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Martha Chase, Max Delbruck, and the American Phage Group

Martha Chase is a bit of an enigma. Her career started promisingly enough working in a productive field amongst productive scientists; but following her PhD, her health precipitated setbacks in both her career and her home life from which she did not recover.

She received her bachelor’s degree in 1950 from the College of Wooster. Later that same year, she joined the laboratory of Alfred Hershey in Cold Spring Harbor to work as his lone laboratory assistant asking questions about the mechanisms of life using a viral model system. The virus they used was the bacteriophage T2, a fascinating conglomeration of proteins and DNA that specifically infects bacteria. Through her work with Hershey, she became linked to the remarkable influence of a network of biologists nucleated around the German-American biologist, Max Delbrück, called the ‘American Phage Group.’

Delbrück, himself a Nobel Prizewinner, led a rich intellectual life amongst an elite group of academic luminaries. The Chemist, Karl Friedrich Bonhoeffer, was a close friend and mentor to him during his younger years steering him into the study of physics where he became associated with Wolfgang Pauli and Niels Bohr. It was Bohr’s influence that put him on the path to Biology through its relationship with Physics. Again, not to be on the outside looking in, he became assistant to Lise Meitner who had worked with Nobel Laureate Otto Hahn to discover fission of Uranium (Meitner is often regarded as missing out in the Nobel for anti-Semitic reasons), and with Otto Frisch, who recognized that fission must be accompanied by a massive energy release tying it to both the potential for energy production and a potential massive destructive power. Delbrück initially came to the States to study genetics in drosophila, but made a deeper mark studying viruses, eventually earning a Nobel Prize in 1969 for his work with Salvador Luria and Alfred Hershey, largely thanks to the diligent work of Martha Chase.

Many of the Phage Group’s members are credited with landmark advances in our understanding of molecular biology. Luria, working with Delbrück, demonstrated that mutation of bacteria occurred in a strictly Darwinian sense, i.e. that bacteria could mutate to resist viruses even without the virus being present. This is a fundamental distinction from Lamarck’s notion that evolution was driven by need, rather than by selection of completely random events. It was at this time that he took on and trained his first PhD student, James Watson (who also did something important – I forget what).

In 1949 Renato Dulbecco came to Caltech to join Delbrück’s group with the focus of understanding how some viruses would lead to tumors. Along with David Baltimore and Howard Temin, Dulbecco shared the 1975 Nobel Prize for discovering how these viruses would reverse transcribe their RNA genome into DNA and integrate it into the host’s chromosome.

Matthew Meselson and Franklin Stahl, also working with the phage group, demonstrated that DNA replication is a semi-conservative process retaining one strand from the ‘parent’ DNA and one ‘new’ strand synthesized as a complement to the ‘parent.’ This work did not earn them a Nobel Prize, although it provided early support for Watson and Crick’s DNA structure and remains a landmark experiment in biology that every student is taught.

As evidenced by the sheer number of Nobel Prizes shared by members of this group, the Phage Group and its associates dominated the fields of bacterial genetics and molecular biology. But before those experiments were performed and Prizes collected, the physical molecule carrying genetic material was yet to be discovered.

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Frederick Griffith (see reference 1)

Frederick Griffith was the first to point the way to this molecule by showing bacteria’s mysterious ability to transfer new characteristics (Darwin’s Traits) between organisms. But, tragically, left his work incomplete due to his death in London during WWII. A number of stories exist regarding his whereabouts when he died. Regardless of its veracity, I personally like the one that suggests that he was working late in the lab when it was bombed by the Nazis.

 

Before his death, in the 1920s, Frederick Griffith demonstrated that some element of a bacterium, that is released upon its death, was sufficient to carry genetic information from one strain of bacteria to another. Specifically, he demonstrated that ‘smooth’ pneumococcus, which secreted a glycocalyx, could transfer this trait to ‘rough’ bacteria that lacked the glycocalyx. Clinically, this was very important because the rough type pneumococcus was easily handled by the immune system, while the smooth type colonized the heart and killed the host. He called this element the ‘Transforming Principle,’ but died before he could identify it specifically.2

 

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see reference-2

Experiments showing that the transforming principle was probably DNA were performed by Avery, MacLeod, and McCarty at the Rockefeller Institute in 1944. Despite being both elegant and thorough, many thought these experiments lacked the appeal needed to be convincing.

