1. Alfred Hershey and Martha Chase:
Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage
Chelsea Bishop
Emily Bonnell
Leanne Dawe
Stephanie Mayne
Emily Porter

2. Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage
Background
Experiments Conducted
8 in total
Discussion & Summary
How Paper Impacted Future

3. ALFRED DAY HERSHEY

4. ALFRED DAY HERSHEY

5. ALFRED DAY HERSHEY

6. MARTHA CHASE

7. MARTHA CHASE

8. MARTHA CHASE

9. NOW FOR THE PAPER…

10. What is a Bacteriophage?
“Bacteria eater”, viruses that attack bacteria
Composed of proteins, which make up protective coat, that encapsulate DNA (or RNA) genome
Discovered by Félix d'Herelle and Frederick Twort
Tail injects DNA into bacteria

11. VS OR

12. Viral Protein and Nucleic Acid in Bacteriophage Growth
How was Adsorption of Isotope to Bacteria Measured?
Mix sample in adsorption medium with bacteria from 18hr broth cultures
Washed, warmed, diluted and centrifuged
Assays of sediment and supernatant components were made

13. Viral Protein and Nucleic Acid in Bacteriophage Growth
How was the precipitation of the isotopes measured?
Mixed with saline, non-radioactive phage and anti-phage serum then centrifuged
How were tests with DNase Performed?
Diluted in veronal buffer with crystalline enzyme and warmed
How were acid-soluble isotopes measured?
Chilled sample precipitated with trichloroacetic acid and serum albumin then centrifuged

14. The Chemical Morphology of Resting Phage Particles
- Anderson 1949
Salt Water
OSMOTIC SHOCK

16. OVERALL FOR EXPERIMENT ONE…
The ghosts represent protein coats that surround the DNA of the intact particles
The ghosts react with antiserum, protect the DNA from DNase and carry the organ of attachment to the bacteria
This experiment characterizes the virus

17. Removal of Phage Coats from Infected Bacteria
The following experiments show that it is possible to separate the phage from the bacterial cell to determine what components of the phage are preserved in the bacterial cell.

18. Methods Bacteria were infected with radioactive phage in a medium.
The first experiment was run using S35 labeled phages.
The second experiment was conducted with P32 labeled phages.
Anything not absorbed by the bacterial medium was removed with the use of a centrifuge.
The cells were re-suspended in MgSO4, CaCl2 and gelatin containing water and spun in the Waring blendor.

19. Note
S35 represents the protein, which makes up the exterior of the phage. Sulfur is a component of every protein.
P32 represents the phage’s DNA, as the DNA backbone is composed of phosphate and deoxyribose.

20. After 60 seconds of blending, the samples were cooled briefly and titrated to measure the bacteria capable of yielding phages.
Samples were centrifuged and the amount of radiolabeled substance was measured.

21. Results

22. The amount of extracellular S35 is highest in the supernatant at 80%
The amount of extracellular S35 is highest in the supernatant at 80%. Extracellular P32 is expected to be present at much lower values, measured between 20-30%.
The P32 that was extracted comes from the DNA that has been damaged. This means that the isotope could leak from the cell, whereas intact cells will keep the P32 inside.

23. Why was the sulfur isotope not 100% extracted ?!?!

24. S35 should theoretically be at 100% as none of the exterior bacteriophage shells would enter the bacterium cells.
It is most likely that some of the phage became stuck to the E.coli.
Anything that will come off in the minute time frame it was spun will be all that is released. The remainder will stay stuck to the bacteria cell, thus explaining why 100% of sulfur was not extracted in the supernatant.

25. The results of this experiment indicate that the phage inserts its DNA into bacterial cells and remains inside the cell, while the exterior protein coat of the phage remains on the outside of the cell; The viral DNA remains in the bacteria.

26. The blender does not break the bacteria
The blender does not break the bacteria. This is indicated by the low levels of P32 in the extracellular material. Knowing that the blender does not break the bacterial cell but removes the phage (high levels of extracellular S35) proves that it is a successful technique for separating the phage from the bacterial cell after the phage’s DNA has been incorporated with the bacterial DNA.

