)
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Figure 13.4
Experiment
Batch 1: Radioactive sulfur (35S) in phage protein
1 Labeled phages
3 Centrifuged cells
2 Agitation frees outside
infect cells.
form a pellet.
phage parts from cells.
4 Radioactivity
Radioactive
protein
(phage protein)
found in liquid
Centrifuge
Pellet
Batch 2: Radioactive phosphorus (32P) in phage DNA
Radioactive
DNA
Centrifuge
4 Radioactivity (phage
Pellet
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DNA) found in pellet
Watson-Crick Model predicted….
Each of two daughter molecules would have one parental strand and
one newly made!
Meselson and Stahl-clever experiment…What did they do??
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Figure 13.11
Experiment
1 Bacteria
2 Bacteria
cultured in
medium
with 15N
(heavy
isotope)
Results
3
transferred
to medium
with 14N
(lighter
isotope)
DNA sample
centrifuged
after first
replication
Conclusion
Predictions:
First replication
Conservative
model
Semiconservative
model
Dispersive
model
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4 DNA sample
centrifuged
after second
replication
Less
dense
More
dense
Second replication
Figure 13.1
Watson and Crick
http://www.ted.com/talks/james_watson_on_how_he_discovered
_dna.html
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What do these terms refer to…
How does this replication thing work??
•origin of replication
•helicase
•topoisomerase
•replication fork
•primase and the RNA primer
•single stranded binding proteins
•DNA polymerase
Search online for stronger and weaker video clips-which one is the
very best and the very worst?
1.Email me the links to your very best and worst (with your group
members names)
2.Jot down on the board enough of the web address that we can
distinguish which ones are the same
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Figure 13.7b
5 end
3 end
1
2
T
A
G
C
G
C
A
T
3 end
5 end
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Figure 13.12
Primase
Topoisomerase
3
3
5
5
RNA
primer
Replication
fork
3
Helicase
5
Single-strand binding
proteins
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Figure 13.15
Overview
Leading
strand
Origin of replication
Lagging
strand
Primer
Lagging strand
Overall
directions
of replication
Leading
strand
Origin of replication
3
5
5
RNA primer
3
3
Parental DNA
Sliding clamp
5
DNA pol III
3
5
Continuous elongation
in the 5 to 3 direction
5
3
3
5
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Figure 13.16a
Overview
Lagging Origin of replication
Leading strand
Lagging
strand
strand
Overall directions
of replication
What is going to latch on at #1?
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Leading
strand
Figure 13.16b-1
3
1 Primase makes
RNA primer.
5
Template
strand
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3
5
Figure 13.16b-2
1 Primase makes
3
RNA primer.
5
Template
strand
3
3
5
RNA primer
for fragment 1
2 DNA pol III
makes Okazaki
fragment 1.
5
3
5
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Figure 13.16b-3
1 Primase makes
3
RNA primer.
5
Template
strand
3
3
5
RNA primer
for fragment 1
2 DNA pol III
makes Okazaki
fragment 1.
5
3
5
3
3 DNA pol III
detaches.
5
Okazaki
fragment 1
3
5
Where is DNA pol III going to go next??
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Figure 13.16c-1
5
3
RNA primer for fragment 2
Okazaki
4 DNA pol III
fragment 2
makes Okazaki
fragment 2.
3
5
Now you have all these bits what has to happen next? And
who does that?
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Figure 13.16c-2
5
3
RNA primer for fragment 2
Okazaki
4 DNA pol III
fragment 2
makes Okazaki
fragment 2.
3
5
5
3
5 DNA pol I
replaces RNA
with DNA.
3
5
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Figure 13.16c-3
5
3
RNA primer for fragment 2
Okazaki
4 DNA pol III
fragment 2
makes Okazaki
fragment 2.
3
5
5
5 DNA pol I
3
replaces RNA
with DNA.
3
5
6 DNA ligase forms
5
3
bonds between
DNA fragments.
3
5
Overall direction of replication
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Figure 13.16
Overview
Lagging Origin of replication
Leading strand
Lagging
strand
strand
Overall directions
of replication
5
3
RNA primer
for fragment 2
Okazaki
fragment 2
1 Primase makes
3
RNA primer.
5
Template
strand
3
Leading
strand
3
5
5
5 DNA pol I
3
2 DNA pol III
replaces RNA
with DNA.
makes Okazaki
fragment 1.
3
5
5
3
6 DNA ligase forms
5
5
3
3
makes Okazaki
fragment 2.
3
5
RNA primer
for fragment 1
4 DNA pol III
bonds between
DNA fragments.
3 DNA pol III
detaches.
5
Okazaki
fragment 1
3
5
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3
5
Overall direction of replication
Figure 13.17
Leading strand
template
Single-strand
binding proteins
Leading strand
Helicase
Overview
Origin of replication
Lagging
Leading strand
strand
Lagging strand
Leading strand
Overall directions
of replication
DNA pol III
5
3
3
Parental DNA
Lagging strand
template
Primer
5
3
Primase
5
DNA pol III
3
5
Lagging strand
DNA pol I
DNA ligase
3
5
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Figure 13.17a
Overview
Origin of replication
Leading strand
Lagging
strand
Lagging strand
Leading strand
Overall directions
of replication
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Figure 13.17b
Leading strand
template
Single-strand
binding proteins
Leading strand
Helicase
DNA pol III
5
3
3
Parental DNA
Lagging strand
template
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Primer
5
3
Primase
Figure 13.17c
5
DNA pol III
3
5
Lagging strand
DNA pol I
DNA ligase
3
5
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Figure 13.14
New strand Template strand
5
3
Sugar
A
T
C
G
C
G
G
C
G
C
T
A
3
DNA
polymerase
A
P Pi
3
Pyrophosphate
C
Nucleotide
C
5
5
2 Pi
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3
T
A
Base
Phosphate
5
Question 1.
True of Leading strand, Lagging strand, or Both????
Daughter strand elongates away from replication fork
Synthesizes 5’ to 3’
Multiple primers needed
Made in segments
Made continuously
Daughter strand elongates toward replication fork
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True of Leading strand, Lagging strand, or Both????
Daughter strand elongates away from replication fork Lag
Synthesizes 5’ to 3’ Both
Multiple primers needed Lagg
Made in segments Lag
Made continuously Lead
Daughter strand elongates toward replication fork from Lead
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Question 2. The diagram below shows a replication bubble with
synthesis of the leading and lagging strands on both sides of the
bubble. The parental DNA is shown in dark blue, the newly
synthesized DNA is light blue, and the RNA primers associated
with each strand are red. The origin of replication is indicated by
the black dots on the parental strands.
Rank the primers in the order they were produced. If two primers
were produced at the same time, overlap them.
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Question 3. The lagging strand is synthesized as a series of
segments called Okazaki fragments Fragment A is the most
recently synthesized and Fragment B will be synthesized next in
the space between primers A and B.
-----Start DNA polymerase III binds to 3’ end of primer B
A. DNA polymerase I replaces primer with DNA
B. DNA polymerase I binds to 5’ end of primer A
C. DNA polymerase III moves 5’ to 3’ adding DNA nucleotides to
primer B
D. DNA ligase links fragments A and B
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In an analysis of the nucleotide composition of DNA, which
of the following will be found?
A = G and C = T
G +C =T+A
A=C
A+C=G+T
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Cytosine makes up 42% of the nucleotides in a sample of DNA
from an organism. Approximately what percentage of the
nucleotides in this sample will be thymine?
31%
42%
8%
16%
It cannot be determined from the information provided.
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