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Thursday, July 19, 2007

The Story of DNA (Part 1)

Where Rosalind Franklin Teaches Jim and Francis Something about Basic Chemistry

What we now know as DNA was first isolated in 1868 by Johann Friedrich Miescher, a student in the lab of Ernst Felix Hoppe-Seyler. A later student of Meischer’s named Richard Altmann called the material "nucleic acid."

Nucleic acids are composed of pentose sugars and four bases. Guanine was discovered in bird droppings in 1848 (guano means excrement of sea birds). Adenine was identified in beef pancreas in 1885. Thymine originally came from calf thymus, hence its name.

By the 1920’s it was clear that there were two kinds of nucleic acid, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), although they weren’t called by those names until much later. Chromosomes were known to contain DNA. It was thought that DNA was some sort of structural element that provided stiffening. The four bases were believed to be present in equimolar quantities and they repeated endlessly along the chain. This idea was referred to as the "Tetranucleotide Hypothesis." It was advanced by leading chemists and widely believed to be correct.

William (Bill) Astbury took the first X-ray diffraction pictures of DNA in the 1930’s. These first images, and Astbury’s interpretation of them, dominated thinking for fifteen years. Astbury noted that there were strong reflections at 0.34 nm and he interpreted this to mean that the phosphate groups were 0.34 nm apart. In other words, the repeating nucleotides were spaced at 0.34 nm intervals. Astbury concluded from his images that the bases were stacked on top of each other like a pile of coins. Both of these assumptions were correct. However, Astbury saw an important reflection at 2.7 nm suggesting the structure repeated every 2.7 nm. If DNA was helical then this would indicate that there were about eight bases in each turn of the helix and each turn was 2.7 nm. This turned out to be very misleading. Astbury also thought that the sugar moiety and the base were in the same plane and this is incorrect.

In 1944, Oswald Avery at the Rockefellar Institute in New York showed that bacteria could be transformed with pure DNA. This clearly indicated that DNA was the genetic material but the data was not widely accepted. The lack of acceptance was not due to Avery’s reputation since he was a recognized and highly respected scientist. It’s just that the concept of DNA as the genetic material didn’t fit with other data and couldn’t be reconciled with the supposed structure of DNA according to the Tetranucleotide Hypothesis. Nucleotides were known cofactors in metabolism and complex carbohydrates are usually structural as in cellulose and bacterial cell walls. Proteins, one the other hand, are special and unique.

Throughout the 1940’s everyone knew of Avery’s experiment but they set it aside as unexplainable. This is an excellent illustration of how science works. Usually it is a good idea to reserve judgment when a single experiment conflicts with the current paradigm.

Avery’s work stimulated Erwin Chargaff to take up the study of the chemistry of DNA. He carried out careful analyses of DNA from many sources and discovered that the base composition varied considerably. Some species had more guanosine and cytosine and less adenosine and thymidine while in others the relative compositions were quite different.

Chargaff also noted that the amount of adenosine was equal to the amount of thymidine and gaunosine equaled cytosine. It followed that the numbers of purines equaled the number of pyrimidines. For the most part, these molar ratios were not thought to be significant. What WAS important was the discovery that in DNA the four bases were not present in the same amounts. This destroyed the Tetranucleotide Hypothesis and paved the way to an important understanding: DNA could contain information now that the amounts of the bases could vary. It’s safe to say that only a small number of scientists appreciated this point. An even smaller number, not including Chargaff himself, appreciated the significance of A = T and G = C.

About this time James Dewey Watson was a graduate student at Indiana University in Bloomington, Indiana, He had gone there because of a famous geneticist, Hermann Muller, but Watson ended up in the lab of Salvador Luria working on bacteriophage. Luria, along with Max Delbruck, founded the ‘phage group—an elite group of scientists dedicated to discovering the secrets of life by working with the simplest organisms. They met every summer in Cold Spring Harbor and they visited each other often.

Watson’s Ph.D. thesis was unremarkable except for the fact that he completed it within four years and became Dr. Watson when he was only 21 years old. He set off to Europe on a post-doc. After a stint in Denmark with members of the phage group, he ended up in Cambridge.