28. The results from the blender experiment allow for other experiments to be conducted on just the bacteria with the adsorbed phage DNA.

30. Two different levels of infection were used along with two different times in the blender.
One infection contained ten times the viral load than the other.
The blender was either not used or used on high for 2.5 minutes.

31. Initially, when the blender is not utilized, the amount of P32 that is removed after a saline wash is 10% and the survival of the bacteria is high.
When the bacteriophage is ten times the concentration, the survival of the bacteria decreases to 89% and the amount of P32 that is removed during the wash is about the same.
Slightly more P32 is removed from the blended washings. Again, this is due to damaged bacteria, therefore, the DNA can leak out.

32. Looking at the S35 isotope with a low multiplicity of infection, very few of the protein bacteriophage wash out in the saline solution, instead, they remain attached to the bacteria.
However, when the blender is used the majority of the S35 isotopes are eluted. With the highest multiplicity, the S35 elutes 46%. This number is considered high as the blender was not used to shake the protein coat off the bacteria. It is most likely in this case that the high concentration of the virus causes the shells to fall off the bacterial cell at a high rate as the bacteria can no longer take such a high infection.

33. Important Findings
75-80% of the sulfur can be stripped away from the bacteria utilizing mechanical force. This number increases with increasing multiplicity of infection (without and mechanical force).
Phosphorus is released in much smaller quantities than the sulfur. Phosphorus can be released without mechanical force, however, slightly more is released with mechanical force. The phosphorus release is due to damaged bacterial cells, which leak the internal DNA.
Methods used do not inactive intracellular phage.
Finally, the majority of the sulfur (protein) remains at the bacterial cell surface and does not aid in the multiplication of intracellular phage. The phosphorus (phage DNA) enters the bacterial cell soon after absorption of the phage and is protected from removal by the bacteria’s cell wall.

34. Morphology and How Phages Work:
Sensitization of Phage DNA to DNase by Absorption to Bacteria.
This table indicates that the removal of the viral protein coat occurs before the multiplication of the virus, that bacterial cells protect viral DNA and the temperature at which the virus structure is stable.

35. Sensitization of Phage DNA to DNase by Absorption to Bacteria: Live Bacteria
•P32 and S35 labelled phage absorbed to bacteria using absorption medium. This was then washed and centrifuged.
•This is the "control" of the experiment where there is little difference in non-sentimental isotopes.
•The percentage of isotopes is almost equal both before and after treated with DNase, indicating that the infected live bacteria protects the viral DNA from being broken down.

36. Sensitization of Phage DNA to DNase by Absorption to Bacteria: Heated Before and After Infection
•P32 and S35 labelled Phage absorbed to bacteria using absorption medium either before or after heating bacteria to 80°C. This was then washed and centrifuged.
•Heated before: The infected phage DNA gets broken down by DNase (susceptible), indicating that the bacteria has been damaged by heat before infection, leaving the phage DNA open to breakdown by DNase.
•Heated after: Results are similar to the heated before infection sample, indicating that the infected phage is relatively equally susceptible to DNase if the bacteria was heat killed before or after infection.
•“No DNase” indicates non-sedimental DNA that is intact before DNase is added.
•However, there should have been no change in S35 compared to the live bacteria sample, indicating that there was some protein contamination—A crude experiment.

37. Sensitization of Phage DNA to DNase by Absorption to Bacteria: Heated Unabsorbed Phage
•This shows that the intact phage is not sensitive to heat treatment up to 80°C, where only 13% of DNA is lost.
• P32 and S35 labelled Phage unabsorbed to bacteria, heated to various temperatures, and treated with DNase.
•The importance of this is to show that in later experiments where the bacteria is heated to 80°C, only the infected bacteria will break and expose phage DNA. Whole viruses will not affect the results because 80°C does not readily break down the viral protein coat.
•However, it is important to note that 13% of DNA is broken down by DNase, indicating that there is still going to be some amount of contamination from the uninfected viruses that may alter results.

