Dr. Pnina Abir-Am, Resident Scholar

Pnina Abir-Am

Dr. Pnina Abir-Am, historian of science at Brandeis University’s Women’s Studies Research Center, is the first individual to complete a term as Resident Scholar in the OSU Libraries for the 2012-13 school year.  An accomplished scholar, Abir-Am has authored and edited a number of noteworthy publications, including the influential book Uneasy Careers and Intimate Lives: Women in Science, 1789-1989 (Rutgers University Press, 1987, 1989) co-edited with Dorinda Outram.

Abir-Am traveled across the country to conduct research in support of another book, DNA at 50: A Revisionist History of the Discovery of DNA Structure, scheduled for publication in 2013.  Delving into the Pauling Papers, the Jack Dunitz Papers, the David and Clara Shoemaker Papers and the History of Science Oral History Collection, Abir-Am sought “to better explain Pauling’s failure with solving the structure of DNA by examining in greater detail his deployment of a group known as ‘Pauling’s boys.’”

In her Resident Scholar presentation, Abir-Am argued – as have many others – that Pauling was ideally positioned to solve the DNA structure, given his great successes in protein research from 1936-1951 and culminating in his elucidation of the alpha-helix.  The question then, is why did he fail to discover the double helix?  Why did he lose the “race” to James Watson and Francis Crick?

The reasons for the failure are manifold, and Abir-Am acknowledges many that have been pointed out by other researchers.  For one, Pauling was very casual in his approach, believing protein structures to be of more importance than DNA.  He also underestimated the research being conducted by certain of his peers, including Erwin Chargaff, J.T. Randall and Rosalind Franklin.

In particular, Abir-Am argues that Pauling disregarded the work being conducted at Kings College, London, believing that physicists like J.T. Randall and Maurice Wilkins could not be expected to solve a complex biological structure like DNA, as their training left them ill-equipped for the task.  By the time Pauling did get serious about the DNA structure, he was too far behind the competition, using poor quality data and rushing a structure to print. Indeed, in the end, Pauling’s attitude toward DNA could be summed up as “too little too late,” a situation further reinforced by the political problems – culminating in the revoking of his passport – that he faced throughout 1952.

Abir-Am sheds new perspective by focusing on the social structure surrounding Pauling at Caltech during the early 1950s. In examining the story from this perspective, Abir-Am wonders what “Pauling’s boys” – understudies, peers and other colleagues including Alexander Rich, Robert Corey, Eddie Hughes, Verner Schomaker, Jerry Donohue, David Harker and Pauling’s second-born son, Peter – could have done to render Pauling’s attempt at DNA more successful.

Abir-Am posits that “the boys” could have done plenty: collect x-ray crystallographic data, collaborate on model building, make calculations, serve as delegates at conferences and even collect intelligence on rivals.  To some extent all of this did occur, but never to the point where Pauling shied away from his manifestly wrong triple-helical structure.

In thinking about what could have gone differently, Abir-Am offers three possible conjectures as to why “the boys,” all hugely talented, didn’t steer Pauling down a more productive path:

1. They did voice their objections but Pauling ignored them since, after the success of the alpha-helix, he was no longer seeking advice;
2. Long accustomed to accepting Pauling’s ways, “the boys” lost the ability to criticize his work;
3. Pauling did not inform “the boys” of his interest in DNA because he wanted to surprise them.

By the conclusion of her stay, Abir-Am was still wrestling with these questions and evaluating her conjectures.  An entire chapter of her DNA book will be devoted to Pauling’s failed structure – we’ll be very excited to read it!

The OSU Libraries Resident Scholar Program offers stipends of up to $2,500 per month to support research using the collections of the Special Collections & Archives Research Center.  For more on the program, check out its homepage. And to read of the work done by past Resident Scholars, see this link.

Update

After seeing this post, Dr. Abir-Am asked that we add some comments of her own, which are included here.

My initial reaction to OSU-SCARC’s (Oregon State University, Special Collections and Archive Research Center) Paulingblog’s entry of 11-21-12, reporting on my lecture “‘Pauling’s Boys’ and the Mystery of DNA Sructure” was “Wow, they did a better job than I might have done on my own!” Indeed, OSU-SCARC’s Program for Resident Scholars is a scholar’s paradise: a spacious reading room flooded by sunlight provides a superb “room with a view” of gorgeous Oregon trees. State of the art equipment scans archival documents straight into your flash drive. Rare, as well as recent, books that scholars might need to complement one’s ongoing archival research, line the reading room’s walls forming tasteful panels. The entrance is flanked by two glass cases for archival exhibits that rotate periodically and give the foyer a museum look.

But above all, SCARC is a paradise because of its angelic people, all eager to help resident scholars make the best of their precious stay. I was amazed at how readily the SCARC personnel not only guided me through the maze of archival documents in their care, but also helped me in preparing essential visuals. By displaying photomontages of Pauling and his associates, I was better able to convey his enigmatic predicament, as a leading molecular structurist who missed the solution of DNA structure, even though he was surrounded by many gifted and loyal associates, or “boys” in his era’s jargon. Along these lines, a slide of attendees at the Pasadena international conference on “Protein and Nucleic Acid Structure” which Pauling organized in September 1953, captured by photo 2 above, (click for enlargement) distinguished between “boys” from rival groups by color circles around their heads. These graphical devices were critical for my new argument that the outcome of competition over DNA structure was a matter of group rather than individual action.

Having spent considerable time in many archives on both sides of the Atlantic ocean, I have to conclude that OSU-SCARC, situated in the remote splendor of the Pacific Northwest, provides greater scholar-friendly opportunities than anything I have seen, including my prior favorite CCAC. (Churchill College Archive Center in Cambridge, UK) I now count SCARC scholars among my cherished colleagues and consider their work to be a valuable resource for my own chapter on Pauling & Co.’s effort with DNA structure. Last but not least, SCARC’s interest in this chapter, as well as in my forthcoming book DNA at 50 proved invigorating in propelling me toward a speedier revision of both chapter and book.