 

Knowledge of Avery’s work supported the case for DNA as the genetic material, but Protein remained a persistent contender because, with its 20 physiological amino acids, its capacity to carry the information associated with genes seemed more reasonable. DNA, on the other hand, was an arrangement of only four bases, a simplicity that obfuscated its coding potential. One compromise hypothesis suggested that perhaps DNA served as a scaffold for the information-carrying proteins, although Avery’s experiments showing that protein-free DNA preps could transform bacteria strongly argued strongly against this model.

So, the issue remained to be effectively demonstrated denying Avery and his co-workers Nobel. A more satisfying answer, reaffirming Avery’s discovery, was to come from the ever-productive phage group in the hands of Martha Chase.

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Martha Hershey and Alfred Chase (see reference 3)

Working together, as laboratory technician for Alfred Hershey, the two performed their eponymous experiment in 1952 with the purpose of identifying what served as the genetic material in phages.

 

Hershey intended to use the T2 bacteriophage to assess this question, in part because it contained no other molecules such as fats or sugars, making it an exceedingly simple model (See illustration of method, panel A) but also because electron micrographs already hinted of protein ‘ghost’ particles left outside of the cell while new phages were being assembled within. Indeed, by involving only DNA and unglycosylated proteins, it was possible to label the DNA and Protein elements individually using radioactive Phosphorous-32 to mark the DNA and radioactive Sulfur-35 to mark proteins. These isotopes worked well because they were trackable by following the radioactivity, while each was specific to its target due to the natural, exclusive distribution of Sulfur and Phosphorous in Protein and DNA, respectively.

 

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A bacterium with phages attached to its surface and phage capsids assembling within the cell. (attribution unknown)

The basic experiment was simple in theory. T2 bacteriophage was grown in media containing either nucleotides with Phosphorous-32 or amino acids with Sulfur-35. In the first condition, only the phage DNA was radiolabeled. In the second condition, only the protein was labelled (see illustration of method, panel B). Once the phage was prepared, it was allowed to attach to fresh bacteria for a time period known to allow for the passage of genetic material. At this time, the bacterial cultures were moved into a kitchen blender and pulsed to remove the material that did not enter the host cells (the ghosts). Centrifugation permits the separation of the ghosts and any other viruses in the supernatant from the (infected) bacterial cells. The only thing remaining was to check to see where the radioactive elements were: the supernatant fraction that did not enter the cell, or within the cell, where the genetic material was (see illustration of method, panel C). Like most experiments, much of the work invested in the project occurred prior to the actual experimentation in order to optimize each condition.4

 

 

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Illustration of Method for Hershey-Chase Experiment

The answer was clear, Radioactive Sulfur was never found inside of infected cells, only in the supernatant. Radioactive Phosphorus was overwhelmingly found within the cell. With this elegant experiment, the question was answered, and DNA was widely recognized as the genetic material setting up the next, obvious Nobel Prize: what is the structure of DNA? And does this structure reveal any of its properties?

 

At the University of Southern California, Martha continued to study phages under Giuseppe Bertani (Joe to his friends), ultimately following him to the Karolinska Institute in Stockholm, Sweden where she completed her PhD thesis on “Reactivation Of Phage-P2 Damaged By Ultraviolet Light” in 1964.5,6,7 Her obituary, which is one of only a few primary sources of information on Chase, describes life after earning her PhD as plagued with personal troubles arising from short-term memory loss that likely contributed to the end of her scientific career and possibly her marriage.

Despite the fact that this work represented the accomplishment of Hershey in the 1969 Nobel Prize along with Delbruck and Luria, Chase did not share in this honor. As a technician in the lab, it may be that her hands performed many (if not all) of the Hershey laboratory’s experiments, but technicians are rarely (if ever?) included in the Prize on the assumption that it is unlikely that they are major theoretical contributors to the work. Her name, however, will forever be associated with this experiment, serving as a lasting reminder of her contribution to molecular biology.

