Post on 18-Mar-2016
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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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http://en.wikipedia.org/wiki/File:Fred_Griffith_and_%22Bobby%22_1936.jpg
GriffithThere are unknown heritable substances…Turned to a bacterial pathogen…Streptococcus pneumoniae
Is it gram positive or negative?
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Figure 13.2
LivingS cells(control)
Mouse healthyResults
Experiment
Mouse healthy Mouse dies
Living S cells
LivingR cells(control)
Heat-killedS cells(control)
Mixture ofheat-killedS cells andliving R cells
Mouse dies
Work by Avery identified the transforming substance as DNA
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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
Labeled phagesinfect cells.
Batch 1: Radioactive sulfur (35S) in phage proteinExperiment
Agitation frees outsidephage parts from cells.
Centrifuged cellsform a pellet.
Radioactivity(phage protein)found in liquid
Batch 2: Radioactive phosphorus (32P) in phage DNA
Radioactivity (phage DNA) found in pellet
Radioactiveprotein
RadioactiveDNA
Centrifuge
Centrifuge
Pellet
Pellet
1 2 3
4
4
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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
Conservativemodel
Semiconservativemodel
Dispersivemodel
Predictions: First replication Second replication
DNA samplecentrifugedafter firstreplication
DNA samplecentrifugedafter secondreplication
Bacteriacultured inmediumwith 15N(heavyisotope)
Bacteriatransferredto mediumwith 14N(lighterisotope)
Less dense
More dense
Experiment
Results
Conclusion
1
3
2
4
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Figure 13.1
Watson and Crickhttp://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
3 end
5 end
3 end
5 end
T A
C G
CG
TA
12
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Figure 13.12
Single-strand bindingproteins
Helicase
Topoisomerase
Primase
Replicationfork
5
5
5
3
3
3
RNAprimer
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Figure 13.15
Parental DNA
5
3
5
3
5
3
Continuous elongationin the 5 to 3 direction
53
5
3
DNA pol III
RNA primerSliding clamp
53
Origin of replication
Origin of replication
Lagging strand
Laggingstrand
Overalldirections
of replication
Leadingstrand
Leadingstrand
Overview
Primer
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Figure 13.16a
Origin of replicationLagging strand Lagging
strand
Overall directionsof replication
Leadingstrand
Leadingstrand
Overview
What is going to latch on at #1?
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Figure 13.16b-1
5 3
5
3 Primase makesRNA primer.
Templatestrand
1
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Figure 13.16b-2
5 3
5
3 Primase makesRNA primer.
RNA primerfor fragment 1
Templatestrand
DNA pol IIImakes Okazakifragment 1.
53
5
3
1
2
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Figure 13.16b-3
5 3
5
3 Primase makesRNA primer.
RNA primerfor fragment 1
Templatestrand
Okazakifragment 1
DNA pol IIImakes Okazakifragment 1.
DNA pol IIIdetaches.
53
5
3
5
35
3
1
2
3
Where is DNA pol III going to go next??
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Figure 13.16c-1 RNA primer for fragment 2Okazakifragment 2 DNA pol III
makes Okazakifragment 2.
5
35
3 4
Now you have all these bits what has to happen next? And who does that?
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Figure 13.16c-2 RNA primer for fragment 2Okazakifragment 2 DNA pol III
makes Okazakifragment 2.
DNA pol Ireplaces RNAwith DNA.
5
35
3
5
35
3 4
5
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Figure 13.16c-3 RNA primer for fragment 2Okazakifragment 2 DNA pol III
makes Okazakifragment 2.
Overall direction of replication
DNA pol Ireplaces RNAwith DNA.
DNA ligase formsbonds betweenDNA fragments.5
35
3
5
35
3
5
35
3 4
6
5
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Figure 13.16
5 3
5
3
Origin of replicationLagging strand Lagging
strand
Overall directionsof replication
Leadingstrand
Leadingstrand
Overview
Primase makesRNA primer.
RNA primerfor fragment 1
Templatestrand
Okazakifragment 1
DNA pol IIImakes Okazakifragment 1.
DNA pol IIIdetaches.
53
5
3
5
35
3
RNA primerfor fragment 2
Okazakifragment 2 DNA pol III
makes Okazakifragment 2.
Overall direction of replication
DNA pol Ireplaces RNAwith DNA.
DNA ligase formsbonds betweenDNA fragments.
5
35
3
5
35
3
5
35
31
2
3
4
5
6
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Figure 13.17
3
5
Origin of replication
Lagging strand
Laggingstrand
Overall directionsof replication
Leading strand
Leading strand
Overview
5 3
5
3
Leading strand
Lagging strandDNA ligaseDNA pol I
DNA pol III
Primase
DNA pol IIIPrimer
53
5
3
Lagging strandtemplate
Parental DNA
Helicase
Single-strandbinding proteins
Leading strandtemplate
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Figure 13.17a
Origin of replication
Lagging strand
Laggingstrand
Overall directionsof replication
Leading strand
Leading strand
Overview
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Figure 13.17b
35
3
Leading strand
DNA pol III
PrimasePrimer
5
3
Lagging strandtemplate
Parental DNA
Helicase
Single-strandbinding proteins
Leading strandtemplate
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Figure 13.17c
55
3
5
3
Lagging strand
DNA ligaseDNA pol IDNA pol III
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Figure 13.14
Pyro-phosphate
New strand
Phosphate
Nucleotide
5 3Template strand
SugarBase
5
3
5
3
5 3
DNA poly-
meraseT
A T
C G
AT
CG
CPP P
P
P iP
i2
A T
C G
A
CG
C
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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 DNAB. DNA polymerase I binds to 5’ end of primer AC. DNA polymerase III moves 5’ to 3’ adding DNA nucleotides to primer BD. 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.