DNA The Discovery of the Double Helix
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StudyGuideHistoryofDNATheGeneticMaterialdraft4.docxThe History of DNA, the Genetic Material
Introduction
Friedrich Miescher
Deoxyribonucleic acid was discovered in 1869 by Friedrich Miescher (Mirsky, 1968, p132). Miescher was the first to extract DNA from cells and identify it as a unique chemical substance. It may be surprising to learn that DNA was discovered so long ago. Perhaps this is because we tend to view DNA in the light of the many important recent discoveries beginning with the elucidation of its molecular structure by Watson and Crick in 1953 and continuing through the modern age of molecular biology and genetic engineering. In this section we will look at the scientific work leading up to this momentous discovery and the contributions of the many investigators who made it possible.
Miescher was a Swiss physician. As a medical student, he became interested in histochemistry. After attending the University of Tűbingen, he began to carry out research in the laboratory of the biochemist Felix Hoppe-Seyler. This laboratory, which was the first laboratory devoted to research in biochemistry, was located in an ancient castle overlooking the Neckar River in Germany. It was here that Miescher made the discovery of nuclein, or as it became known later, deoxyribonucleic acid (DNA).
As a source of cells for his experiments, Miescher used pus. Pus is composed largely of white blood cells. He extracted the pus from bandages that he obtained in the Tübingen surgical clinic. Miescher began to analyze the chemical composition of the intact white blood cells by treating them with various solutions, including salt solutions, acid, alkali and alcohol. When he treated the pus cells with alkali, he obtained a substance that he attributed to the nucleus. He then began experiments designed to isolate the nucleus of the white blood cells, a method that had not been attempted before. Miescher isolated the nuclei of white blood cells by treating them with dilute hydrochloric acid. He removed the protein from the nuclei by treating them with pepsin and then treated them with dilute alkali. He extracted from the isolated nuclei a material that could not be assigned to any of the chemical groups known at that time. He called the substance that he had discovered nuclein. We know it now as deoxyribonucleic acid.
Albrecht Kossel
Kossel also worked in the laboratory of Hoppe-Seyler. Kossel analyzed nucleins, and discovered key components of what is now called DNA. These included the nitrogenous bases adenine, thymine, cytosine, guanine, and uracil. Kossel was awarded the 1910 Nobel Prize in Physiology or Medicine for research in cell biology, especially his work on proteins and nucleic acids.
Phoebus Levene
Phoebus Aaron Theodor Levene was a Russian-born American chemist and pioneer in the study of nucleic acids (todayinsci.com). “Phoebus Levene was born in Sagor in Russia. He grew up in St. Petersburg and studied medicine at the Imperial Military Medical Academy (modares.ac).” Phoebus Levene was a former student of the Russian chemist and composer Alexander Borodin (chemsoc.org). Borodin was the composer of Polovtsian Dances. After receiving his M.D. degree from the St. Petersburg Imperial Medical Academy in 1891, Levene fled from Russian anti-Semitism. Along with his family, he emigrated to the United States. Appropriately, they arrived in New York City on July 4, a day synonymous with independence and freedom.
In 1905, Levene began to carry out research on DNA at the Rockefeller Institute of Medical Research. By 1900, it was known that the basic building blocks of DNA were phosphate, a sugar, and four nitrogenous bases. The nitrogenous bases included adenine (A), thymine (T), cytosine (C) and guanine (G). The adenine and guanine were large double-ring molecules known as purines. The thymine and cytosine were smaller single-ring molecules known as pyrimidines. In 1909, Levene found that the carbohydrate present in nucleic acid from yeast is the pentose sugar ribose. In 1929, he succeeded in identifying the carbohydrate in the nucleic acid from the thymus of an animal. It is also a pentose sugar but lacks one oxygen atom of ribose and was therefore called deoxyribose. The nucleic acid from yeast was named ribonucleic (RNA) and the nucleic acid from the thymus gland was named deoxyribonucleic acid (DNA). The basic building block of the DNA molecule is a nucleotide, which is made up of a phosphate group, a deoxyribose sugar and one of four nitrogenous bases: adenine, thymine, cytosine, or guanine. Levene determined how the nucleic acid components combine to form the nucleotides. He also proposed that nucleic acids were composed of linear chains of purines and pyrimidines linked to each other through the sugar-phosphate-ester backbone (Olby1994).