References

  1. Photograph of Frederick Griffith, photographer unknown
  2. “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Deoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III.” January 1944. Exp. Med., 79: 137-158.
  3. photo: Martha Chase and Alfred Hershey, 1953. Attribution unknown. I found both of these images at https://varietyofrna.wikispaces.com/Hershey+and+Chase
  4. Link to Hershey and Chase’s J. Exp Med paper: http://jgp.rupress.org/content/jgp/36/1/39.full.pdf
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827068/#
  6. http://www.the-scientist.com/?articles.view/articleNo/22403/title/Martha-Chase-dies/
  7. http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll18/id/368326/rec/7
  8. See http://www.nobelprize.org/ for a listing of Nobel Prizes, Biographies, Acceptance Speeches, and even games.
 
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Posted by on October 3, 2016 in Uncategorized

 

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Thinking about thinking.

I’ve often taught Science as a way of thinking critically. That is, science education has (at least) two aspects. First, is the content knowledge. This is necessary because it’s not always necessary to reinvent the wheel. If every person had to start with their own tabula rasa and fill it themselves, without the help of those who came before, progress would be non-existent. Further- and this leads into the second aspect, prior knowledge provides a proving ground for developing critical thinking.

For example, every introductory biology class spends a decent amount of time talking about photosynthesis and cell respiration. Just memorizing the pathways is not enough to actually learn anything. In fact, it’s probably the quickest way to ensure that you don’t learn. Instead, it’s useful to talk about how this pathway was discovered.

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von Helmont

Instead, it’s useful to talk about how this pathway was discovered. What was the question that people sought to answer? What was known /thought / assumed initially? What were the first (apparently unsuccessful) experiments done to address the question?

 

Jan Baptist von Helmont did one of the first good experiments to ask the question: Where does a tree’s mass come from?

He used a willow tree for his experiment and monitored the mass of the tree, the mass of the soil, and the mass of the water he gave it. Because the mass of the soil changed very little, while the mass of the tree grew enormously, he concluded that the tree’s substance came from the water he provided. In his own words, “But I have learned by this handicraft-operation that all Vegetables do immediately, and materially proceed out of the Element of water onely. ”

(It is notable that von Helmont recognized, in other experiments, that carbon dioxide was released from burned wood. He called this ‘gas sylvestre,’ referring to the Latin term for wood / forest, silva. This is important because the majority of a tree’s mass comes from the carbon dioxide in the air. von Helmont didn’t do just one experiment in his lifetime, after all.)

The importance of these historical experiments is that it allows the student to consider, ‘if I were in this person’s position, knowing what he or she did, how would I go about asking such a question?’

It was with this in mind that I came across this video on critical thinking, which I would say is the true value of science.

 

The topics we ask questions about depends on our interests. Perhaps today we are interested in where the mass of a tree comes from and we’ll be biologists. Perhaps most of the time we have a driving interest in the way that molecules interact, so we are primarily chemists. Regardless of the topic, we use the same critical thinking and experimental procedures to answer our questions, so we are really all scientists.

 

 

 

 
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Posted by on September 9, 2016 in Uncategorized

 

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A Genetics Riddle

Along with his brothers, a soldier goes off to war leaving behind his wife and two sons. Six years later he returns to his family after losing both his brothers in action. Something is different though. His wife suspects something, but can’t put her finger on it. She just knows that something is different about her husband. Over the next two years, the family grows by twins (a boy and a girl) and then another girl. Then, in an auto accident, the husband dies and his widow decides that she can now investigate a hunch she has had for some time without upsetting her husband.

That month, she takes all of her children in for their annual checkup and vaccines, and also asks the doctor to check her blood type along with all of the children.

The results, mailed to her (see below) later that week, give her a start as she realizes her hunch was correct.

What was her hunch? How did she arrive at her conclusion?

