seema & pallvi
TRANSCRIPT
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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