we will focus on these 3 classic experiments highlighted in this chapter…… griffith (frederick)

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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)


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?


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


Transformation-did not really understand mechanism

•Can we do this-pick up DNA from our environment?


Hershey and Chase (1952)Their work pointed to DNA rather than proteins…

Bacteriophages what are they???? (worked with one called T2)


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)


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


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??


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


Figure 13.1

Watson and Crickhttp://www.ted.com/talks/james_watson_on_how_he_discovered_dna.html

http://www.ted.com/talks/james_watson_on_how_he_discovered_dna.html

http://www.ted.com/talks/james_watson_on_how_he_discovered_dna.html


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


Figure 13.7b

3 end

5 end

3 end

5 end

T A

C G

CG

TA

12


Figure 13.12

Single-strand bindingproteins

Helicase

Topoisomerase

Primase

Replicationfork

5

5

5

3

3

3

RNAprimer


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


Lagging strand

Laggingstrand

Overalldirections

of replication

Leadingstrand

Leadingstrand

Overview

Primer


Figure 13.16a

Origin of replicationLagging strand Lagging

strand

Overall directionsof replication

Leadingstrand

Leadingstrand

Overview

What is going to latch on at #1?


Figure 13.16b-1

5 3

5

3 Primase makesRNA primer.

Templatestrand

1


Figure 13.16b-2

5 3

5


RNA primerfor fragment 1

Templatestrand

DNA pol IIImakes Okazakifragment 1.

53

5

3

1

2


Figure 13.16b-3

5 3

5



Templatestrand

Okazakifragment 1


DNA pol IIIdetaches.

53

5

3

5

35

3

1

2

3

Where is DNA pol III going to go next??


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?




DNA pol Ireplaces RNAwith DNA.

5

35

3

5

35

3 4

5




Overall direction of replication


DNA ligase formsbonds betweenDNA fragments.5

35

3

5

35

3

5

35

3 4

6

5


Figure 13.16

5 3

5

3

Origin of replicationLagging strand Lagging

strand


Leadingstrand

Leadingstrand

Overview

Primase makesRNA primer.


Templatestrand

Okazakifragment 1


DNA pol IIIdetaches.

53

5

3

5

35

3


Okazakifragment 2 DNA pol III


Overall direction of replication


DNA ligase formsbonds betweenDNA fragments.

5

35

3

5

35

3

5

35

31

2

3

4

5

6


Figure 13.17

3

5


Lagging strand

Laggingstrand


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


Figure 13.17a


Lagging strand

Laggingstrand


Leading strand

Leading strand

Overview


Figure 13.17b

35

3

Leading strand

DNA pol III

PrimasePrimer

5

3

Lagging strandtemplate

Parental DNA

Helicase

Single-strandbinding proteins

Leading strandtemplate


Figure 13.17c

55

3

5

3

Lagging strand

DNA ligaseDNA pol IDNA pol III


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


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


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


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.


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


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


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.

we will focus on these 3 classic experiments highlighted in this chapter…… griffith (frederick)

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