Tetranucleotide Hypothesis
Although Levene contributed much to our knowledge of the chemistry of nucleic acids, he is best remembered for an incorrect hypothesis of the structure of DNA known as the Tetranucleotide Hypothesis. This theory, which gained many adherents, misled DNA researchers into believing that DNA had a simple repetitive structure. This of course, turned out not to be the case. But acceptance of his theory led many to believe that DNA was an unlikely source of the genetic material. In order to serve as the genetic material, a chemical would have to be large and complex. This was necessary because the genetic material had to contain a vast amount of information, the information needed to code for all of the genetic traits of the organism. As a result, other scientists began to direct their attention to protein as the genetic material. Proteins were large molecules with a complex, varied structure.
Frederick Griffith
The Discovery of Bacterial Transformation in Pneumococcus
Frederick Griffith was a British microbiologist in the Ministry of Health’s pathology laboratory in London (Olby, 1994). Griffith was trying to develop a vaccine that was effective against pneumonia. The disease Pneumonia occurs in two forms; one is caused by a virus and the other is caused by Pneumococcus bacteria. Griffith worked with the bacterial form of pneumonia. Pneumococcus bacteria, which are responsible for the bacterial form of Pneumonia, exist in two forms, known as rough (R) and smooth (S). The terms rough and smooth describe the appearance of the bacteria when grown on Petri plates. Bacteria on Petri plates grow as colonies, which are rounded masses composed of thousands of bacterial cells. The smooth organisms produce colonies that have a smooth outline, are dome-shaped, and regular. The rough organisms produce colonies that are granular, flat and irregular. The smooth organisms are virulent (that is, capable of causing disease); the rough organisms are avirulent.
In 1928, Griffith performed a series of experiments designed to develop a vaccine that was effective against pneumonia. Although he did not succeed in developing a vaccine, he unexpectedly discovered the phenomenon known as bacterial transformation, a discovery that proved to be crucial to the eventual identification of DNA as the genetic material.
Griffith’s experiments consisted of the following steps:
1) Griffith injected one group of mice with the smooth virulent strain and these mice died after a few days.
2) He then injected another group with the rough non-virulent strain and these mice continued to be healthy.
3) Griffith took a heat-killed strain of the virulent bacteria and injected it into mice and observed that they did not die.
4) The fourth experiment proved to be the crucial one. He injected the living cells of the R form of pneumococcus into a mouse, together with heat-killed cells of the S form of the bacteria. The mice succumbed to the infection and died. From their blood Griffith isolated colonies of The S form of pneumococcus.
Griffith explained his findings by concluding that the R type bacteria had been transformed into S type pneumococcus. Apparently, a heat-stable substance derived from the dead S type bacteria had been able to travel from those cells to the R cells, enter them, and transform them into S form bacteria. The substance which he called the transforming principle gave the affected bacteria the ability to synthesize a new polysaccharide coat. This was a genetic characteristic. The bacteria also changed from nonvirulent to virulent. So apparently, the transforming principle was genetic material.
Avery, MacLeod and McCarty
The Identity of the Transforming Substance
Avery, MacLeod and McCarty performed experiments in the early 1940s that identified the transforming principle as DNA and provided the first evidence for the genetic role of DNA (Olby, 1994). Avery and McCarty concentrated first on isolating biologically active “transforming principle” from samples of pneumococci. Then attention turned to its chemical analysis.
The approach used by Avery, MacLeod and McCarty to identify the transforming substance was to employ enzymatic destruction of its activity. Using a process of elimination, they tried enzymes that inactivate various types of molecules. They exploited the specific nature of enzymes. A specific enzyme acted upon a specific substrate. They found that treating the transforming principle with the protein-digesting enzymes trypsin and chymotrypsin did not inactivate the substance. This indicated that the transforming principle was not a protein. Next they tried ribonuclease, an enzyme that breaks down RNA. When used on the transforming principle, ribonuclease also failed to inactivate it, showing that the transforming principle was not RNA. Similarly, lipases, enzymes that break down lipids, did not inactivate the transforming principle. However, when they used DNA polymerase, an enzyme that broke down DNA, it did inactivate it, providing evidence that the transforming principle was composed of DNA.