The Paulingblog’s Photo 2 conveys the civilized environment of OSU Libraries’ Willamette Lecture Room. For the sake of completeness, I wish to remind future applicants that the environment outside OSU’s library can also become a much cherished memory, especially the wild rapids of the McKenzie River which we survived during the Labor Day weekend preceding my 9-5-12 talk. Hopefully, the treasures I left untouched, whether in the archive or in the nearby Oregonian wild nature (e.g. Upper Klamath – I signed a petition to open it for rafting – Crater Lake, Sunset Bay) will soon cheer additional beneficiaries of SCARC’s Program for Resident Scholars.

Rafting on the McKenzie River, Labor Day weekend, 2012.

Pauling Predicts the Process of Gene Replication

A segment of the original Watson and Crick DNA model. 1953.

“…I realized that I myself might discover something new about the nature of the world, have some new ideas that contributed to better understanding of the universe. For seventy years the motive to obtain greater understanding has dominated my life.”

-Linus Pauling. “The Nature of Life, Including My Life. Chapter 1 – How I developed an Interest in the Question of the Nature of Life.” May 5, 1992.

On May 28, 1948, Linus Pauling gave the 21st Sir Jesse Boot Foundation Lecture at the University of Nottingham. His talk, “Molecular Architecture and the Processes of Life,” presented many interesting examples of the important roles that certain molecules play in the human body. In so doing, Pauling discussed topics such as respiration, genetics and the immune system, and in typical Pauling fashion, displayed a knack for providing simple yet fascinating explanations of complicated subject matter. Although the entirety of his speech is interesting, Pauling’s comments concerning the gene were clearly well ahead of his time, and that is the focus of today’s post.

By 1948 it had already been suggested, through experimentation by Oswald Avery, that DNA was the genetic material. However most major scientists, including Pauling, still thought it more likely that proteins, being more complex and versatile substances than DNA, would carry the building blocks of heredity. As a result, DNA didn’t gain much importance until James Watson and Francis Crick discovered its structure in 1953. But scientists concerned themselves with trying to understand the gene long before they were aware of its place in the DNA molecule.

Pauling and two colleagues in Glasgow, Scotland, April 1948.

Included among these interested researchers was Pauling, who in his Boot Lecture predicted both the basic manner in which genes act as templates for proteins as well as the means by which gene replication might occur.

 I believe that the same process of molding of plastic materials into a configuration complementary to that of another molecule, which serves as a template, is responsible for biological specificity. I believe that genes serve as the templates on which are molded the enzymes that are responsible for the chemical characters of the organisms, and that they also serve as templates for the production of replicas of themselves.

As it turned out, Pauling’s simple statement had outlined the basics of the now familiar mechanism for the transcription of a protein from an RNA molecule. At the time of his talk, he may not have known the specific elements of the procedure, but the bulk of his prediction was more or less spot-on.

So an impressive start, but Pauling wasn’t done there. Continuing, he commented on how he imagined the gene might replicate itself.

The detailed mechanism by means of which a gene or a virus molecule produces replicas of itself is not yet known. In general the use of a gene or virus as a template would lead to the formation of a molecule not with identical structure but with complementary structure. It might happen, of course, that a molecule could be at the same time identical with and complementary to the template on which it is molded. However, this case seems to me to be too unlikely to be valid in general, except in the following way. If the structure that serves as a template (the gene or virus molecule) consists of, say, two parts, which are themselves complementary in structure, then each of these parts can serve as the mold for the production of a replica of the other part, and a complex of two complementary parts thus can serve as the mold for the production of duplicates of itself.

Again, Pauling hit the nail right on the head. We are now aware that DNA replication occurs precisely in this manner, and the fact that he was able to logically deduce the essentials of the mechanism without knowing the site or the structure of the gene is rather remarkable.

To read Pauling’s entire speech, click this link. For more information on Linus Pauling ranging from his attempts at elucidating the structure of DNA to his prolific peace work, please visit the Linus Pauling Online portal.

The Triple Helix

“We have seen Paulings paper on Nucleic Acid. Have you? It contains several very bad mistakes. In addition, we suspect he has chosen the wrong type of model.“

-James Watson, letter to Max Delbrück, February 20, 1953.

We were very interested to see that a model of the Pauling-Corey “triple helix” structure of DNA has been built by Farooq Hussain.  As Hussain notes on his website, the model was constructed based on drawings published by Linus Pauling and Robert Corey in their paper detailing the incorrect structure.

The proposed triple helix structure of DNA. Model by Farooq Hussain.

Hussain’s representation of the triple helix is striking; especially so when compared with Watson and Crick’s far more elegant and intuitive double helix, surely the most famous molecule in history.

Double helix model, courtesy of P. Shing Ho.

Indeed, Watson was well within his right to feel confident in his February letter to Delbrück.  As he noted before closing, “Today I am very optimistic since I believe I have a very pretty model, which is so pretty I am surprised no one has thought of it before.”

For more on the triple helix, see our write-up on the subject, published in April 2009.

DNA: The Aftermath

Pastel depiction of the DNA base pairs by Roger Hayward.

The solving of the double helix structure of DNA is now considered to be one of the most important discoveries in modern scientific history. The structure itself suggested a possible mechanism for its own replication, and it also opened up a huge window of opportunity for advances in multiple fields ranging from biology to genetics to biochemistry to medicine. Almost immediately after James Watson and Francis Crick announced their structure, new research began based on the structure’s specifications.

