Transcript Independent Functions of Viral Protein and Nucleic Acid in Growth of

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
Independent Functions of Viral Protein and Nucleic Acid
in Growth of Bacteriophage
● Background
● Experiments Conducted
● 8 in total
● Discussion & Summary
● How Paper Impacted Future
ALFRED DAY HERSHEY
ALFRED DAY HERSHEY
ALFRED DAY HERSHEY
MARTHA CHASE
MARTHA CHASE
MARTHA CHASE
NOW FOR THE PAPER…
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
VS
OR
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
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
The Chemical Morphology of
Resting Phage Particles
- Anderson 1949
Salt
Water
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
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.
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.
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.
●
●
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.
Results
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.
Why was the sulfur isotope not 100%
extracted ?!?!
- 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.
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.
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.
The results from the blender experiment allow for other experiments to be
conducted on just the bacteria with the adsorbed phage DNA.
● 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.
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.
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.
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.
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.
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.
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.
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.
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.
• 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
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.
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.
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.
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.
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.
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
•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.
•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
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
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
•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
•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
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”
Experiment 7:
A Progeny of S35-Labeled Phage
Nearly Free from the Parental Label
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
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.
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
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.
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
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!
Findings
•
Stripping reduces almost all of the S35-content of all fractions
•
With regards to the fraction containing most of the phage progeny, the S35content 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
Experiment 8:
Properties of Phage Inactivated by Formaldehyde
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
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!
Properties of Inactivated Phage
1) It is absorbed to sensitive bacteria
• ~70%
2) The absorbed phage kills bacteria
• ~35% effective
3) The DNA of the inactive particles is resistant to DNase
• However, is sensitive to osmotic shock
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.
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
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?!
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.
Unanswered Questions
1) “Does any sulfur-free phage material other than DNA enter the
cell?”
2) “If so, is it transferred to the phage progeny?”
3) “Is the transfer of phosphorus to progeny direct or indirect?”
Discussion
• Hershey and Chase did successfully show that:
– Physical separation of bacteriophage T2 into genetic and non-genetic parts is
possible!
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.
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
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
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
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
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!
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.
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
-
"for their discoveries concerning the replication
mechanism and the genetic structure of
viruses"
•
Nobel Prize Organization
Where are they Now?
Alfred D. Hershey (1908 – 1997)
• 1945
– 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
Where are they Now?
Martha Chase (1927 – 2003)
• 1950’s
– 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
American Phage Group
• What was is?
• 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
Where are we Today?
• Genome mapping
– 23andMe
• Genetic engineering
– Designer children
• Disease prediction models
– ex: Huntington’s disease
“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
References
Hershey, A., & Chase, M., (1952). Independent Functions of Viral Protein and
Washington, Cold Spring Harbor, Long Island.
Nucleic Acids in Growth of Bacteriophage. Department of Genetics, Carnegie Institution of
"The Nobel Prize in Physiology or Medicine 1969". Nobelprize.org. Nobel Media AB 2014. Web. 26 Jan 2016. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/1969/>
"Alfred D. Hershey - Biographical". Nobelprize.org. Nobel Media AB 2014. Web. 26 Jan 2016. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/1969/hershey-bio.html>
"Chase, Martha Cowles (1927- )." World of Microbiology and Immunology. 2003. Encyclopedia.com. 26 Jan. 2016 <http://www.encyclopedia.com
BrainyQuote,. "Alfred Day Hershey Quotes At Brainyquote". N.p., 2016. Web. 27 Jan. 2016.
Carr, Dr. Steve. "Hershey And Chase 1952". Bio 4241- Advanced Topics in Genetics. N.p., 2016. Web. 24 Jan. 2016.
Carr, Dr. Steve. "Bio4241 - Advanced Topics In Genetics". Mun.ca. N.p., 2016. Web. 24 Jan. 2016.
Encyclopedia.com,. "Chase, Martha Cowles (1927- ) – FREE Chase, Martha Cowles (1927- ) Information | Encyclopedia.Com: Find Chase, Martha Cowles (1927- ) Research". N.p., 2016.
Web. 24 Jan. 2016
“Gender Bias in Science, Part IV: Martha Chase”. The Mad Science Blog. http://www.themadscienceblog.com/2013/10/gender-bias-in-science-part-iv-martha.html. Oct. 28, 2013
Gutenberg, Project. "American Phage Group | Project Gutenberg Self-Publishing - Ebooks | Read Ebooks Online". Gutenberg.us. N.p., 2016. Web. 24 Jan. 2016.
Molecular Genetics. (2016). Retrieved January 26, 2016, from http://biology.tutorvista.com/cell/molecular-genetics.ht