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Posted by on March 27, 2016 in Uncategorized

 

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What’s all the fuss about Zika Virus?

Check out the Cloudy Media Blog for a good discussion about the Zika Virus and it’s Uber transit pal, the Mosquito. Consider why some people are raising the alarm and others are trying to reassure us that it’s not the end of the world if you do contract Zika.

microcephaly-comparison-triple-350px.jpgFor most people Zika is not all that harmful. the symptoms are often described as similar to a mild case of the flu. But, like rubella, the major risk may be to women who contract the disease while pregnant. It has been found that a rise in microcephaly cases in Brazil has coincided with a rise in Zika cases (increasing since April 2015). However, whether there is a causal link between the virus and the birth defect has been more difficult to demonstrate. Correlation is not Causation, otherwise the link between sunburns and the dramatic rise in ice cream sales seen every summer might make us consider shuttering the Dairy Queen to prevent skin cancer.

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So, why think there is a link between Zika and Microcephaly? The immediate reason is the spike in Zika cases just before the spike in microcephaly.

Why doubt the connection? One reason is that Brazil is not the only country seeing a spike in Zika infections. In fact, Columbia has many more confirmed cases of Zika than Brazil (however it is estimated that Brazil may have a larger population of unconfirmed cases) but has not yet seen the spike in microcephaly (or at least I haven’t been able to find good data on it). So, shouldn’t Zika be causing birth defects in children regardless of where they live?

To answer this question, some have decided to look directly at the brains of children born (or fetuses not born) with microcephaly to see if there is any evidence of the virus there. In work performed by the CDC, four of four cases were positive by RT-PCR (a technique looking for virus-specific RNA), and sequence analysis provided further evidence of Zika virus infection, revealing highest identities with Zika virus strains isolated from Brazil during 2015. Workers in San Paulo, Brazil found that the mothers of 23 of 29 infants (79.3%) with microcephaly reported signs of Zika virus infection. The majority of these reported that the symptoms occurred during the first trimester of the pregnancy.

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Now, if you’ll excuse me, I have to get myself into a vector for the airport. Seriously, check out Cloudy Media.

 
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Posted by on March 18, 2016 in Uncategorized

 

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Back from the Dead

Halloween seems like a good time to resurrect old blog posts that haven’t seen the sunlight for several years. Creeping out of the tomb is my first blog post about Genes, DNA, Memes, and GMO foods. Rather than post it here, I decided to post it over on my Medium site to see if it can catch some new eyes.

Take a look: Linked Memes

 
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Posted by on October 26, 2015 in Uncategorized

 

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The Skinny on Cancer Immunotherapy: focus on CAR T Cells

Screen Shot 2015-10-22 at 9.47.44 AMOne of the more interesting modern therapies being used to fight cancer aims to coax, or engineer a patient’s own T Cells to fight disease.
In very basic terms, the principle is not dissimilar to vaccine strategies used against infectious disease. That is, they direct and boost the patient’s immune system against target cells. One reason vaccinations have been so successful in fighting disease is that they leave much of the hard work to nature – the same nature that has been keeping you and your ancestors healthy enough to successfully reproduce for millions of years. Give the immune system a push in the right direction with a well designed, safe vaccine and the body does the rest leading to (at least theoretically) life-long protection. At this point, the most limiting factor to how long protection lasts is because we live so much longer than humans have ever lived before.

William-Coley_206x236Immunotherapy against cancer has been an area of interest since the 1890s, when William Coley observed that cancer patients who had infections at the site of surgical resection fared better than those without infections. Rather than dismissing this observation as uninformative, he speculated that the immune system plays an active role in preventing or regressing tumors.

In fact, the immune system is constantly performing ‘immune surveillance’ to prevent newly-generated cancer cells from developing into tumors. Direct evidence for this involves ‘knocking out’ elements of the immune system and watching for cancer. As predicted by the theory, immunodeficient animals develop spontaneous tumors at a higher rate, and earlier than do immune-competent animals.