An Early Idea from George Gamow

The Pauling Papers contain an interesting example of research done on the structure of DNA mere months after its discovery. On October 22, 1953, the Russian-born physicist (and founder of the “RNA Tie Club“) George Gamow sent a letter to Linus Pauling that mentioned some work he had been doing with DNA. Gamow explained that he had found a manner by which the twenty amino acids that make up proteins could be related to different combinations of the four nucleotides found in DNA.

At this time, it wasn’t known that the DNA strands unwind during replication, and Gamow assumed that protein synthesis occurred directly on the double helix. He suggested that a “lock and key relationship” might exist between each amino acid and that the “holes” formed between each complementary base pair in the DNA chain. Science is now aware that this is not the case, but Gamow’s letter is nicely demonstrative of the innovative research ushered in by Watson and Crick’s solving of DNA.

Excerpt from Gamow's letter to Pauling, October 22, 1953.

Click here to view Gamow’s entire letter, and here to read Pauling’s response.

RNA

As the buzz around DNA started to die down, scientists began to move toward the next logical step: RNA. By then, Watson and Crick’s structure was widely accepted, and it had been clear for some time that DNA was the site of the gene. So, then, how did DNA transfer its information to RNA, and finally on to proteins?

Gamow’s above suggestion was a possibility, but it didn’t even involve RNA. Watson spent some time playing with the matter, but was not able to equal his luck with DNA. Unfortunately, it would be quite some time before this mechanism was elucidated. Even now, some of the finer details of how this is accomplished are not completely understood.

Four members of the RNA Tie Club, 1955. Clockwise from upper left: Francis Crick, Leslie Orgel, James Watson and Alexander Rich. Founded by George Gamow, the RNA Tie Club met twice a year in pursuit of greater understanding of RNA.

Eventual Honors

Unsurprisingly, as time went on, Watson and Crick began to accumulate awards for their work with DNA. On December 15, 1959, Linus Pauling responded to a previous letter sent to him by Sir William Lawrence Bragg soliciting Pauling’s support of the nomination of Watson and Crick for the Nobel Prize. In this letter, Pauling stated that he would indeed be willing to write the requested letter of support. However, contrary to Bragg’s suggestion that they be nominated for the prize in chemistry, Pauling stated his belief that a prize in physiology or medicine would be much more fitting.

Several months later, on March 15, 1960, Pauling finally sent his letter to the Nobel Committee.  By the time of its authorship, Pauling’s feelings about the importance of Watson and Crick’s work had become even more tepid.

While acknowledging that “the hydrogen-bonded double-helix for DNA proposed by Watson and Crick has had a very great influence on the thinking of geneticists and other biologists,” Pauling notes that their work was, at least to some degree, “stimulated” by his and Robert Corey’s incorrect triple-helix structure, and abetted by Maurice Wilkins‘ x-ray photographs.  Pauling also points out that Wilkins, Corey, Karst Hoogsteen and himself had already tweaked the Watson-Crick model a bit, “which suggests the possibility that a further change in the structure of nucleic acid may be found necessary.”

In the end, Pauling couldn’t bring himself to go through with the promised nomination.

It is my opinion that the present knowledge of the structure of polypeptide chains in proteins is such as to justify the award of a Nobel Prize in this field in the near future, to Robert B. Corey for his fundamental investigations of the detailed molecular structure of amino acids and the polypeptide chains of proteins or possibly divided between him and Kendrew and Perutz. On the other hand, I think that it might well be premature to make an award of a Prize to Watson and Crick, because of existing uncertainty about the detailed structure of nucleic acid. I myself feel that it is likely that the general nature of the Watson-Crick structure is correct, but that there is doubt about details.

Pauling’s hesitations served only to delay their inevitable receipt of a Nobel Prize for a short time. In 1962, Francis Crick, James Watson, and Maurice Wilkins shared the award in Physiology or Medicine “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.”

The discovery of the structure of DNA was clearly one of the most important discoveries in the modern scientific era. Not only was it a huge breakthrough in itself, but it also opened the door for major advances in numerous other science-related fields. For more information on DNA, check out the rest of the posts in our DNA series or the website on which they are based, “Linus Pauling and the Race for DNA: A Documentary History.” For more information related to Linus Pauling, please visit the Linus Pauling Online portal.

Letters to Peter

Linus and Peter Pauling at Warwick Castle, England. 1948.

“You know how children are threatened ‘You had better be good or the bad ogre will come get you.’ Well, for more than a year, Francis and others have been saying to the nucleic acid people at King’s ‘You had better work hard or Pauling will get interested in nucleic acids.’”

-Peter Pauling. Letter to Linus Pauling, January 13, 1953.

Normally, when Linus Pauling became interested in something, he would dive headlong into it. Hours and hours of his time, over weekdays and weekends, would be committed to research in pursuit of fleshing out every last useful detail. This arduous process is best illustrated by his work on the nature of the chemical bond, work which would later win him a Nobel Prize in Chemistry.

Pauling’s experience with DNA, however, was not an example of this typical approach.

First, it should be noted that Pauling did not have years to spend working on DNA. Its importance was fully realized in the summer of 1952, less than a year before Watson and Crick elucidated its structure, and although Pauling actually began studying nucleic acids as early as 1933, he wasn’t able, or willing, to spend a significant amount of time on a molecule that was perceived to be relatively unimportant.

Even after learning of the importance of DNA, Pauling still didn’t make time for it. As emphasized in earlier posts on Linus Pauling and DNA, Pauling remained very much preoccupied with his work on the nature of proteins.

An examination of Pauling’s correspondence with his son Peter – a man uniquely positioned in the middle of the DNA story – reveals that other matters, many of them trivial, also took precedence over Pauling’s pursuit of the structure of DNA.

