Michigan, Alfred Hershey grew up attending several different public schools in Owosso and

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1 Brett Dempsey Dr. Arneson 25 April 2016 Alfred Hershey: 1969 Nobel Laureate Introduction Born on December 4, 1908 to Robert Day Hershey and Alma Wilbur Hershey in Owosso, Michigan, Alfred Hershey grew up attending several different public schools in Owosso and Lansing. His father worked for an auto-manufacturing firm, and instilled the importance of hard work in his son from an early age. Hershey graduated from Owosso High School in 1925, the same year he enrolled at Michigan State University (AAI, 2016). He was always interested in the sciences, but developed a keen curiosity for the field of bacteriology during his undergraduate years. After earning his B.S. in Chemistry from Michigan State in 1930, Hershey entered a doctorate program in Bacteriology at the same university. His doctoral dissertation was centered on the chemistry of Brucella, a pathogenic genus of bacteria responsible for Brucellosis or undulant fever. Hershey s thesis detailed the chemical mechanisms responsible for the separation of bacterial components (DNA FTB, 2011). Following his doctorate training, Hershey accepted a position as an instructor in the Department of Bacteriology and Immunology at the Washington University of Medicine at St. Louis. While at Washington University, he worked alongside Jacques Jacob Bronfenbrenner, who is considered by many to be a key innovator in the field of bacteriophage research. From 1936 to 1939, Hershey and Bronfenbrenner published multiple papers on the growth of bacterial cultures and the lysogenic properties of bacteriophages (Encyclopedia of World

2 Biography, 2004). Throughout the early 1940s, he performed his own research involving the immunologic reaction of phages and other factors that influence phage activity. In 1943, Hershey began a collaboration with Max Delbruck and Salvador Edward Luria regarding similar areas of phage study. The three scientists went to Nashville and formed what is known as a phage group where they would come together once a year at Cold Spring Harbor to discuss their work and encourage research on particular strains of bacteriophages (Encyclopedia of World Biography, 2004) proved to be a successful year, as Hershey and Luria both discovered that phage viruses, along with the bacteria they infect, could experience spontaneous mutations. Later that year, he and Delbruck discovered that differing strains of bacteriophage could exchange their genetic material within a dually infected bacterial cell. Hershey referred to this process as genetic recombination. In 1950, he moved to Cold Spring Harbor, New York with his assistant Marth Chase and became a staff scientist in the Carnegie Institution of Washington s Department of Genetics. This would set the scene for his renowned Hershey-Chase Experiment (Encyclopedia Britannica, 2016). The Awarding of the Nobel Prize The Hershey-Chase experiment, widely known as the blender experiment, was conducted in 1952 and gave sufficient evidence to allow Hershey to conclude that DNA, rather than protein, is the genetic material of life. This established the framework for modern molecular genetics, earning Alfred Hershey a share of the 1969 Nobel Prize in Medicine. Thanks

3 to this discovery, scientists now knew that DNA is the blue print of all lifeforms on the planet. This catapulted all areas of biological research, opening a myriad of possibilities for scientists to investigate. Hershey not only advanced the field of molecular genetics with this discovery, he uncovered a large piece of the foundation of modern molecular and cellular biology. With how crucial this finding proved to be, one would have a difficult time devising reasons as to why Hershey didn t deserve a Nobel Prize (Berkeley, 2016). Specifically, Hershey and Chase worked with a DNA virus called T2, a bacteriophage that infects E. coli. Bacteriophages that are produced within bacteria growing in radioactive mediums will themselves be radioactive. If radioactive sulfur ( 35 S) is present, they will be included in the protein coats of the bacteriophage; if radioactive phosphorus ( 32 P) is present, then the DNA will become radioactive, not the protein. Hershey found that when bacteriophages containing radioactive phosphorus were allowed to infect nonradioactive bacteria, all of the infected cells also exhibited radioactivity. Additionally, he found that much of this radioactivity was passed on to the offspring in the next generation of bacteriophages. When the bacteria were infected by bacteriophages containing radioactive sulfur on the other hand, little to no radioactivity could be detected in the infected cells. The radioactive sulfur detection followed the removal of the virus coats by an electric blender (Kimball, 2014). These experiments made it clear that the DNA component in the bacteriophages was injected into the bacterial cells while the protein component remained outside. Furthermore, they allowed Hershey to conclude that the injected DNA component is the only component that has the capacity to direct the formation of new virus particles that include protein coats. This provided even more evidence that DNA is the genetic material. Hershey s experiments built on

