Independent Function Of Viral Protein And Nucleic Acid In Growth Of Bacteriophage

Frederick Griffith and Oswald Avery, Colin MacLeod, and Maclyn McCarty had shown that DNA  was the biomolecule that carried genetic information.

Alfred Hershey and Martha Chase were testing two competing hypothesis. DNA was the genetic materialProtein was the genetic material.

Hershey shared the 1969 Nobel Prize in Physiology or Medicine  for his “discoveries concerning the genetic structure of viruses.”

Experiment that inspire :- Which substance directed this takeover- DNA or Protein

Percent of isotope Whole phage labeled with Plasmolysed phage labeled with

P32 S35 P32 S35

Acid soluble - - 1 -

Acid soluble after treatment with DNase

1 1 80 1

Adsorbed to sensitive bacteria 85 90 2 90

Precipitated by antiphage 90 99 5 97

Conclusion :-

Most of the DNA and Protein are still in the virus particle.

Radioactive virus that are lysed release radioactive DNA into the media but not

the

protein.

Protein specifically adsorb to phage susceptible bacteria but DNA does not.

Radioactive virus that lysed keep their protein-based ghost attached to the

bacteria

but the DNA is not associated with the bacterial cells.

Composition of Ghost and Solution of plasmolysed Phage

Percent of isotope Phage

labeled withNon-sedimentable isotope ,per cent After DNase No DNase

Live bacteria

Live bacteria

S35

P32

2

8

1

7

Bacteria heated before infectionBacteria heated before infection

S35

P32

1576

1113

Bacteria heated after infectionBacteria heated after infection

S35

P32

1266

1423

70O

Heated unabsorbed phage: 80O

acid-soluble P32

90O

100O

P32

P32

P32

P32

5138188

Sensitization of phage DNA to DNase by Adsorption to bacteria

Conclusion:-

Phage DNA largely sensitive to DNase after adsorption to heat-killed bacteria. Phage DNA adsorbed to live bacteria, then heated to 80oC for 10 minutes, at which temperature the unadsorbed phage is not sensitized to DNase.

The Phage DNA adsorbed to unheated bacteria - resistant to DNase

heat-kille

d

bacteria

do not

shield

DNA

heat-kille

d bacteria

do not shield

DNA

Unabsorbed phage frozen, thawed,

fixed

Infected cells frozen, thawed, fixed

Infected cells fixed only

Low speed sediment fraction

Total P32 - 71 86

Acid soluble - 0 0.5

Acid-soluble after DNase - 59 28

Low speed supernatant fraction

Total P32 - 29 14

Acid soluble 1 0.8 0.4

Acid-soluble after DNase 11 21 5.5

Sensitization of intracellular phage to DNase by Freezing,Thawing, and Fixation with Formaldehyde

Conclusions:-

Freezing and thawing makes the intracellular DNA labile to DNase under without causing too much of it to leach out of cells. Cell membrane may be permeable to DNase under conditions that do not permit escape of majority of phage DNA or cell contents. DNA is not merely in solution, part of organized structure at all times.

Phage labeled withS35 P32

Sediment fractionSurviving phage 16 22

Total isotope 87 55

Acid-soluble isotope 0 2

Acid-soluble after DNase 2 29

Supernatant fractionSurviving phage 5 5

Total isotope 13 45

Acid-soluble isotope 0.8 0.5

Acid-soluble after DNase 0.8 39

Release of DNA from phage Adsorbed to Bacterial Debris

Conclusion:-The sediment contains phage adsorbed to bacterial debris and the supernatant contains unabsorbed phage particlesa)DNA is DNase resistant while inside the phage coat.b)DNA was found in both the fractions in an DNase labile form (29% and 39%) which indicates that it was released from the protective coatings of phages when they contacted bacterial debris.c)87% of the phage protein was in the sediment (i.e adsorbed) but only 55% of the phage DNA.

This shows that the DNA was released from the Phage particle

DNA

protein

Location of radioactive DNA (32P) or protein (35S). After 5 minutes of bacteria exposure to the virus, Hershey and Chase quantified the effects of blending duration (X-axis) on the percent of total radioactive material (Y-axis) released from the bacteria.

Removal of Phage Coats from infected Bacteria

protein 80% proteins outside bacteria

DNA35% DNA inside bacteria

Removal of S35 and P32 from bacteria infected with radioactive phage, and survival of the infected bacteria, during agitation in a Warnig blendor.

DNA

protein

protein not heritable material?

Bacteria were grown in glycerol –lactate medium

Sub cultured , Sedimented & Resuspended

Added S35-labeled phage T2

Left overnight at 37°C

Fractionation of Lysates

Low speed sediment (2500g for 20 minute)

High speed sediment (12,000g for 30 minutes)

Second low speed sediment

Bacteria were grown in glycerol –lactate medium

Sub cultured , Sedimented & Resuspended

Added S35-labeled phage T2

Left overnight at 37°C

Fractionation of Lysates

Low speed sediment (2500g for 20 minute)

High speed sediment (12,000g for 30 minutes)

Second low speed sediment

Transfer of Sulfur and Phosphorus from Parental Phage to Progeny

FRACTION Lysis at t = 0 S35

Lysis at t = 10

S35

1st low speed sediment 79 81

2nd low speed sediment 2.4 2.1

High speed sediment 8.6 6.9

High speed supernatant 10 10

RECOVERY 100 100

Percent Distribution of Phage and S35 among centrifugally Separated Fractions of Lysates after infection with S35 labeled T2

CONCLUSION :- The result of this experiment is that the distribution of S35 among the fractions is the same for early and late lysates that do not contain phage progeny. This suggests that little or no S35 is contained in the mature phage progeny.

Conclusion :-

Sulfur containing protein is confined to protective coat responsible for adsorption to bacteria and injection of phage DNA.

Protein has no function in phage’s intracellular growth but potentially does. “

When asked what his idea of happiness would be, [Hershey] replied, ‘to have an experiment that works, and do it over and over again.’”- Jonathan Hodgkin, 2001

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