In the fall of 1952, Peter Pauling, an aspiring crystallographer and the second oldest of the four Pauling children, began his graduate studies at the University of Cambridge. Coincidentally, James Watson and Francis Crick were also at Cambridge at this time, and not long after his arrival, Peter had met them, become an office-mate, and was spending off-hours time with the duo.

Because Linus Pauling and the Watson-Crick tandem were both attempting to solve the structure of DNA, Peter’s arrival at Cambridge gave his father an excellent opportunity to keep tabs on the work being done by his competitors in England. A close examination of the voluminous father-son correspondence from this era suggests, however, that DNA was far from a pressing topic in Pasadena.

Also, as to your curtains: will you check the dimensions and let us know. You say in your letter two windows 6’ 6” high, 50” and 37” wide respectively, in other words four curtains each 48” wide. Mama thinks that you probably mean four curtains each 36” wide. It would be hard to get the wider material.

Also, would you write us as to the exact points between which the vertical dimensions are measured. What is the distance from, say, the top of the window frame (or some other exactly specified locus) to the floor, and also to the bottom of the window frame? Mama thinks that probably the curtains should reach all the way to the floor, but in any case they should extend from the top of the window frame to the bottom of the window frame (if you have window frames), or from a point a little below the opening at the bottom. She suggests that one of your old curtains might serve for one of the windows, and that she would then have to make only a pair for the larger window.

I sympathize with you about the bed. I remember sleeping on a bed which had a two by four across under my ear; it was not very comfortable.

-Linus Pauling, letter to Peter Pauling, October 22, 1952.

Linus first wrote to Peter in England on October 22, 1952. By this time, the elder Pauling was well aware of the importance of DNA, but had not yet devised a structure. Watson and Crick, on the other hand, had developed a structure for DNA a year earlier. Although their model turned out to be incorrect, the two men continued their work with nucleic acids. Clearly, for Watson and Crick, DNA was becoming extremely important. For Pauling this did not appear to be the case – although Watson and Crick were both mentioned in this first letter, DNA was not.

As it turns out, other subjects – including, but not limited to, curtains for Peter’s new apartment, recent travels and upcoming travel plans, finances, and, of course, cars – were much more prevalent than was DNA in the Paulings’ early correspondence.

As time went on, nucleic acids naturally became a slightly larger topic, though never did they assume center stage. Take, for example, this letter sent from Linus to Peter on February 4, 1953. By the time of its authoring, Linus Pauling had completely developed his structure, and had also sent off his manuscript for publication, a development which merited one paragraph worth of description. The rest of the letter is used to discuss, in great detail, Pauling’s plans to travel to England and also his keen interest in purchasing a new Riley from the U.K.-based International Motors. (Being something of a family obsession, cars were a very popular subject in many of the letters between Linus and Peter.)

In another letter from Pauling to Peter written on March 10, 1953, DNA plays a much larger role. This time, about half of the three-page document is dedicated to discussing various aspects DNA; the remainder focuses on travel plans and automobiles.

Peter Pauling, December 1954.

The other letters follow this same trend. Clearly, Linus and Peter’s lengthy discussions on subjects such as cars, traveling, curtains, and other aspects of science suggest that Pauling wasn’t interested in DNA on the level of certain other scientific pursuits.

Another interesting aspect of the correspondence between Linus and Peter Pauling is the opportunity that it provides for tracking the evolution of the consensus response to Pauling’s structure.

As might be expected, Peter’s reaction stayed upbeat throughout all of their letters. However, as time progressed, it is clear that Peter became less-confident that his father had solved DNA. For example, in a few of the earlier letters, Peter mentions that Watson and Crick earlier devised and discarded a structure similar to the Pauling-Corey triple helix, but that the opinion at the Cavendish Laboratory is that Pauling’s structure is a good one, albeit “pretty tight.”

From that point on though, Peter begins talking less about Pauling’s structure, and more about work being done by Watson, Crick, and Rosalind Franklin. One might deduce that, although Peter didn’t specifically issue a disagreement with his father’s structure, he did develop a certain degree of skepticism as time progressed. Peter also does not often mention other opinions of his father’s structure, most likely because, upon further examination, it was not well-received by the English contingent.

Peter Pauling Discusses His Father’s Strengths and Personality

For more information on DNA, please visit the Race for DNA website. For more information on Linus Pauling, check out the Linus Pauling Online portal.

Chargaff’s Rules

Erwin Chargaff, 1930.

“We have created a mechanism that makes it practically impossible for a real genius to appear. In my own field the biochemist Fritz Lipmann or the much-maligned Linus Pauling were very talented people. But generally, geniuses everywhere seem to have died out by 1914. Today, most are mediocrities blown up by the winds of the time.”

-Erwin Chargaff, 1985.

Erwin Chargaff, (1905-2002) a biochemist born in Austria, became interested in DNA earlier than most. In the 1930s, while he was working with the bacteria Rickettsi, he became aware of nucleic acids, and decided to educate himself about them.

In 1944, after Oswald Avery published his paper detailing the transforming principle of the Pneumococcus bacteria, Chargaff decided to devote his laboratory almost entirely to the chemistry of nucleic acids. Experimenting with these delicate substances was not an easy task, but eventually a chromatographic technique was developed that would allow for the separation and analysis of the base rings in DNA. This work would later lead to the development of Chargaff’s Rules, the topic of today’s post.

The guanine-cytosine base pair.

DNA has two main structural components – a backbone made up of sugar and phosphate groups, and a series of bases found in the middle of the molecule. There are four different bases found in DNA: Adenine (A), Cytosine (C), Guanine (G), and Thymine (T). These four bases can be divided into two categories, pyrimidines and purines. The pyrimidine bases, Cytosine and Thymine, contain only one ring, while the purine bases, Guanine and Adenine, contain two rings. In the DNA structure, the bases pair complementarily, meaning that a purine base will bind with a pyrimidine base. More specifically, Adenine binds with Thymine and Cytosine binds with Guanine.

