Chapter 1 DNA RNA Historical Experiments Structures

BMS 424 Chapter 1
DNA, RNA, Historical Experiments &
Structures

Frederick Griffith Experiments with
Streptococcus pneumoniae
S. pneumoniae comes in two strains, smooth and rough strains
S  Smooth : Secrete a polysaccharide capsule; Protects bacterium from immune system of hosts; Produce smooth colonies on solid media.
R  Rough : Unable to form capsule; Produce colonies with a rough appearance.
Griffith conducted experiments in 1928 using two strains of S. pneumoniae: type IIIS and type IIR :
1. Inject mouse with live type IIIS bacteria - Mouse died and Type IIIS bacteria were recovered from the mouse’s blood.
2. Inject mouse with live type IIR bacteria - Mouse survived; No living bacteria isolated from the mouse’s blood.
3. Inject mouse with heat-killed type IIIS bacteria - Mouse survived; No living bacteria isolated from the mouse’s blood.
4. Inject mouse with live type IIR + heat-killed type IIIS cells - Mouse died; Type IIIS bacteria recovered from the mouse’s blood

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Living type S bacteria were injected into a mouse.

Living type R bacteria were injected into a mouse.

Heat-killed type S bacteria were injected into a mouse.

Living type R and heat-killed type S bacteria were injected into a mouse.
Live
type R

Dead type S

After several days

Mouse died

After several days

Mouse survived

After several days

Mouse survived

After several days

Mouse died

Type S bacteria were isolated from the dead mouse.

No living bacteria were isolated from the mouse.

No living bacteria were isolated from the mouse.

Type S bacteria were isolated from the dead mouse.

(a) Live type S

(b) Live type R

(c) Dead type S

(d) Live type R + dead type S

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Conclusion form Griffith’s experiment
The formation of the capsule is guided by the bacteria’s genetic material
1. Transformed bacteria acquired information to make the capsule
2. Variation exists in ability to make capsule
3. The information required to create a capsule is replicated and transmitted from mother to daughter cells
Griffith concluded that something from the dead type IIIS bacteria was transforming type IIR bacteria into type IIIS and called this process transformation. The substance that allowed this to happen was termed the transformation principle.

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Avery, MacLeod and McCarty experiments
Avery, MacLeod and McCarty realized that Griffith’s observations could be used to identify the genetic material. They carried out their experiments in the 1940s. At that time, it was known that DNA, RNA, proteins and carbohydrates are the major constituents of living cells
They prepared cell extracts from type IIIS cells and purified DNA, RNA, proteins and carbohydrates
They found that :-

Only the extract that contained purified DNA was able to convert type IIR bacteria into type IIIS
– Treatment of the DNA extract with RNase or protease did not eliminate transformation
– Treatment with DNase did

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Avery et al. conducted the following experiments
Objective : To further verify that DNA, and not a contaminant (RNA or protein), is the genetic material
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1

Type R cells 2

3

Type S
DNA
extract

Type R cells Mix

4

Type S
DNA
extract
+
DNase

Type R cells Mix

5

Type S
DNA
extract
+
RNase

Type R cells Mix

Type R cells Type S
DNA
extract
+
protease
Mix

Allow sufficient time for the DNA to be taken up by the type R bacteria. Only a small percentage of the type R bacteria will be transformed to type S.
Add an antibody that aggregates type R bacteria (that have not been transformed). The aggregated bacteria are removed by gentle centrifugation.

Plate the remaining bacteria on petri plates. Incubate overnight.

Transformed

Transformed

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Transformed

Hershey and Chase Experiment with Bacteriophage
T2
•

In 1952, Alfred Hershey and Marsha Chase provided further evidence that DNA is the genetic material. Bacteriophage T2 is a composed of only two macromolecules; DNA and protein.

DNA
(inside the capsid head)

Inside the capsid Head
Sheath

Tail fiber

Base plate
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Made up of protein

Summary of Hershey and Chase experiment :1) Used radioisotopes to distinguish DNA from proteins
• 32P labels DNA specifically
• 35S labels protein specifically
2) Radioactively-labeled phages were used to infect non-radioactive Escherichia coli cells
After allowing sufficient time for infection to proceed, the residual phage particles were sheared off the cells.
Phage ghosts and E. coli cells were separated.
Radioactivity was monitored using a scintillation counter.
The Hypothesis
Only the genetic material of the phage is injected into the bacterium. Isotope labeling will reveal if it is DNA or protein.

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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Experimental level
Conceptual level
1.Grow bacterial cells. Divide into two flasks.

Suspension of
E. coli cells
35S-labeled

protein capsid

32P-labeled

DNA

2.Into one flask, add 35S-labeled phage; in the second flask, add 32P-labeled phage.

35S-labeled

32P-labeled

T2 phage

T2 phage

3.Allow infection to occur.
After blending
Bacterial cell
4.Agitate solutions in blenders for different lengths of time to shear the empty phages off the bacterial cells.
Suspension of
Suspension of
E. coli infected withE. coli infected with
35S-labeled phage 32P-labeled phage
5.Centrifuge at 10,000 rpm.
Supernatant
6.The heavy bacterial cells sediment to the with pellet, while the lighter phages remain 35 inS-labeled the supernatant. (See Appendix for empty phage explanation of centrifugation.)
Pellet with unlabeled DNA in infected E. coli cells

Supernatant with unlabeled empty phage
Pellet with
32P-labeled
DNA in infected E. coli cells

Viral genetic material Sheared empty phage (labeled)
Bacterial
cell
Viral
genetic material (labeled)
Sheared
empty phage Sheared empty phages (labeled)
Sheared
empty phages (unlabeled)

7.Count the amount of radioisotope in the supernatant with a Geiger counter.
Compare it with the starting amount.

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The Data
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Total isotope in supernatant (%)

100
Extracellular 35S

80

80%
Blending removes 80% of 35S from E. coli cells.

60

40

Extracellular 32P

35%
Most of the 32P (65%) remains with intact
E. coli cells.

20
0
0

1

2

3

4

5

6

7

8

Agitation time in blender (min)

Data from A. D. Hershey and Martha Chase (1952) Independent Functions of Viral Protein and Nucleic Acid in Growth of
Bacteriophage. Journal of General Physiology 36, 39–56.

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9-17

Interpreting the Data
Most of the 35S was found in the supernatant Total isotope in supernatant (%)

But only a small percentage of 32P

100
80

Extracellular 35S

80%
Blending removes 80% of 35S from E. coli cells.

Extracellular 32P

35%
Most of the 32P (65%) remains with intact
E. coli cells.

60
40
20
0
0

1

2

3

4

5

6

7

8

Agitation time in blender (min)
Data from A. D. Hershey and Martha Chase (1952) Independent Functions of Viral Protein and Nucleic Acid in Growth of
Bacteriophage. Journal of General Physiology 36, 39–56.



These results suggest that DNA is injected into the bacterial cytoplasm during infection

Data is not conclusive since less than 100% of the DNA or protein ended up in the cell or supernatant 
Data is consistent with the hypothesis that DNA is the genetic material
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