4 Avery s 1945 demonstration that genes were DNA, providing further support of this claim. Many argue that without his work, Watson and Crick would not have been able to determine the molecular structure of DNA, which earned them a Nobel Prize as well. Hershey s approach was so successful due to the fact that he was willing to stray from the classic visual approach using microscopes. He and his colleagues were true pioneers in the field, focusing on the molecular level and working with bacteriophages invisible to the naked eye (Kimball, 2014). Hershey had to overcome the fact that no one in the scientific community knew what molecule in the cell actually passes on genetic material from parent to offspring. It was hypothesized that proteins accomplished this task, based on the fact that they are more complex molecules than DNA. Hershey ardently attacked this question without bias, looking for evidence regarding the genetic material of life. His discovery transformed the way we ask questions in biological research, and changed the field of molecular biology forever. Now that scientists knew that DNA was the genetic material in the body, they could investigate disease transmission and treatment at many different angles that they once thought not possible. Referring to Hershey, Luria, and Delbruck (who shared the 1969 Nobel Prize) in her book Scientific Elite: Nobel Laureates in the United States, Dr. Harriet Zuckerman asserted that, ''before the prize finally came to the three founding fathers in 1969, it had gone to 15 molecular biologists and biochemists for investigations built on foundations the three pioneers had laid down'' (Altman, 1997).

5 Post-Nobel Prize Activity Retiring from active research in 1972, Hershey performed most of his post- blender experiment research before winning the Nobel Prize in In the 17 years between the Hershey-Chase experiment and the awarding of the Nobel Prize, Hershey continued his work on phage recombination and genetics. Although he published a few works in these years, none received the notoriety of the groundbreaking 1952 experiment. In 1962, he became the Director of the Genetics Research Unit of the Carnegie Institution at Cold Springs Harbor, where he stayed in this position for three years until his retirement. After 1969, Hershey received no other notable scientific awards, however his career included memberships in the prestigious National Academy of the Sciences and the American Academy of Arts and Sciences, an Albert Lasker Basic Medical Research Award, and the Kimber Genetics Award. Although he retired from active research in 1974, Hershey frequently visited his former research laboratory and provided guidance and assistance to those working there. During his free time, Hershey enjoyed reading, planting trees, and spending time in a woodworking shop. After his retirement, he pursued other interests including music and computers (Altman, 1997).

6 Primary Paper Analysis Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage Hershey, A. D. and Chase, Martha. Journal of General Physiology, Volume 36, Issue 1, Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage is without argument the most instrumental work in the career of Alfred Hershey. This paper singlehandedly won him a share of the 1969 Nobel Prize in Physiology or Medicine. It is the direct result of the notorious 1952 Hershey-Chase or blender experiment. The findings detailed in this paper are considered by many as some of the most crucial scientific discoveries in the last 100 years (Altman, 1997). This piece of primary literature details the methods employed by Hershey and Chase to examine the role of DNA and protein in molecular genetics and bacteriophage growth. Hershey bases the investigation that is detailed in this paper on previous work by Doermann, Dissoway, and Anderson. These researchers have shown that bacteriophages T2, T3, and T4 multiply in a non-infective form. Hershey takes this further by examining the molecular mechanism involved in T2 growth. He asks whether DNA and protein have different roles in the growth in bacteriophage T2. Specifically, Hershey uses a variety of bacteriophage T2 called T2H. H refers to the host range mutant of T2, with UV-phage meaning a phage that is irradiated with ultraviolet light from a germicidal lamp. He also used Esherichia coli as a

7 bacteria model with varying sensitivities to T2 strains. Resistant bacteria B/2 is resistant to T2 but sensitive to its H mutant, while resistant bacteria B/2h is resistant to both. These particular bacteria will not adsorb the phages to which they are resistant. The adsorption of either isotope 35 S or 32 P to bacteria was measured by mixing the sample in adsorption medium with bacteria from 18-hour broth cultures that were previously heated. The mixtures were warmed, diluted with water, and centrifuged. Various assays were then performed with sediment and supernatant fractions of the mixtures. The radioactivity of cells was measured using an end-window Geiger counter and dried samples in order to prevent losses by self-adsorption. For standards regarding the radioactivity of 32 P and 35 S, reference solutions were obtained from the National Bureau of Standards. Additionally, glycerol-lactate medium was chosen over other mediums due to its ability to permit bacterial growth without causing undesirable ph changes at the low concentrations of phosphorus and sulfur that were employed. Hershey acknowledges that the radiochemical purity of the prepared samples is somewhat uncertain. Some might see this as a weakness of the study, but I would argue that it is a strength. He acknowledges where the uncertainty lies early in the paper, and proposes that this could be due to the presence of inactive phage particles and empty phage membranes. A critical aspect of the paper is the implementation of 35 S and 32 P. 35 S was used because it was known that they would be incorporated into the protein coats of the bacteriophages and therefore will be detectable by end window Geiger counter analysis. It is incorporated into protein because two of the amino acids, cysteine and methionine, contain sulfur. There are no sulfur atoms in DNA, so this allows Hershey to differentiate the two molecules. 32 P was used