The adenine-thymine base pair.

Although this information is now considered fundamental biology, it wasn’t fully understood until after Watson and Crick discovered the structure of DNA in 1953. However, Chargaff’s research in the late 1940s had suggested that the four bases paired in the manner described above.

When Chargaff first decided to devote his laboratory to nucleic acids, he allowed a postdoctoral student named Ernst Vischer to choose his research program from a list of suggested topics. Vischer decided to analyze the purines and pyrimidines in nucleic acids, and went to work developing the chromatographic technique so crucial to isolating the bases. Although his technique was rather crude, it did the trick and Vischer achieved great success. The results of the base analysis showed that the amounts of Adenine and Thymine were about equal, and also that the amounts of Guanine and Cytosine were about equal. Eventually, Chargaff came to the conclusion that in a single molecule of DNA, Guanine/Cytosine = Adenine/Thymine = 1. This concept would later become known as Chargaff’s Rules.

Chargaff’s Rules were officially announced in a lecture delivered in June of 1949 and were first published in May of 1950. However, Linus Pauling had heard about the ratios much earlier – straight from Chargaff in late 1947, while traveling to England for his six-month stay as a professor at Oxford University. Pauling, who considered the trip by ship across the Atlantic Ocean with his family to be a vacation, did not pay attention to what Chargaff told him.

Crellin Pauling, the youngest child of Linus and Ava Helen Pauling, mentioned the remarkable background to the incident in a speech given during a symposium to celebrate Pauling’s life that was held here at Oregon State University in 1995.

[Click here to view the rest of Crellin's talk]

Over time Chargaff mentioned his work to individuals beyond Pauling. In the spring of 1952, Chargaff met James Watson and Francis Crick.  A prickly character, it is clear that Chargaff didn’t think much of the duo. In his truly remarkable autobiography Heraclitean Fire: Sketches from a Life before Nature, Chargaff calls Watson and Crick “a variety act” and further describes them as:

One 35 years old (Crick), with the looks of a fading racing tout. . .an incessant falsetto, with occasional nuggets gleaming in the turbid stream of prattle. The other (Watson), quite undeveloped. . .a grin, more sly than sheepish. . .a gawky young figure.

He further notes that:

I never met two men who knew so little and aspired to so much. They told me they wanted to construct a helix, a polynucleotide to rival Pauling’s helix. They talked so much about ‘pitch’ that I remember I wrote it down afterwards, ‘Two pitchmen in search of a helix.’

[More samples from Chargaff's acid pen are available here]

Regardless of what he thought of them, Chargaff still mentioned his work to Watson and Crick. The information, although published almost two years earlier, seemed to be new to the pair.

Though Chargaff himself didn’t speculate much on his rules, and Pauling completely ignored them, they did prove to be extremely useful to Watson and Crick. With this new knowledge, the feedback they had received from Rosalind Franklin and Maurice Wilkins, and data obtained through their own research, Watson and Crick were soon able to correctly deduce the structure of DNA.

For more information on DNA, please visit the Race for DNA website, or check out the other posts in the DNA series. For more information on Linus Pauling, visit the Linus Pauling Online portal.

The Hershey-Chase Blender Experiments

Martha Chase and Alfred Hershey, 1953.

“When asked what his idea of happiness would be, [Hershey] replied, ‘to have an experiment that works, and do it over and over again.’”

- Jonathan Hodgkin, 2001

In 1944 the Avery-MacLeod-McCarty experiments demonstrated that DNA, rather than proteins, is the carrier of genetic information.  Though the work appeared to be well-supported, and was endorsed by other researchers, the trio met with resistance from much of the scientific community.  For nearly a decade, the Avery group was forced to repel attacks on the validity of their experiments, defending both their findings and their reputations.

Finally, in 1952, Alfred Hershey, a Carnegie Institution researcher working at Cold Spring Harbor Laboratory, set out to conclusively settle the issue.  Like many of his contemporaries, Hershey believed that proteins, with their complicated structures, were more likely to be the carriers of genetic information than was the simple DNA molecule.  Hershey, however, was about to make a discovery that would turn his own notions on end.

In order to show that proteins carry genetic information, Hershey and his lab technician, Martha Chase, decided to track the transfer of proteins and DNA between a virus and its host.  For their experiment, they chose to use the T2 bacteriophage as the vehicle for delivering genetic material.  Like all bacterial viruses, the T2 is comprised of only a protein-based outer wall and a DNA core, its simple structure making it the perfect research candidate.  The phage reproduces by injecting its genetic material into a bacterium, leaving its protein shell attached to the host.  Then, through a microscopic takeover, the virus seizes control of the bacterium’s reproductive mechanisms and uses them to duplicate itself, destroying the host in the process.

Though it was known that the protein shell remained outside the bacterium, researchers thought it possible that certain proteins were transferred from the virus to the bacterium upon attachment. If genetic material was in fact carried by proteins, this would explain how a phage is able to reproduce within a bacterium without the entirety of the protein shell penetrating the bacterium’s membrane.  In order to prove that proteins are the carriers of genetic information, Hershey and Chase needed to demonstrate that at least a portion of the phage’s protein mass was transferred to the interior of the bacterium.

In their first experiment, Hershey and Chase tagged the T2 phage DNA with Phosphorous-32, a radioactive form of the element.  Because phosphorous can be found in large quantities in DNA, but in only trace amounts in protein, the researchers could track the location of DNA and protein according to the radiation concentrations.  They then allowed the tagged phages to begin infecting samples of E. coli.  After introducing to the phage culture to the bacterial sample, they used a Waring blender to violently disturb the infected bacteria, causing the protein shells to detach from their hosts.  Then, using a centrifuge, they separated the bacterium from the phages and protein.