They then learned that the substance would precipitate in alcohol, forming fibrous strands that wound around the stirring rod. Analysis revealed the presence of phosphorous, it absorbed ultraviolet light at a maximum in the region 2600 Ǻ, and it had a molecular weight of at least half a million. It gave a strong Dische reaction for DNA, but also a weak Bial reaction for RNA. As all of these are characteristics of DNA, this provided strong evidence that the transforming principle was DNA. Avery, MacLeod, and McCarty published their discovery that the transforming principle was DNA in 1944 in the Journal of Experimental Medicine.
Hershey and Chase
Blender Experiment
In 1952 an experiment using bacteriophage was carried out by Alfred Hershey and Martha Chase that has since become known as the blender experiment. They knew that proteins contain sulfur but not phosphorous. DNA contains phosphorous but not sulfur. They were trying to determine which component, DNA or protein was the genetic material. Whatever the genetic material was, it had to enter the cell. In the first experiment they grew bacteriophage virus in the presence of radioactively labeled sulfur (35S). This procedure labeled the protein coat of the virus. They then allowed the labeled viruses to infect bacterial cells. After allowing enough time for the viruses to infect the cells, they separated the cells using a kitchen blender. This procedure separated the protein coats of the virus, which remained outside the cells, from the core of the virus, which had been injected into the cells. Next they placed the mixture into a centrifuge tube and spun it using a centrifuge. This device spins the tubes around in a circle at high speed. As centrifugal force acts on the particles and liquids in the mixture, the heavier particles fall to the bottom of the tube; the lighter particles remain near the top. The heavy particles at the bottom of the tube constitute the pellet, the lighter particles in the liquid at the top of the tube constitute the supernatant. The heavy particles represent the cells. The lighter protein coats of the virus remained near the top in the supernatant. They then analyzed each component for radioactivity. The radioactivity was found in the supernatant. This argued that protein was not the genetic material. Remember that the genetic material has to enter the cell.
In another experiment, they grew the viruses in the presence of radioactive phosphorus (32P). This labeled the DNA of the virus. Again, they allowed the viruses, this time labeled with radioactive phosphorus, to infect the bacterial cells. They separated the protein coats from the cells using the blender. They centrifuged the cells to separate the protein coats in the lighter supernatant from the heavier cells in the pellet. This time when they analyzed each component for radioactivity, they found the radioactivity in the pellet. This indicated that the DNA had entered the cells and produced evidence that DNA was the genetic material, and not protein.
Erwin Chargaff
Chargaff analyzed the nitrogenous bases in DNA. Chargaff found that the amount of adenine present in DNA always equals the amount of thymine, and the amount of guanine always equals the amount of cytosine. These findings are commonly referred to as Chargaff’s rules.
Chargaff also analyzed and compared DNA from a number of different species to one another. Chargaff constructed ratios of A + T When this ratio was examined from several individuals within a species,
C + G
there was little variation. The ratios were quite close. However when the average of the ratios from one species, say the Human, was compared with the ratios from another species, such as an Ox, the average of the ratios was much different. This was exactly what one would expect if DNA was the genetic material.
Watson and Crick
The Discovery of the molecular structure of DNA was made by James D. Watson and Francis Crick and was reported in an article in the Journal Nature published on April 25, 1953. They described a molecule composed of two strands wound around one another, the now famous double helix. Key pieces of information that led to the discovery were also gained from Maurice Wilkins and Rosalind Franklin.