8 because it will be taken up by the DNA because of its many phosphorous atoms, causing it to become radioactive. Additionally, radioactive phosphorous will not be incorporated into the protein, further allowing Hershey to differentiate between the two molecules. By employing the method described above regarding the absorption of radioactive isotopes, Hershey analyzed the sensitization of Phage DNA to DNase by adsorption to bacteria. The main purpose of this area of investigation was to provide further insight into the molecular mechanism by which the virus multiplies, including the alteration or removal of the proactive protein coats of the particles. Hershey derived three main conclusions from this analysis: 1. phage DNA becomes highly sensitive to DNase after adsorption to heat-killed bacteria, 2. DNA of phage adsorbed to live bacteria becomes highly sensitive to DNase after adsorption, and 3. the DNA of phage adsorbed to unheated bacteria is resistant to DNase. These results suggest that the DNA of phage adsorbed to live bacteria is protected by cellular structures immune to DNase activity, most likely a protein coat. Further experimentation confirmed this claim through microscopy after cycles of freezing and thawing of the DNA. Hershey also analyzed the respective levels of survival of infected bacteria after removal of radioactive sulfur and radioactive phosphorus during agitation in a Waring blender. He found that removal of radioactive phosphorus resulted in an approximately 30% survival rate while removal of radioactive sulfur yielded an 80% survival rate. Since 32 P is incorporated into DNA and 35 S is incorporated into protein, these results provide evidence that the DNA is more important for cell survival than the protein. This provides support for the claim that DNA provides molecular instructions for the rest of the cell and is the blueprint. Further investigation with assays of the extracellular isotopes from suspension with agitation and no

9 agitation illustrated that the bulk of the phage sulfur remains at the cell surface during infection, and takes no part in the multiplication of intracellular phage. However, he found that the bulk of the phage DNA enters the cell quickly after adsorption of phage to bacteria. This provides further evidence that DNA is the genetic material, since it seems to be so much more involved in the phage multiplication process. In order to determine whether the DNA or protein is passed from parental phage to their progeny, Hershey employed a glycerol-lactate medium growth process followed by sedimentation analysis using centrifugation in order to determine the levels of 35 S or 32 P in the supernatant or pellet. He found that in the radioactive sulfur preparation, the radioactivity was found in the supernatant in the parental phage and little to none in the progeny, suggesting that the protein was not passed from parent to offspring. In fact, Hershey found that less than 1% of the phage progeny contained the parental radioactivity. On the other hand, he concluded that 32 P was found in the phage progeny at a high rate and was found in the pellet of the parental phage preparation. This is a groundbreaking finding due to the fact that it suggests that DNA is in fact the viral genetic material, not protein as previously thought. The main weakness of this paper is the vague nature by which Hershey describes his assays. Frequently, he mentions that an assay was used, but never returns to this or provides details as to what assay was employed. This could be due to the fact that this paper was written almost 70 years ago and there probably wasn t very many different types of assays used in science, however I still think it would improve the paper if he were to go into more depth regarding the exact methods used.

10 A strength of this paper is Hershey s clear portrayal of the results. It was relatively easy to analyze his results because of the way that he summarized them in numbered lists. This made the main conclusions from each portion of the experiment and each figure very clear and easy to recognize. Additionally, Hershey does a great job of identifying where error and contamination might lie in the experiment and where this could have skewed the results. This adds to the validity of the experiment and makes it easier to accept the methods that he used. Summary Alfred Hershey, a hard-working man from Owosso, Michigan, proved to be a giant in the field of molecular biology. His discovery provided countless avenues of investigation for researchers for years to come. Although he began his career in research focused on bacteriology, his research has been applied across many species including humans. The Hershey-Chase blender experiment proved to be the most important work of his career, and is the main reason that Alfred Hershey received a share of the 1969 Nobel Prize in Medicine or Physiology. Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage is considered to be the most instrumental publication for his receiving the prize (Altman, 1997). Hershey completed a variety of valuable research throughout his career, but none came close to the grandiosity of the blender experiment. Alfred Hershey passed away in 1997, but will always be remembered for his contributions to our understanding of molecular biology and genetics. 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 Hershey (FamousPeople, 2016).

11 References Authors Title Source Altman, Lawrence K. Alfred D. Hershey, Nobel The New York Times Laureate for DNA Work, Dies at 88 The American Association of Alfred Day Hershey AAI: Nobel Laureates Immunologists 1-3. DNA from the Beginning Alfred Day Hershey DNA from the Beginning. ( ) The Editors of the Encyclopedia A.D. Hershey: American Encyclopedia Britannica. Britannica Biologist Encyclopedia of World Biography Alfred Day Hershey Encyclopedia of World Biography

12 Hershey, A. D., and Chase, Martha Independent Functions of Journal of General Viral Protein and Nucleic Physiology ,1, Acid in Growth of Bacteriophage Kimball, John W. The Hershey and Chase Kimball Biology. 19 April Experiment The Famous People Alfred Day Hershey TheFamousPeople.com University of California Berkeley The Hershey-Chase Blender University of California Experiment Berkeley Biology

Lesson Overview Identifying the Substance of Genes

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