After the war, an X-ray crystallography group was set up in Cambridge, England, to study the structure of proteins. The group was headed by Lawrence Bragg, a Nobel Laureate who developed the original theory of X-ray diffraction. The chief members of the group were Max Perutz, working on the structure of hemoglobin, and John Kendrew who worked with myoglobin. They later received Nobel Prizes for solving the first protein structures.

In the Fall of 1947 Francis Crick moved to Cambridge as an overage graduate student. He was 31 years old and had served in the Admiralty during the war. In the beginning he was associated with the biochemists and was in close touch with Fredrick Sanger. At the time Sanger was sequencing insulin. Although the results came in slowly over the next six years, Crick was aware of them instantly because he attended the monthly seminars. Sanger showed that every insulin molecule in beef pancreas had the same sequence of amino acids. This was the first direct evidence that proteins had a defined amino acid sequence and Crick realized right away that the sequence had to be encoded in the genes. Sanger got eh Nobel Prize for this work and later on received a second Nobel Prize for developing the technique of sequencing DNA.

1948: Linus Pauling was in Oxford as a visiting Professor on leave from the Californian Institute of Technology in Pasadena, USA. He discovers the α-helix and publishes the model with Richard Robert Cory on his return to California. This is the first clear indication of helices in macromolecules and soon after the model was published Max Perutz proves that hemoglobin contains α-helices. Pauling was the pre-eminent chemist at that time and his influence was enormous. He was in contact with the phage group but for the most part he was their major competitor. The fact that Pauling scooped the Cambridge group by coming up with a model for the &alpha:-helix will have an influence on later events..

1949: Crick moves to the Cavendish lab in Cambridge to study under Perutz. Crick had realized that it was important to learn X-ray diffraction in order to study structure. He had come to recognize the importance of information in the gene (whatever it was) and he hoped to discover how this information (sequence) gave rise to three dimensional structure. At this time Crick was interested in DNA but was not by any stretch convinced that it was the genetic material.

In the late 1940’s another structure group was established at King’s College in London, England. Maurice Wilkins joined the group and began to look at DNA fibers. In May of 1950, he had been given an excellent preparation of DNA containing large intact molecules (from Rudolph Signer in Bern, Switzerland). Wilkins’ first pictures were better than the fifteen-year-old images of Astbury. However, Wilkins’ interest in DNA was a sideline. The main focus of his work was proteins and he turned the DNA project over to a graduate student, Raymond Gosling.

Later on the group in London decided that they needed to hire someone more senior to work on the structure of DNA. They found Rosalind Franklin, a chemist who had been working on the structure of complex chemicals in Paris, France. She was anxious to return to England and when she arrived in January 1951 she immediately took over the DNA project, presumably under the direction of Maurice Wilkins. Wilkins gave Rosalind Franklin the excellent DNA samples that he had obtained from Signer the previous summer.

In retrospect, it is clear that Wilkins never meant to assign complete control of the project to Franklin. He was looking for a collaborator even though he assigned Gosling to Franklin as her graduate student. This conflict between Franklin and Wilkins became more intense over the next two years until they were barely speaking to one another.

Meanwhile, the evidence that DNA was the genetic material was mounting to those who were paying attention. The famous Hershey-Chase experiment was being completed and word was spreading among the insiders. In this experiment, Hershey had labeled bacteriophage DNA with radioactive phosphorus and the protein were labeled with radioactive sulphur. After the phage adsorbed to the bacteria, the culture was put in a Waring blender and the resulting agitation knocked off the empty phage particles. The bacteria could then be separated from the radioactive sulphur labeled proteins. When the bacteria were concentrated by centrifugation the DNA was found to be in the bacteria.

After a short time the bacteria lysed producing a new burst of phage. The experiment clearly indicated that the injected DNA carried the information to produce new phage particles. The Waring blender experiment was much more sloppy than Avery’s earlier experiment but it was confirmation and it got quite a lot of people thinking about DNA as the genetic material, especially those who were associated with the phage group and understood the significance of a phage head stuffed with DNA. Watson was one of those who realized how important the experiment was.

During the winter of 1950-51 Watson was in Naples doing some experiments when he attended a seminar by Maurice Wilkins who was visiting from London. Wilkins showed his X-ray diffraction images of DNA fibers. This impressed Watson who then decided that he had to learn about diffraction techniques in order to solve the structure of DNA. He managed to obtain a fellowship, with the help of his phage buddies, to study under Bragg in Cambridge.