The Hershey-Chase Blender Experiment. Diagram by Eric Arnold.

Once the separation was complete, they measured the radiation concentrations in the E. coli cells and the protein shells.  The phosphorous tracer appeared in large quantities only in the bacterial sample, demonstrating that DNA was transferred from the bacteriophage to the host organism.  Further, despite the protein shells being detached while reproduction of the phage should have been taking place, the virus was still copied in each of the host cells. This, in turn, suggested that the proteins shell itself was not necessary to the replication process following the initial insertion of genetic material.

Shocked by their findings, Hershey and Chase decided to perform the test once again, this time using a different tracer molecule.  They chose sulfur for the second test, because it appears in the amino acids that make up proteins, but is not present in DNA.  This allowed them to track the same process as in the first experiment, but in reverse.  After tagging the proteins, infecting the E. coli cells, and separating the shells from the host, the researchers tested for the presence of sulfur.  In accordance with their previous results, the sulfur could only be found in the protein shells and not in the bacteria. And again, the phage’s genetic material was replicated despite the protein shell being disconnected from the bacteria via the blending process.

Sufficiently impressed by the significance of his findings, Hershey returned to the phosphorous-tagged batch to engage in some follow-up research.  Upon examining the offspring of the phages, the researchers found that the young bacteriophages also possessed phosphorous-tagged DNA, but their protein lacked any trace of radioactivity.   The implications of their first experiments were reinforced.

At first, the pair was inclined to believe that the experimental or data-collection procedures were flawed.  They rechecked the experiment design, the equipment, and the bacterial cultures.  It was all in vain, though.  Hershey was a notoriously cautious researcher and his experiments were always well-planned and precisely executed. The results were no mistake and the import of their work was clear: Hershey and Chase had elucidated direct, irrefutable evidence that DNA, not protein, is the source of genetic material.

Alfred Hershey, 1960.

Later that year, the pair reported their findings in a short paper in The Journal of General Physiology titled “Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage.”  This publication catalyzed a storm of activity in the scientific community, with researchers all over the world clamoring for details on the experiments.  Alfred Hershey’s lectures on the subject were attended by the greatest scientific minds in Europe and North America; Pauling was one of hundreds to hear him speak.  In the years following the discovery, DNA became a major focus for researchers all over the world, resulting in Pauling’s own attempts to deduce its structure and the eventual success of Watson and Crick.  Even today, our genetic research traces its roots from the work of Alfred Hershey and Martha Chase.

For more on the story of the quest for DNA, see our documentary history website on the subject.  For more information about Linus Pauling, visit the Linus Pauling Online portal.

The Passport Imbroglio

Ava Helen and Linus Pauling's passport photo. 1953.

A quick glance at the “Today in Linus Pauling” widget found at the top of the left sidebar of the Pauling Blog gives an excellent representation of the span and influence of Linus Pauling’s career. Rarely does a day go by where he didn’t write at least one manuscript or give a speech at a university or some other institution. Most days, readers will also note that he won some sort of award – including, of course, his two Nobel Prizes in chemistry and peace. Basically, Pauling’s career fits very well with the old cliché that anything can be done if the mind is simply set on it.

However, if one looks closely enough, a few failures can still be picked out of Pauling’s illustrious career. One of these failures is undoubtedly his attempt at determining the correct primary structure of DNA. Pauling first started working with DNA in the early 1950s, right around the time when his scientific career was reaching its peak. During this time, Pauling’s pursuits had also taken a controversial political shift – work which caused him to be denied a passport for a short period of time. This passport denial, because it is believed by many to be the reason why Pauling was beaten to the structure of DNA, is the topic of today’s post.

Near the end of 1951, Pauling received an invitation to attend a meeting of the Royal Society in England; a meeting that was specially designed for him to address questions about his protein structures. The meeting was scheduled for May 1, 1952, and promised to give Pauling an opportunity to visit King’s College in London, where he knew Maurice Wilkins had some excellent X-ray patterns of DNA.

However, when Pauling sent in his passport renewal application in January 1952, he was upset but unsurprised to find it denied by Ruth B. Shipley, the head of the State Department’s passport division. Shipley didn’t give Pauling a good reason for the denial, stating only that “the Department is of the opinion that your proposed travel would not be in the best interests of the United States.” Reading between the lines, Pauling’s liberal views had clearly earned him the label of “possible Communist,” and Shipley, who was a fervent anti-Communist, had the authority to deny passports at her discretion.

Video Link: Pauling discusses his reaction to the refusal of his passport.

Fortunately for Pauling, the delay caused by the situation was not a long one. In the summer of 1952, he sent in another passport application. Again, Shipley immediately denied it, but her decision was overruled – after much deliberation – by higher-level employees of the State Department. Eventually, Pauling was notified that he would be granted a limited passport to travel for a short period of time in England and France if he agreed to sign an affidavit stating that he wasn’t a Communist. Surprised and pleased by the news, Pauling immediately agreed and received his new passport within days.

Ruth B. Shipley. Image extracted from "Commies Step Up Attacks on Passport Curbs", Chicago Tribune.

Thus equipped with the necessary papers, Pauling traveled to England, where he stayed for a month. He visited the same places and talked with the same people that he would have earlier in the year, but he did not visit King’s College to view Wilkins’ X-ray data. As it turns out, Pauling wasn’t even thinking about DNA during his time in England.

After England, Pauling traveled to France, where he learned of the results of the Hershey-Chase blender experiment:  DNA was in fact the site of the gene, not proteins, as Pauling had believed. Upon learning of the keen importance of DNA, he decided that he would solve the structure of the molecule.