38. Sensitization of Intracellular Phage DNase by Freezing, Thawing and Fixation with Formaldehyde
Acid soluble:Free DNA in solution before DNase is added
This table shows that a series of freezing and thawing phage infected bacteria (during the latent phase) cause the intracellular DNA to be susceptible to DNase without much DNA leaching out, more so than infected bacteria that have only been fixed with formaldehyde.

39. • These numbers indicate that freezing, thawing and fixation allow more intracellular DNA to be susceptible to DNase than formaldehyde fixation alone, while remaining attached the bacterial cell, even if the cell is lysed and other components released.
• Although fixed only cells absorbed more phage DNA than the frozen and thawed samples, it also had less DNA susceptible to DNase and although formaldehyde allows DNA to not leach out, it causes some damage to it.
• The unabsorbed phage DNA is almost completely unaffected (protected by its protein coat). However, there is some discrepancy in results: all of the total P32 should have been broken down by DNase and none of the unabsorbed phage should have been broken down.
•This information indicates that intracellular phage DNA is part of an organized structure in the infected cell during its latent period

40. Liberation of DNA from Phage Particles by Absorption to Bacterial Fragments
This table aims to show that the sensitization of phage DNA after absorption to bacteria is then followed by the ejection of phage DNA from its protein capsid using phages attached to bacterial fragments. This is done using S35 & P32 labelled T2 phages that were mixed with bacterial debris, heated to 37°C, and then centrifuged.

41. Liberation of DNA from Phage Particles by Absorption to Bacterial Fragments: Sediment
• Of the total 55% P32, 22% is protected surviving phage while 29% is absorbed and susceptible to DNase, indicating that it is no longer protected when attached to bacteria fragments.
• S35 is labelled protein fragments found with the bacterial debris and show virtually no change after DNase is added.

42. Liberation of DNA from Phage Particles by Absorption to Bacterial Fragments: Supernatant
• A similar situation is seen here, where out of 45% of the total labelled DNA, 5% was surviving phage and 39% was attached to bacterial cell debris and susceptible to DNase.

43. Liberation of DNA from Phage Particles by Absorption to Bacterial Fragments: Overview
• Surviving phages and S35 labelled proteins are found mostly in the sediment, indicating that it was not absorbed by the bacteria fragments.
• 40% of the total phage DNA (indicated by DNA breakdown via DNase) as well as the unabsorbed surviving phage DNA is found in the supernatant.
• Sedimental DNA is either surviving phage or unabsorbed DNA liable to DNase.
• Therefore, indicating that phage DNA is released from its protein capsid after injection into bacteria because of its sensitivity to DNase.

44. Liberation of DNA from Phage Particles by Absorption to Bacterial Fragments: An Unreliable Experiment
• It is indistinguishable whether the freed DNA is all of the released phage DNA or only a portion of it.
• A subsequent experiment was performed where bacteria saturated in UV killed T2 absorbed T4 better, but when bacterial debris was saturated with T4, it absorbed T2 better. This experiment also showed that some of the absorbed phage was still active and some of the inactive DNA was not released from the debris (indicated by use of DNase). Also, when a phage attaches to one receptor, it inactivated the attachment of another type of phage to the bacteria.
-This experiment indicated that if there is contamination of other viruses, they may inactivate the receptors and skew the results.

45. Percent Distribution of Phage and S35 among Centrifugally Separated Fractions of Lysates after Infection with S35-Labeled T2
•Bacteria were grown in glycerol-lactate medium overnight, then a subculture was taken in same medium
•Infected with S35 labelled phage
•After 5 min the culture was diluted with water and sedimented to remove unabsorbed phage and S35 then suspended in glycerol-lactate medium

46. •Phage growth was ended at times 0, 10 and at max yield by adding HCN (cyanide) to stop intracellular phages from maturation
•For the max yield of phage, lysing phage was added 25 mins after putting the infected cells in the culture medium and HCN was added after 2 hrs. Thus, causing lysis of infected cells to happen slowly; Cultures were left overnight.

