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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1

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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

2

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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

9

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Conservative Semiconservative Dispersive

10

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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

12

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5´

3´

5´

5´ 5´3´

3´

RNA polymerase makes primer DNA polymerase extends primer

13

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Replisome

Replisome Origin

Origin Origin

Origin

Termination Termination TerminationOrigin TerminationTermination

14

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Supercoiling

Replisomes

No Supercoiling

Replisomes

DNA gyrase

15

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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´

16

17

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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

18

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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

19

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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

20

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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

21

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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

22