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 material Protein was the genetic material. Experiment that inspire :- Which substance

directed this takeover- DNA or Protein

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

Composition of Ghost and Solution of plasmolysed Phage

Percent of isotope Whole phage labeled with
P32 Acid soluble Acid soluble after treatment with DNase Adsorbed to sensitive bacteria Precipitated by antiphage 1 85 90 S35 1 90 99

Plasmolysed phage labeled with

P32 1 80 2 5 S35 1 90 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

Sensitization of phage DNA to DNase by Adsorption to bacteria

Percent of isotope Phage labeled with
S35 P32 S35 P32

Non-sedimentable isotope ,per cent

After DNase
2 8 15 at- ria e t he 76 act no b
do A DN ll ki ed sh ld ie

No DNase
1 7 11 13

Live bacteria Live bacteria Bacteria heated before infection Bacteria heated before infection

Bacteria heated after infection Bacteria heated after infection

S35 P32

a eri act NA b 12 -killed ield D h t 66a not s he do

14 23

70O Heated unabsorbed phage: acid-soluble P32 80

O

90O 100O

P32 P32 P32 P32

5 13 81 88

Conclusion:Phage DNA largely sensitive to DNase after adsorption to heat-killed bacteria. Phage DNA adsorbed to live bacteria, then heated to80oCfor10 minutes, at which temperature the unadsorbed phage isnotsensitized to DNase. The Phage DNA adsorbed to unheated bacteria - resistant to DNase

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

Unabsorbed phage Infected cells frozen, thawed, fixed thawed, fixed frozen,Infected cells fixed only

Low speed sediment fraction

Total P32 Acid soluble Acid-soluble after DNase

1 11

71 0 59 29 0.8 21

86 0.5 28 14 0.4 5.5

Low speed supernatant fraction

Total P32 Acid soluble Acid-soluble after DNase

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.

Release of DNA from phage Adsorbed to Bacterial Debris

Phage labeled with
S35 P32

Sediment fraction
Surviving phage Total isotope Acid-soluble isotope Acid-soluble after DNase 16 87 0 2 22 55 2 29

Supernatant fraction
Surviving phage Total isotope Acid-soluble isotope Acid-soluble after DNase 5 13 0.8 0.8 5 45 0.5 39

Conclusion:The sediment contains phage adsorbed to bacterial debris and the supernatant contains unabsorbed phage particles a)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. a)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

Removal of Phage Coats from infected Bacteria

protein

DNA

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.

80% proteins outside bacteria

protein

35% DNA inside bacteria

DNA

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

protein

protein not heritable material?

DNA

Transfer of Sulfur and Phosphorus from Parental Phage to Progeny

Bacteria were grown in glycerol lactate medium Sub cultured , Sedimented & Resuspended Added S35-labeled phage T2 Left overnight at 37C Fractionation of Lysates Low speed sediment (2500g for 20 minute) High speed sediment (12,000g for 30 minutes) Second low speed sediment

Percent Distribution of Phage and S35 among centrifugally Separated Fractions of Lysates after infection with S35 labeled T2 FRACTION Lysis at t=0 S35 Lysis at t = 10 S35

1st low speed sediment 2nd low speed sediment High speed sediment High speed supernatant RECOVERY

79 2.4 8.6 10 100

81 2.1 6.9 10 100

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.

Sulfur containing protein is confined to protective coat responsible for adsorption to bacteria and injection of phage DNA. Protein has no function in phages 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.

THANKS