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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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DNA Replication

DNA Replication occurs during the S (Synthesis) phase of cell cycle. The purpose of DNA replication is to create an identical copy of all the DNA in the cell so that, following cell division, both daughter cells will have complete copies of all the information required to build a cell and do all the things the cell does.

Data from several laboratories were elegantly integrated by the work of Watson and Crick to describe the structure of DNA as comprised of two anti-parallel strands bound together by polar (hydrogen) bonds between one purine and one pyrimidine.  Including:

1. Erwin Chargaff ‘s observations that

a) DNA was 50% purine (A and G) and 50% Pyrimidine (C and T) and

b) the proportion of A = the proportion of T; the proportion of C = the proportion of G .

2. Rosalind Franklin’s X ray crystallography data that indicated that DNA had a regular, repeating pattern and the molecule was of a specific width.

3. Oswald Avery’s group along with Hershey and Chase established that DNA was the genetic material (therefore making the structure of this molecule of high importance)

4. Knowledge of the distance between molecules engaged in hydrogen bonds.

5. Knowledge of the chemical properties of nucleotide molecules, comprised of hydrophilic deoxyribose sugars and phosphate groups and hydrophobic bases.

Altogether, this information provided enough background for the pair of researchers to arrive at the structure of DNA by engaging in model building.

How this all leads into the mechanism of DNA replication comes down to the following brief statement at the end of Watson and Crick’s Seminal Paper of the structure of DNA:

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”1

 

What did they mean by this?

“The novel feature of this structure is the manner in which the two chains are held together by the purine and pyrimidine bases…joined together in pairs, a single base from one chain being hydrogen-bonded to a single base from the other chain… [O]nly specific pairs can bond together. These pairs are: adenine…with thymine…, and guanine… with cytosine.”1

So, if the sequence of DNA bases on one strand dictates the sequence of the other, then each of the strands can be used as a template to make another. When this is done with each of the two strands, the result is two identical DNA molecules.

It’s one thing to say that it hasn’t escaped your notice that there is a mechanism for duplicating DNA inherent in its structure, but quite another to say that you know how it works.

This was the question that Matthew Meselson and Franklin Stahl were to solve in 1958.2 They imagined three possibilities:

  1. A Conservative method of replication – the original DNA splits open and new strands are made based on that information, then the original strands comes back together and the new strands zip together. We conserve both strands of the original copy.
  2. A Semi-Conservative method of replication – The original DNA splits open and new strands are synthesized to pair with each of the originals, the new DNA then exists with one original strand and one new one.
  3. A Non-Conservative / Dispersive method of replication – Frankly, I don’t know how this would work, but the result would be two new DNA molecules where bits of each strand of each molecule may be from the original or the new DNA.

How to distinguish between these methods?

Meselson and Stahl devised an experiment that in which they grew the bacteria, E. coli in broth containing DNA made of two different isotopes of Nitrogen. In one broth, let’s call it the ‘light’ broth, they had the light form of DNA with 14N, in the other, ‘heavy’ broth, they had the heavy form of DNA with 15N.

One really is heavier than the other. When they are centrifuged, they will come to rest at different ‘heights’ in the tube.

If the bacteria is grown in broth containing only the heavy DNA, and that DNA is harvested and spun down, you would see a tube like (a) containing a single band of the heavy DNA.

If than bacteria was moved into a new medium containing light DNA, and DNA was allowed to replicate once,

Assuming  semi-conservative or dispersive models of development – you would see (b) a single band of intermediate density – because all new DNA would be partly heavy and partly light.

Assuming the conservative model – you would see (c) two distinct bands – one heavy and one light.

So this immediately tests for or against the conservative model.

The actual result was a single intermediate band was found. This eliminates the conservative model of replication, but a second round of replication in the light broth is required to discriminate between those two models.

If the Semi-Conservative model is correct, then the intermediate band would remain, but a new light band would show up (d).

If the Dispersive model is correct, then the intermediate band would inch upwards (become lighter) as more light elements are mixed in randomly within the strands. (e)

What they found was exactly like that pictured in figure d. Further, if the bacteria were allowed to grow for more generations, the ‘light’ band of DNA would become larger as more light DNA is created, while the intermediate band will remain indefinitely.

 

 

Meselson and Stahl with Chase

 

 

References

  1. Watson. J. D. and Crick F.H.C.  “A Structure for Deoxyribose Nucleic Acid”  Nature 171, 737-738 (1953).
  2. Meselson, M. and Stahl, F.W. (1958). “The Replication of DNA in Escherichia coli”. PNAS 44: 671–82.
 
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Posted by on November 27, 2012 in Education

 

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