The pudding: (from : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1857231/)

Evidence for Immuno Surveillance

Evidence for Immuno Surveillance

But vaccinations used against infectious diseases are given before the patient is infected (known as prophylactic vaccination).

How can we immunize people against all the cancers that may crop up in all their various forms?

The answer is – we don’t. In the case of cancer, we perform vaccinations ‘therapeutically’, or after disease has started. Otherwise there really would simply be too many possible targets.
So, we wait, and help the body to fight the challenges that actually do arise.
A number of methods have been developed and tested to accomplish this, here, I want to specifically address a personalized therapy that takes cells from the patient, ‘aggravates’ and expands them, and then re-infuses them into the same patient.
Currently, there are several ways this is being done with various outcomes.

One method involves immunizing the patient against killed cancer cells isolated from the themselves (via surgery), then harvesting the reacting cells and expanding them to numbers much higher than those reached in vivo, and then re-administering to the patient as a jump-start to immunity. The advantages are that these immune cells are ‘self’ and therefore do not have to be ‘matched’ to the recipient a la transplantation surgery. It is also possible to remove any regulatory cells (T regs), that often impair immune responses, prior to re-administration.
A more engineered response has been investigated by investigators such as Carl June, of the Abramson Cancer Center at the University of Pennsylvania. These cells, known as CAR T Cells express ‘Chimeric Antigen Receptors’ directly target tumor cells using transgenic antibodies that incorporate the intracellular signaling domains of up to three immune-activating receptors. See the illustration below for details of this receptor’s design (taken from ‘Breakthroughs in Cancer Immunotherapy webinar by Dr. June )
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In the case of CAR T Cells, most have been made to fight B Cell Chronic Lymphocytic Leukemia (B Cell CLL). These cells are a good test case for the technique for a number of reasons, including the fact that they uniformly* express a marker called CD19 on their surface and also because they are a ‘liquid tumor’ – meaning that the cancer cells are individual cells moving through the body (at least many are). Treatment of solid tumors can bring added complications such as the need to infiltrate the tumor in order to find target cells.
As I said, CD19 is a common protein expressed on these cells. Therefore, at least the CAR receptor part is standardized between patients – this is the piece that is added to cells transgenically so that they will bear a receptor known to engage the target cells with high affinity. Because it must be added to the patient’s own cells, this is accomplished using a viral vector that infects the T Cells in culture and provides the DNA required to make the receptor. (In case you’re worried about the virus, these are engineered to only infect the first cell they encounter, they cannot reproduce themselves and continue an infection)
So, let’s walk through it:

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1. Blood cells are isolated from a patient
2. T Cells are purified (i.e. isolated)
3. T Cells are infected with virus in culture.
4. T Cells grow up with the chimeric antigen receptor expressed on their surface
5. These cells are then re-injected into the patient via I.V. drip over about 30 minutes time.
6. Let the cells do the work

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This therapy has an impressive track record so far with studies with success rates from ~60%- 90% of patients responding and remaining disease free for years (Maude et al).
Following the initial infusion of cells, CAR T Cells proliferate in vivo to very high numbers and can even form immunological memory cells to come to the rescue in the event of a relapse.
So, what next?
A number of startup companies have emerged to tackle the logistics of bringing this type of therapy – an extreme example of personalized medical care – into being. Unlike traditional drug therapies where a single compound is mass produced and distributed world-wide, each patient must have their own cells processed and returned to them for infusion. This therapy is much more of a service, and as such, will require physical locations across the country that can manage the handling of cells.
The up side, however, is potentially transforming fatal diseases into manageable ones with a high quality of life after therapy.
Just ask Emma:
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*Well, most do, anyway.

 
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Posted by on October 22, 2015 in Uncategorized

 

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Vaccines

To all my former students (as well as everyone else who reads this blog): please check out “Vaccines” a PBS documentary about the challenges faced by society revolving around maintaining society’s immunity against a number of vaccine-preventable diseases. Vaccines airs on PBS stations on August 26th at 9pm. You can also watch the film online here.

 
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Posted by on August 26, 2015 in Uncategorized

 

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