October 1951: Jim Watson arrives in Cambridge and meets Francis Crick. Watson was 23 years old. Crick was a 35 year old graduate student. Watson convinces Crick that genes are made of DNA and together they resolve to discover the structure of DNA and the secret of life. The two became fast friends and spent hours talking about biology. They are moved to a separate office of their own in order not to bother anyone else.

It’s important to note how crucial this meeting was. Watson had convinced himself that DNA was the stuff of life and he needed to solve the structure. Watson had the biological background from hanging out with the phage group and the bacterial geneticists. Crick had taught himself about structure and X-ray crystallography and was certain that structures would provide clues to the secret of life. Watson and Crick were thinkers and talkers rather than experimentalists, especially Crick. At the time, Watson and Crick were among the few people in the world who really “knew” that genes were made of DNA. They may have been the only scientists who desperately wanted to solve the structure of DNA and achieve fame and glory.

Crick was a personal friend of Maurice Wilkins, the man who had taken pictures of DNA fifteen months earlier. Crick knew about the Wilkins’ pictures and he knew that Rosalind Franklin was making slow progress on solving the structure. Within a few weeks of Watson’s arrival they had constructed a model based on Astbury’s data; what Crick and Watson remembered of the Wilkins data; and what Watson had learned from Rosalind Franklin. Here’s how it came together.

Alexander Todd in Cambridge had just worked out the chemical structure of DNA. The backbone consists of alternating sugar/phosphate groups joined through the 3′ carbon of one sugar and the 5′ carbon of the adjacent sugar residue. Crick was also aware of the unpublished results of Sven Furberg, a graduate student in London. Furberg had solved the three-dimensional structure of cytidylate, one of the nucleotides in DNA. He learned that the base and the sugar were at right angles to each other. Recall that Astbury’s conclusion was that they were in the same plane and that view had dominated thinking for fifteen years. Furberg had proposed that DNA formed a single-stranded helix with the bases sticking out and stacked on top of one another.

Watson went to a seminar by Franklin in London on Wednesday, November 21, 1951. While there, he learned that DNA contained several chains, that the chains were probably joined by hydrogen bonds between the phosphate groups, and that the structure was a helix. Watson also thought that Franklin had said that each unit of DNA (i.e., one turn of the helix) contained eight water molecules. In fact, Franklin had said that each nucleotide was associated with eight water molecules.

In a few short days of feverish activity Watson and Crick had built a model of DNA. It had three chains and the phosphate groups were on the inside with the bases projecting outward. Watson and Crick invited Wilkins, Franklin, and Gosling up from London to see their triumph. Unfortunately for them, the first model was destroyed in a few minutes as Franklin demonstrated that it was impossible. She showed them that their structure did not have enough water and that there was no way to form the phosphate-phosphate interactions that they had modeled. Watson and Crick admitted defeat.

Shortly after this fiasco, Lawrence Bragg (their boss) ordered Watson and Crick to stay away from DNA. The problem belonged to Wilkins and Franklin in London.

Clayton, J. and Denis, C. eds. (2003) 50 Years of DNA. Nature/Pallgrave/Macmillan

Judson, H.F. (1996} The Eighth Day of Creation: Makers of the Revolution in Biology. expanded ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. USA

Maddox, B. (2002) Rosalind Franklin: The Dark Lady of DNA. Perennial/HarperCollins

Watson, J.D. and Berry, A. (2003) DNA: The Secret of Life. Alfred A. Knope, New York, USA

Watson, J.D. (1168) The Double Helix. Atheneum, New York USA


Steve Reuland said...

"What we now know as DNA was first isolated in 1968..."

Did you mean 1868?

Torbjörn Larsson said...

Oh noes! The story grew on me, so by the time I got to the end of the post I had forgotten about it being part 1. But I guess you had to break it up, as this is a story with a twist, eh? :-P

PS. I had forgotten about Furberg. So close from so little information, but still failing to synthesize all of the pertinent... DS

Steve LaBonne said...

I didn't know Judson had put out an expanded second edition. I read the original way back then. I still regard it as one of the best popular history-of-science books ever written, on a par with the finest books written for a broad public by "regular" historians.

Steve LaBonne said...

"Way back WHEN", that is.