James Watson
James D. Watson was born in Chicago, Illinois on April 6th, 1928 to James D. Watson, a businessman, and Jean Mitchell who did secretarial and administrative work. He was raised in Chicago. He attended Chicago public schools, beginning with Horace Mann Grammar School for eight years. It soon became apparent that Watson was remarkably bright. During the wartime years, he was discovered by Louis Cowan, producer of the Chicago Quiz Kids, and appeared on his radio program. Students were chosen for this program based upon their high IQs, academic ability, and personality. Watson’s major interest as a young man was ornithology; he was an avid birdwatcher. After attending South Shore High School for two years, Watson received a scholarship to the University of Chicago. At the young age of 15, he was admitted to the University in an experimental program that accepted capable students two years earlier than the normal high school graduation date. It was as a senior in college that Watson’s interest in DNA grew and he expressed a desire to learn what the gene was (Watson, 1968). An important experience that motivated him toward understanding the nature of the gene was his reading of Schrödinger’s book “What is Life?” (Olby, 1994, p 297). In this book Schrödinger postulated the existence of an aperiodic crystal that contained genetic information in its chemical structure and raised the possibility that the chemical structure of the gene could be determined. In 1947, at the age of 19, Watson received a Bachelor of Science degree in Zoology. He then received a Fellowship for graduate study in Zoology at Indiana University in Bloomington. He received his Ph.D. degree in Zoology in 1950 at the age of 22.
Watson spent the summer of 1948 in Cold Spring Harbor working on phage virus experiments. Watson’s colleagues at Cold Spring Harbor recognized the importance of Avery’s work pointing to DNA as the genetic material. It is likely that they influenced Watson into becoming convinced of the role of DNA in heredity. Watson’s Ph.D. supervisor Salvador Lauria then directed him to continue his studies by doing postdoctoral work under the direction of Herman Kalckar in Copenhagen. Watson then spent a year, from September 1950 to September 1951 working on a postdoctoral program in the University of Copenhagen, as a Merck Fellow of the National Research Council, to learn the biochemistry of DNA (Watson, 1968). At the end of the year Kalckar invited Watson to accompany him to Naples where he was going to spend two months at the Zoological Station. It was during this time that Watson met Maurice Wilkins. Their meeting occurred in May, 1951, at a scientific meeting Watson went to at the Zoological station in Naples. Maurice Wilkins presented a talk on DNA and displayed a photograph showing the X-ray diffraction pattern of crystalline DNA. In Watson’s words, “It was Wilkins who first excited me about X-ray work on DNA.” In early August 1951, Luria, arranged with John Kendrew for Watson to work at the Cavendish Laboratory, where he started work in early October 1952 (nobel.se/medicine/laureates). It was at Cambridge University that Watson met Francis Crick.
Francis Crick
Francis Crick, along with James Watson was a co-discoverer of the molecular structure of DNA and one of the founders of the field of Molecular Biology. After discovering the molecular structure of DNA with Watson, Crick developed the Central Dogma concept, which codified the key functions of DNA and made significant contributions to the study of Protein Synthesis. In later years, Crick began to investigate another scientific frontier, the study of consciousness.
Francis Harry Compton Crick was born on June 8th, 1916, near the town of Northampton, England. His parents were Harry Crick and Anne Elizabeth Crick. Crick attended Northampton Grammar School and later, at the age of 14, he received a scholarship to attend Mill Hill School, a private boys’ school in London. In 1937, at the age of 21, Crick received a Bachelor of Science degree in physics from University College London. Following his graduation, Crick began a Ph.D. research program at University College on the viscosity of water, working under Edward Andrade. Crick’s research was disrupted by World War II, which Britain entered in 1939. In 1940, he was given a civilian job at the British Admiralty and he worked on developing mines.