However, when he returned to Caltech in September of 1952, he continued to work almost exclusively with proteins. It wasn’t until November that Pauling would finally take a serious stab at the structure of DNA. And, as has been well-documented, even with his excellent knowledge of structural chemistry, Pauling’s data – presented in the form of blurry X-ray patterns created by William T. Astbury – was insufficient. He ended up creating a model that was nearly identical to one Watson and Crick had made over a year earlier. Of course, Pauling soon learned that his structure was incorrect, and before he could make another attempt, Watson and Crick had solved DNA.

The importance of Pauling’s passport imbroglio is, as it turns out, counter to the popular mythology of the DNA story. Although the denial of Pauling’s passport caused minor delays in his travels, it surely did not keep him from determining the structure of DNA. Even if he had traveled to England as originally planned, it is unlikely that he would have visited Wilkins to view his X-ray data. Pauling, even after finding out that DNA was extremely important, made no effort to obtain better data, nor did he even work specifically with DNA for quite some time. One is forced to conclude then, that the reason that Linus Pauling was not able to solve DNA is that he never really put his mind to the matter, not because of a pesky passport denial that delayed his travels a mere ten weeks.

For more information on Linus Pauling’s DNA pursuits, please visit the website Linus Pauling and the Race for DNA: A Documentary History. For other information on Pauling, check out the Linus Pauling Online portal.

The X-Ray Crystallography that Propelled the Race for DNA: Astbury’s Pictures vs. Franklin’s Photo 51

Rosalind Franklin, March 1956

During their so-called race to discover the structure of DNA, Linus Pauling and the unlikely pair of James Watson and Francis Crick utilized remarkably similar approaches in attempting to solve the riddle of the genetic material. In fact, one of the main tactics used by Watson and Crick was to approach the problem in the same manner that they assumed Pauling would. Although Pauling and Watson and Crick did, at one point, come up with nearly identical, yet incorrect, structures, it was Watson and Crick who would eventually solve DNA. Why then, if the pair were thinking like Pauling, were they able to beat him to the structure?

Although there were a variety of reasons behind Watson and Crick’s success, a good portion of it can be attributed to the relative superiority of resources available to them. Watson and Crick obviously had each other to keep themselves in check, but they also benefited from other voices of criticism such as Rosalind Franklin, Maurice Wilkins, and later Jerry Donohue. Linus Pauling also shared his ideas with his colleagues, but none of them were very familiar with DNA, and therefore couldn’t offer much feedback. (And they were largely ignored even when they did offer criticisms of Pauling’s structure.)

Another vital resource available to Watson and Crick was an excellent X-ray crystallography pattern, the famous photo 51, taken by Rosalind Franklin. Although, in all likelihood, Pauling could have also viewed Franklin’s photographs had he tried, he settled on using blurry patterns published by William T. Astbury several years before Franklin’s superior images. These X-ray photographs are the main topic of today’s post. In particular, the factors accounting for the difference in quality between Franklin’s and Astbury’s patterns will be discussed. Before delving into this subject, however, a brief overview of X-ray crystallography is necessary.

William T. Astbury, ca. 1950s.

X-ray crystallography, also sometimes known as X-ray diffraction, is used to determine the arrangement of atoms within a crystalline molecule. It is a rather complicated procedure, and the photos taken in the process can be interpreted only by a person with significant training. The steps to obtaining these photos are as follows.

First, an adequate crystal must be obtained. This is a very difficult step because the crystal must be large enough to observe and also sufficiently uniform. If it does not meet these specifications, errors – such as blurriness – will occur, often rendering the resulting crystallographic patterns useless, at least for purposes of determining atomic arrangement.

After an adequate crystalline specimen is obtained, a beam of X-rays is shined through it. When the beam strikes the electron clouds of the atoms in the crystal, it is scattered. These scattered beams can then be observed on a screen placed behind the crystal. Based on the angles and intensities of the scattered beams, a crystallographer can create a three dimensional picture of the electron density of the crystal.

Finally, from the electron density information, the mean positions of the atoms within a crystal can be determined, and the structure of the molecule can be considered “solved.” That said, just one image is not nearly enough to determine the structure of an entire crystal. Therefore, the crystal must be rotated stepwise through angles up to and even slightly beyond 180 degrees, depending on the specimen. Patterns are required at each step, and complete data sets may contain hundreds of photos.

Clearly, because the process of X-ray crystallography is so cumbersome, there are many opportunities for mistakes that may have led to the poor quality of Astbury’s photographs. However, Astbury’s techniques seem to have been excellent. He was a very experienced crystallographer, and had achieved great success in his earlier work with X-ray diffraction on substances such as keratin.

As it turns out, Astbury’s photos were of poor quality because of the DNA sample he was using. In the early 1950s, Rosalind Franklin had discovered that DNA came in two forms – a dry condensed form and a wet extended form. Astbury’s DNA sample was well prepared from calf thymus, but it contained a mixture of the two forms. This turned out to be the major reason why Astbury’s photographs were so blurry

Astbury's images, from "X-Ray Studies of Nucleic Acids," 1947. Plate 1.

Astbury's images, 1947. Plate 2.

It is important to note that, even if Astbury had known he was using a poor crystalline sample of DNA, he probably still wouldn’t have been able to compete with the quality of Franklin’s photos. In 1950, three years after Astbury’s images were published, Maurice Wilkins developed a way to obtain much better X-ray patterns of DNA through the use of a solution of sodium thymonucleate. This solution is highly viscous, and Wilkins found that thin strands could be drawn out by gently dipping a glass stirring rod into a sample and slowly pulling it out. These thin strands were pure DNA, and Wilkins was able to get excellent X-ray patterns from them.

Before long, Wilkins had also acquired better equipment and had also hired Rosalind Franklin to run it. Franklin, essentially working independently, used the same basic technique developed by Wilkins. She did, however, add several of her own smaller experimental refinements, which made the photographs even better. Eventually, she developed photo 51, which would later be shown to Watson and Crick. The rest, as they say, is history.