47. •Lysates were centrifuged into low speed sediment (2500 G for 20 min)
•High speed supernatant (12000 G for 30 min)
•Second low speed was done by re-centrifuging resuspended high speed sediment
•The distribution of S35 in these cultures are shown in Table VI

48. 99.4
101
•T=0 is original labelled phage, T=10 and T=Max yield will have phage offspring present
•Virtually no difference in the amount of S35 sedimented at these times, so it can be concluded that the sulfur is not getting passed from parent to offspring

49. Adsorption Tests with Uniformly S35 Labelled Phage and with Products of Their Growth
•2 different strains of the virus: standard and mutant
•3 strains of E.coli:
H (sensitive to both virus)
B/2 (sensitive to mutant virus; h-mutant lysing phage, but not standard; T2 progeny)
B/2h (resistant to both strains)
•UV-h: Virus killed by UV light

50. •Both UV killed and not UV killed have similar amounts of S35
•Unabsorbed phage possibly due to contamination
•When new viral cells are made, same amount of S35 is present in samples. It has not been passed to offspring
•Since offspring are not labelled, we see less S35 than phage in phage progeny

51. •Uniformly labeled phage = seed phage from table VI
•Products = high speed sediment fractions from table VI
•Test to see how much virus is absorbed by each of the different strains of bacteria
•Basically a control test to show bacteria is in fact infected by viruses

52. What about P32?
•The experiments that were conducted in tables VI and VII were also completed using P32 labelled phage instead of S35. The results were not collected in a table, but it is explained that P is shown to be transferred to offspring
•About 30% of P32 was found in the progeny
•”P32 goes in, S35 stays out”

54. Experiment 7: A Progeny of S35-Labeled Phage Nearly Free from the Parental Label

55. Methods – How’d they Complete it?
Infected bacteria with S35-labeled phage at a high ratio to ensure full infection achieved
Washed off any unabsorbed phage
Divided samples: Stripped and Non-stripped
Stripped samples were agitated in a Waring Blendor

56. Methods cont’
Both samples were centrifuged to remove extracellular S35
Cells were then lysed and salt was added to cause S35 to remain attached to the bacterial debris
This would then cause the sulfur to be in the sediment, not the supernatant.

57. Cell Stripping Process
Stripping of the bacterium was achieved by using a Waring Blendor
This method removed phage stuck to the outside of the cell
In non-stripped sample, ghosts would still be adhered

59. What Does This Mean? Stripped Cells:
Most (86%) of the S35 was found in the blendor
fluid. Therefore, it was found in the ghosts removed by the blendor.
Small amount of S35 was found in the sediment
1st low-speed: 3.8
Almost none from inside the cell
Values too close to background to be confident they are not actually zero.

60. What Does This Mean? Non-Stripped Cells:
Almost 40% of the ghosts came off from just suspension in water
Remainder stayed on bacteria as ghosts
Majority of remaining ghosts were removed after the 1st low-speed spin
31% of the S35
More was found in higher spins, unlike as seen in the stripped cells
During the high-speed fraction, 9.4% of the S35 found
Perhaps required to break off any ghosts which were well adhered to the bacterium

61. What Does This Mean?
Majority of phage is recovered in the high-speed sediment
Therefore, the high-speed spin is required to release most of the phage progeny
NOTE: none of the numbers add to 100%
Take away:
there is very little S35 present alongside the phage progeny!