After the war, in 1947, Crick became interested in Biology. During this time, Crick read an article by Linus Pauling on the hydrogen bond, a book by Lord Adrian on the brain, and “What is Life” by Erwin Schrödinger. Crick reassessed where his interests were and decided to switch from Physics to Biology. He narrowed down his interest to two main areas: the borderline between the living and the nonliving and the workings of the brain (Crick, 1988). Crick had to decide between the two areas. Feeling that his current scientific background better qualified him for investigating the borderline between the living and the nonliving, he began to explore opportunities to conduct research in this area. He contacted Maurice Wilkins who was working under John Randall in the Physics Department at King’s College in London. Wilkins was studying cells using ultraviolet microscopes. Although Crick decided against working with Maurice Wilkins (He felt that his work tended too much toward the biological), he established a continued friendship with him. Crick eventually was offered a position at Strangeways Laboratory, headed by Honor Fell at Cambridge. His project involved studying the physical properties of cytoplasm in cultured fibroblast cells. While he was giving a report about his progress to Sir Edward Mellanby the director of the Medical Research Council (MRC), Crick became aware of a new opportunity. A new MRC unit was being established at the Cavendish Laboratory at Cambridge University to study the structure of proteins using the technique of X-ray diffraction. Crick was offered a research position to work toward a Ph.D. on research of protein structure. So in 1949, Crick entered the emerging field of molecular biology to work with Max Perutz and John Kendrew at the Cavendish to study proteins.
Maurice Wilkins
Maurice Wilkins was born in Pongoroa, New Zealand on December 15, 1916; in 1923 the family moved to Ireland and eventually settled in Birmingham. After graduating from King Edward’s High School in Birmingham, he entered St John’s College at Cambridge where, as a student of the physicist Mark Oliphant, he learned the principles of electricity, magnetism and quantum mechanics (Wilkins, 2003, p32). After graduating from Cambridge in 1938, Wilkins returned to Birmingham and became a research assistant in the laboratory headed by John Randall. Working under Randall, Wilkins completed a Ph.D. research program on thermoluminescence.
During the war, Wilkins joined the Manhattan Project working to develop the atomic bomb. At Berkeley, Wilkins worked on vaporizing uranium metal. Success came after Lawrence, the director of the lab suggested using an electric current to accomplish this. Wilkins began to think about his post-war plans. During this time, Wilkins read a book that strongly influenced him. The book was “What is Life?” by Erwin Schrödinger. He was attracted to Schrödinger’s idea of linking the biological idea of a gene with the idea of electrons moving in crystals. He wrote about a gene being an aperiodic crystal (Wilkins, 2003, p 83-84). He decided to accept an offer by Randall to work at St. Andrews University in Scotland (Wilkins, 2003, p 84). Shortly, Randall was offered the Headship of the Physics Department at King’s College in London (Wilkins, 2003, p 97). The Medical Research Council made him Director of a Biophysics Research Unit that combined physicists working together with biologists. Wilkins then began to work under Randall’s direction at King’s College (Wilkins, p 99). Shortly after he began working at King’s, Wilkins met Francis Crick (Wilkins, 2003, P 109). Crick inquired at Randall’s lab for a job but was unsuccessful. Crick went on to Cambridge and got a job there. But Wilkins and Crick became friends.
Wilkins at that time was studying DNA using special reflecting microscopes. He then had a stroke of luck. At a big scientific conference in London, the biochemist Rudolph Signer offered very pure samples of DNA that he had extracted. Wilkins decided to examine the DNA using X-ray diffraction (Wilkins, 2003, p 118-119). In 1950, Randall asked his student Raymond Gosling to take some patterns ‘to complement the studies’ of sperm he was making with his electron microscope (Wilkins, 2003, p 121).” Wilkins was friendly with Raymond Gosling and tried to help him. Gosling and Wilkins soon obtained patterns of DNA that were much sharper and more detailed than any before (Wilkins 2003, p 123). This was the picture that Watson saw at the scientific meeting in Naples that excited him to pursue the structure of DNA.
In the summer of 1950, Randall appointed a new X-ray specialist to work on protein solutions. Her name was Rosalind Franklin, and she had been working in Paris on the structure of coals (Wilkins, 2003, p 127-128). Wilkins recollection of the events surrounding her hiring were as follows: “She had no experience of really crystalline materials, but was an expert in X-ray techniques. Protein solutions seemed to me an unpromising field and, since the DNA work had gone so very well, I thought Randall might agree to Rosalind being transferred to DNA. Generally I had difficulty persuading Randall to change his decisions, but when I saw him I was surprised that he agreed very readily to my suggestion, with merely the proviso that Rosalind would have to be consulted about the change. Randall wrote to her, outlining a new program of work, and I heard that she had agreed to it (Wilkins, 2003, p 128).” Wilkins was away on holiday when Franklin arrived (Wilkins, 2003, p 128). During his vacation, he made up his mind to give up completely his microscope work and concentrate full time on X-ray structure analysis of DNA Wilkins, 2003, p 129).