Crystallographic photo of Sodium Thymonucleate, Type B. "Photo 51." Taken by Rosalind Franklin, May 1952.

Rosalind Franklin and William Astbury were both excellent crystallographers, but Franklin’s experience with DNA gave her a clear advantage when working with the molecule. Her brilliant X-ray patterns would later prove to be a major determining factor in the “race for DNA”. For more information on DNA, please visit the Race for DNA website. For much more on Linus Pauling, check out the Linus Pauling Online portal.

Oswald Avery’s Pneumococcus Experiments: Forerunner of the DNA Story

Portrait of Oswald T. Avery, ca. 1940s.

DNA, although now known to be extremely important, was overlooked for quite some time. Until early 1953, around when the Watson and Crick structure of DNA was published, most major scientists thought that proteins, rather than DNA, were probably the site of the gene.

In the early 1940s however, experiments performed by Oswald T. Avery and his colleagues at the Rockefeller Institute for Medical Research made a strong argument for DNA as the source of the genetic material. Unfortunately, for many years not much attention was paid to Avery’s work.

Streptococcus pneumoniae, also called pneumococcus, was the subject of Avery’s experiment. This bacterium causes a variety of diseases, including pneumonia and peritonitis. The organism can be found in two forms, smooth (S) and rough (R) which are designated as such simply because of their appearance when viewed microscopically. The smooth appearance is a result of the formation of a polysaccharide capsule that encases the bacterial cell. This capsule protects the cell from immunological defenses, which makes the S form virulent. The R form, on the other hand, is mutated so that it does not synthesize the enzyme that creates the polysaccharide capsule, and is therefore not virulent.

The pneucmococcus bacteria can be further characterized into types, which are designated by roman numerals. Although an S form bacterial cell can be experimentally changed to an R form (and vice versa) provided the cell is not too far degraded, change of type never suddenly occurs. For example, a type III S cell can be converted to a type III R cell, but a type II cell will never spontaneously convert to a type III cell.

Although a spontaneous change of type is not possible, a specific experiment had been done showing that a transformation of type can be induced. This experiment was first performed by injecting a live culture of the Type II R form into mice along with a dead culture of the Type III S form. Theoretically, none of the mice should have died because they hadn’t been exposed to a virulent form of the bacteria. However, many of the mice did actually die, and living Type III S form bacteria was extracted from their blood. Later, the same experiment was accomplished by growing the bacteria in a glass dish rather than in mice.

Understandably, this transformation from a non-virulent bacteria to a virulent bacteria was troubling to the medical community. Although some scientists may have been concerned with the mechanism of transformation, Oswald Avery was more concerned with the identity of the agent performing the transformation. He went to work on devising an experiment that would allow him to isolate the transforming agent from the rest of the bacterial cell. Although DNA extraction is now considered a simple process, it was just beginning to emerge during the time when Avery began his work. The fact that Avery did not know that the transforming agent was in fact DNA complicated matters even further.

Nevertheless, after years of hard work, Avery and his colleagues were able to develop an experiment that effectively isolated the transforming agent from the bacterial cells. Type III S form bacteria were grown in large vats of broth made from beef hearts. The bacteria was then killed, and washed with brine in order to remove the polysaccharide capsule and whatever protein would come off in the process. The remainder of the bacteria was then precipitated in pure grain alcohol. After this, the precipitate was washed with chloroform and subjected to a digestive enzyme, both of which functioned to remove the remaining protein. Finally, after no trace of protein was evident, pure grain alcohol was once again added, which allowed the transforming agent to be separated. The process was long and difficult, and in the end only yielded approximately ten to twenty-five milligrams of the agent per seventy-five liters of culture.

Transformation of pneumococcal types, from Avery's 1944 paper.

After obtaining enough of the active transforming agent to conduct his tests, Avery and his colleagues set out to show exactly what the substance was. First, standard qualitative tests for proteins were performed, which came back negative. Qualitative tests for DNA, however, were strongly positive. Chemical analysis of the substance also showed that the ratio of nitrogen atoms to phosphorous atoms was approximately 1.67 to 1. This number is very close to the DNA ratio, and would have been different had there been a significant amount of protein present.

Next, tests with digestive enzymes were performed. The addition of enzymes that digest proteins and RNA left the agent intact, while enzymes that digest DNA completely destroyed the substance.

Finally, immunological tests involving centrifugation and electrophoresis were performed, which also showed that proteins and polysaccharides weren’t present, but that DNA was.

Once Avery was satisfied with the results of his tests, he began writing a manuscript that explained the experiment. In January of 1944 “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types” by Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty was published. Although it may seem that Avery and his colleagues had proven that DNA was the site of the gene, this was not entirely the case. There was still a possibility that, as Avery puts it in the manuscript, “the biological activity of the substance described is not an inherent property of the nucleic acid but is due to minute amounts of some other substance adsorbed to it or so intimately associated with it as to escape detection. . .”

Avery was clearly being very cautious in his conclusions, never stating that he was certain that DNA was the transforming agent. It is possible that his cautiousness with the matter contributed to his lack of attention received. However, there were other reasons why Avery wasn’t given serious attention. As James Watson later stated in 1983:

Both Francis and I had no doubts that DNA was the gene. But most people did. And again, you might say, ‘Why didn’t Avery get the Nobel Prize?’ Because most people didn’t take him seriously. Because you could always argue that his observations were limited to bacteria, or that [the transformation of pneumococcus that he described was caused by] a protein resistant to proteases and that the DNA was just scaffolding.

Although Avery’s manuscript may not have been received with high praise at the time of its publication, it is now considered to be a very thorough account of an expertly accomplished experiment. For more information on DNA, please visit the Race for DNA website. For information on Linus Pauling, a major player in the DNA story, visit the Linus Pauling Online portal.