62. Findings
Stripping reduces almost all of the S35-content of all fractions
With regards to the fraction containing most of the phage progeny, the S35- content is reduced to less than 1% (from nearly 10%) of the initial amount
High-speed sediment spin released most of the phage progeny
There is very little S35 present alongside the phage progeny
Stripped Cells: Sediment did not contain labeled S35
The S35 found in the lysate fractions was composed of the coats of the parental phage particles remains

63. Experiment 8: Properties of Phage Inactivated by Formaldehyde

64. Methods Treated virus with Formaldehyde:
Warmed Phage T2 at 37°C for 1 hour in absorption medium
Contained 0.1% commercial formalin
Dialyzed free from formaldehyde

65. What was the Outcome?
The fixation of DNA into phage with formaldehyde altered the phage replication process
Large reduction in plaque titer number
Decreased by 1000 fold!
Plaque = measure of virus quantity (an area of infection surrounded by uninfected cells)

66. Properties of Inactivated Phage
It is absorbed to sensitive bacteria
~70%
The absorbed phage kills bacteria
~35% effective
The DNA of the inactive particles is resistant to DNase
However, is sensitive to osmotic shock
osmotic shock = sudden change in solute concentration around a cell, causes rapid change in movement of water across cell membrane

67. Properties of Inactivated Phage
4) The DNA of the inactive particles is not sensitized to DNase by absorption to heat-killed bacteria
Also not released into solution by absorption to bacterial debris
5) Absorbed phage DNA can be detached
~70% can be detached
Detached DNA is almost completely resistant to DNase.
Detached from infected cells spun in the Waring Blendor

68. What Do These Properties Show?
T2 Inactivated by Formaldehyde:
Cannot inject its DNA into cells to which it attaches
The behavior observed in this experiment (by inactivated T2) supports previous experiments involving active phage

69. Discussion What Does All of this Suggest?
When T2 attaches to bacteria cell:
A residue of 80% (or more) sulfur-containing protein stays at cell surface
Residue also composed of protective membrane material
Plays no further role in infection
So what about the other 20% sulfur?!

70. Discussion What Does All of this Suggest?
20% Sulfur in question - either does or does not enter cell:
Appears to remain extracellular
However, phosphorus and adenine (derived from DNA of infecting particle) are TRANSFERRED to phage progeny
Showed us that sulfur-containing protein has no function in phage multiplication, yet DNA does.

71. Unanswered Questions
“Does any sulfur-free phage material other than DNA enter the cell?”
“If so, is it transferred to the phage progeny?”
“Is the transfer of phosphorus to progeny direct or indirect?”

72. Discussion Hershey and Chase did successfully show that:
Physical separation of bacteriophage T2 into genetic and non-genetic parts is possible!

73. Summary The sulfur-containing protein of the phage particle:
makes up the protective membrane and guards phage DNA from DNase
Principal antigenic material
Responsible for virus attaching to bacteria
Treating unabsorbed phage by heat-killing, heating or freezing and thawing have little or no sensitizing effect
Neither heating nor freezing/thawing releases phage DNA from infected cells
Thus, phage DNA forms part of intracellular structure.

74. Summary cont’
Absorption of phage T2 to bacterial debris
Part of the phage DNA appeared in solution, phage sulfur still attached to debris
The other half of phage DNA is attached to debris but separates due to action of DNase
Stripped cells release 75% of phage sulfur and 15% of phage phosphorus to solution due to shearing force
Cells still capable of phage progeny

75. Summary cont’ Upon infection:
Most of phage sulfur remains at cell surface
Most of phage DNA enters cell
Bacteria infected with phage labeled radioactive sulfur yielded a phage progeny which contained less than 1% of the parental radioactivity
Those labeled with radioactive phosphorus contained 30% or more of the parental phosphorus

76. Summary cont’
Inactivated phage (by formaldehyde) can absorb bacteria, but does not release its DNA to the cell.
Therefore, release of phage DNA from protective membrane depends on components of phage particle
Sulfur-containing protein of phage is confined to protective “coat”
Responsible for absorption of bacteria
Instrument of injection of phage DNA
No function in growth of intracellular phage
DNA has some unknown function

77. Simplified Conclusion
Absorbed phage:
Inserts DNA into cell
Sulfur-containing protein stays outside cell
Phage:
Release DNA into solution
Phage progeny:
Had parental P32
Little or no parental S35
DNA has “some function” while sulfur-containing protein did not have intracellular function

78. Other Experiments
The results of Hershey and Chase’s experiment sparked the interest of numerous scientists and convinced many that DNA was in fact the molecule of heredity
For example: Watson and Crick
Resultantly, the works published by Hershey and Chase were essential in igniting the race to find the structure of DNA!

