2019 Establishment of a virulent full-length cDNA clone for type I feline coronavirus strain C3663
Mice live + Mice die a.b.c.d. Live Virulent Strain of S. pneumoniae Live Nonvirulent Strain of S....
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Transcript of Mice live + Mice die a.b.c.d. Live Virulent Strain of S. pneumoniae Live Nonvirulent Strain of S....
Mice live
+
Mice liveMice die
a. b. c. d.
Live VirulentStrain of S. pneumoniae
Live NonvirulentStrain of
S. pneumoniae
Heat-killed VirulentStrain of S. pneumoniae
Mixture of Heat-Killed Virulentand Live Nonvirulent
Strains of S. pneumoniae
Mice dieTheir lungs contain live
pathogenic strain of S. pneumoniae
Polysaccharidecoat
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Result: When the experiment is done, only 32P makes it into the cell in any significant quantity.
Hypothesis: DNA is the genetic material in bacteriophage.
Prediction: The phage life cycle requires reprogramming the cell to make phage proteins. The information for this must be introduced into the cell during infection.
Test: DNA can be specifically labeled using radioactive phosphate (32P), and protein can be specifically labeled using radioactive sulfur (35S) . Phage are grown on either 35S or 32P, then used to infect cells in two experiments. The phage heads remain attached to the outside of the cell and can be removed by brief agitation in a blender. The cell suspension can be collected by centrifugation, leaving the phage heads in the supernatant.
Phage grown in radioactive 35S,which is incorporated into phage coat
Virus infectbacteria
Blender separatesphage coat from bacteria
Centrifuge formsbacterial pellet
35S in supernatant
35S-Labeled Bacteriophages
Phage grown in radioactive 32P.which is incorporated into phage DNA
Virus infectbacteria
Blender separatesphage coat from bacteria
Centrifuge formsbacterial pellet
32P in bacteria pellet
32P-Labeled Bacteriophages
Conclusion: Thus, DNA must be the molecule that is used to reprogram the cell.
Further Experiments: How does this experiment complement or extend the work of Avery on the identity of the transforming principle?
SCIENTIFIC THINKING
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Pu
rin
esP
yrim
idin
es
Adenine Guanine
NH2CC
NN
N
C
HN
C
CH
O
H
H
OC
NC
H
N
C
NH2
H
CH O
O
C
NC
H
N
CH3C
CH
H
O
O
C
NC
H
N
CH
CH
NH2
CC
NN
N
C
HN
C
CHH
Nitrogenous Base
4´
5´
1´
3´ 2´
2
8
7 6
39
4
5
1
O
P
O–
–O
Phosphate group
Sugar
Nitrogenous base
O CH2
N N
O
NNH2
OH in RNA
Cytosine(both DNA and RNA)
Thymine(DNA only)
Uracil(RNA only)
OHH in DNA
3
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BaseCH2
O
5´
3´
O
P
O
OH
CH2
–O O
C
Base
O
PO4
Phosphodiesterbond
4
5
6
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5´
3´
P
P
P
P
OH
5-carbon sugar
Nitrogenous base
Phosphate group
Phosphodiester bond
O
O
O
O
4´
5´
1´
3´ 2´
4´
5´
1´
3´ 2´
4´
5´
1´
3´ 2´
4´
5´
1´
3´ 2´
7
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2 nm3´
3´ 5´
0.34 nm
5´
C
C
C
G
G
G
G
G
T
T
T
T
A
A
A
3.4 nm
Minorgroove
Majorgroove
Majorgroove
Minorgroove
8
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A
H
Sugar
Sugar
Sugar
Sugar
T
G C
N
H
N O
H
CH3
H
HN
N N H N
N
N
H
H
H
N O H
H
H N
N H
N
N HN N
Hydrogenbond
Hydrogenbond
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Conservative Semiconservative Dispersive
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Samples are centrifuged
E. coli
0
1
2
0 rounds 1 round 2 rounds
Bottom
15N medium
14N medium
E. coli cells grownin 15N medium
Cells shifted to14N medium andallowed to grow
DNA
Samples taken atthree time pointsand suspended incesium chloridesolution
Rounds ofreplication
Top
0 min0 rounds
20 min1 round
40 min2 rounds
From M. Meselson and F.W. Stahl/PNAS 44(1958):671 11
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P
P
P
P
P
P
P
P
P
P
P
Pyrophosphate
3´
3´
5´
5´
New StrandTemplate Strand
O
HO
OH
O
O
O
O
O
O
O
O
O
OC
C
T
T
T
A
A
A
G
G
A
P
P
P
P
P
PP P
P P
P
P
P
P
3´
3´
5´
5´
New StrandTemplate Strand
O
HO
OH
OH
O
O
O
O
O
O
O
O
O
C
C
T
T
A
A
A
G
G
A
Sugar–phosphatebackbone
DNA polymerase III
TO
P
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5´
3´
5´
5´ 5´3´
3´
RNA polymerase makes primer DNA polymerase extends primer
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Replisome
Replisome Origin
Origin Origin
Origin
Termination Termination TerminationOrigin TerminationTermination
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Supercoiling
Replisomes
No Supercoiling
Replisomes
DNA gyrase
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RNA primer
Open helixand replicate
First RNA primer
Open helix andreplicate further
Lagging strand(discontinuous)
Second RNA primer
Leading strand(continuous)
RNA primer
5´
3´
3´
5´
5´
3´
3´
5´
5´3´
5´
3´
5´
3´
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5´
3´
Primase
RNA primer
Okazaki fragmentmade by DNApolymerase III
Leading strand(continuous)
DNA polymerase I
Lagging strand(discontinuous)
DNA ligase
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Leading strand (no problem)Lagging strand (problem at the end)
Last primer
Replication first round
Shortened template
Primer removal
Replication second round
Origin
5´
3´
3´
5´
3´
5´
5´
3´
3´
5´5´
3´
5´
3´
5´
3´
3´
5´
3´
5´
Removed primercannot be replaced
Leadingstrand
Laggingstrand
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G
GGGGG
T T
TTT TG
TT
GG
GGG
T
TTT
CCCCC AAAA
CCCCC AAAA
Telomere extendedby telomerase
Template RNA ispart of enzyme
Telomerase
Now readyto synthesizenext repeat
5´
3´
5´
3´
5´
3´
Synthesis by telomerase
Telomerase moves andcontinues to extend telomere
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T
A
T
A
A A
A A
TA
TA
TT
T T
Thymine dimercleaved
Photolyase
Helix distorted bythymine dimer
Thymine dimer
DNA with adjacent thymines
UV light
Visible light
Photolyase bindsto damaged DNA
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Damaged or incorrect base
Uvr A,B,C complexbinds damaged DNA
DNA polymerase
Excision of damaged strand
Resynthesis by DNA polymerase
Excision repair enzymes recognize damaged DNA
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