79. Conformation of Experiment
In 1953 Watson and Crick won the race to find the structure of DNA!
Their journal article “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid” suggested the copying mechanism by which DNA acts as hereditary material and that DNA is synthesized by thousands of proteins found in cells.

80. The Nobel Prize
In 1969 Hershey was awarded the Nobel Prize in Physiology or Medicine
The prize was awarded jointly:
1/3 share for Alfred D. Hershey
1/3 share for Max Delbrück
1/3 share for Salvador E. Luria

81. "for their discoveries concerning the replication mechanism and the genetic structure of viruses"
Nobel Prize Organization

82. Where are they Now? Alfred D. Hershey (1908 – 1997) 1945 1962 1965
Married Harriet Davidson
1962
Appointed Director of Genetics Research Unit at the Carnegie Institution of Washington in Cold Spring Harbor, New York
1965
Received the Kimber Genetics Award of the National Academy of Sciences
1969
Awarded Nobel Prize in Physiology or Medicine
1970
Honored with M.D. h. c. from Michigan State University
Born December 4th (Oswosso, Michigan)
Died May 22nd

83. Where are they Now? Martha Chase (1927 – 2003) 1950’s 1964
Took part in meetings of the Phage Group of Biologists in Cold Spring
Harbor, New York
1964
Received doctorate at the University of Southern California
Mid – Late 1960’s
Ended career in science due to personal problems
Born November 30th (Cleveland Heights, Ohio)
Died August 8th

84. American Phage Group What was is? What did they do?
An informal network of biologists in the mid 20th century based at the Cold Spring Harbor Laboratory in New York
What did they do?
Huge contributors to the world of bacterial genetics and molecular biology
Created course on phages that was taught every summer from the 1950’s till the 1960’s
Consisted of some of our favorite scientists!
Centered around Max Delbrück
Included Hershey, Watson, Luria, Benzer, Stent, Stahl, and Dulbecco

85. Where are we Today? Genome mapping Genetic engineering
23andMe
Genetic engineering
Designer children
Disease prediction models
ex: Huntington’s disease

86. “I can only point out a curious fact
“I can only point out a curious fact. Year after year the Nobel Awards bring a moment of happiness not only to the recipients, not only to colleagues and friends of the recipients, but even to strangers”
- Alfred Day Hershey

87. References
Hershey, A., & Chase, M., (1952). Independent Functions of Viral Protein and Nucleic Acids in Growth of Bacteriophage. Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor, Long Island.
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"Chase, Martha Cowles (1927- )." World of Microbiology and Immunology Encyclopedia.com. 26 Jan <
BrainyQuote,. "Alfred Day Hershey Quotes At Brainyquote". N.p., Web. 27 Jan
Carr, Dr. Steve. "Hershey And Chase 1952". Bio Advanced Topics in Genetics. N.p., Web. 24 Jan
Carr, Dr. Steve. "Bio Advanced Topics In Genetics". Mun.ca. N.p., Web. 24 Jan
Encyclopedia.com,. "Chase, Martha Cowles (1927- ) – FREE Chase, Martha Cowles (1927- ) Information | Encyclopedia.Com: Find Chase, Martha Cowles (1927- ) Research". N.p., Web. 24 Jan. 2016
“Gender Bias in Science, Part IV: Martha Chase”. The Mad Science Blog. Oct. 28, 2013
Gutenberg, Project. "American Phage Group | Project Gutenberg Self-Publishing - Ebooks | Read Ebooks Online". Gutenberg.us. N.p., Web. 24 Jan
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