Rosalind Franklin
Rosalind Franklin was born in London, England on July 25, 1920 to Muriel Waley and Ellis Franklin. Franklin excelled at science and studied physics and chemistry at the academically rigorous St. Paul’s Girls’ School in London. When she was 15, she decided to become a scientist and in 1938 she enrolled at Newnham College, Cambridge, graduating in 1941. She held a graduate fellowship for a year, but quit in 1942 to work at the British Coal Utilization Research Association, where she made fundamental studies of carbon and graphite microstructures. This work was the basis of her doctorate in physical chemistry, which she earned from Cambridge University in 1945. After Cambridge, she spent three productive years (1947-1950) in Paris at the Laboratoire Central des Services Chimiques de L’Etat, where she learned X-ray diffraction techniques. In 1951, she returned to England as a research associate in John Randall’s laboratory at King’s College, London.
The Work at King’s
John Randall was the director of the laboratory at Kings College; Wilkins served under him as the assistant director. In the summer of 1950, Randall appointed a new X-ray specialist named Rosalind Franklin to work on protein solutions. Wilkins suggested to Randal that Franklin be transferred to DNA work to which he agreed. In a letter written to Franklin, Randall gave Franklin the impression that she and a graduate student were given the project to work on DNA. This became a source of friction in the laboratory. At the time Maurice Wilkins, the assistant director of the laboratory, was away on vacation. Without informing Wilkins, he wrote a letter to Franklin assigning DNA structural studies to her. According to Wilkins, Randall had given Rosalind the impression that he and Stokes were stopping their work on DNA without consulting them. He also did not warn Franklin about Wilkin’s continuing interest in DNA (physics today.org).” This misunderstanding may have been responsible for a difficult relationship that developed between Rosalind Franklin and Maurice Wilkins, one that led to a lack of cooperation between them.
By Easter of 1951, Rosalind was busy rebuilding the X-ray set with the fine-focus Ehrenberg X-ray tube that had been set up by Stokes and Raymond (Wilkins, 2003, p 135). In the spring of 1951, Wilkins attended a conference in Naples. At the Conference Wilkins showed the first really clear crystalline X-ray pattern of DNA that he had produced with Raymond Gosling (Wilkins, 2003, p 137). In Naples, Wilkins met Jim Watson. Wilkins recollected: “He was very excited about my presentation when I showed our photographs that showed that DNA was crystalline, and he decided to work on the structural chemistry of nucleic acids and proteins (Wilkins, 2003, p 138-139).”
While in Naples, Wilkins obtained samples of Sepia sperm from the Naples Stazione Zoologica. When he returned to London, Wilkins began to compare the Signer DNA to the DNA from a number of different species (Wilkins, 2003, p 140). He found that the DNA from the various sources gave patterns that were basically the same. The patterns all showed a central X. “That feature, as Stokes had urged, was a strong sign of a helix (Wilkins, 2003, p 141).” Wilkins discussed these results in a talk that he gave in Cambridge in July 1951, at a meeting of protein X-ray workers organized by Max Perutz. He described that all DNA had the same unique universal structure that was twisted regularly into a helix (Wilkins, 2003, p 141). As Wilkins left the building after the meeting, Rosalind Franklin came up to him and told him that he should stop doing X-ray work. She concluded with the instruction: “Go back to your microscopes.” (Wilkins, 2003, p 142). Rosalind Franklin’s reaction probably resulted from her understanding that she was given responsibility for the X-ray work on DNA as suggested in Randall’s letter.
In the summer, Wilkins attended the Gordon Conference held in New England in the United States (Wilkins, 2003, p 151). There he met Erwin Chargaff, the biochemist from Columbia University in New York. “He had analyzed the nitrogen bases in DNA, and had found that of the four bases, guanine was present in the same quantity as cytosine, and adenine in the same quantity as thymine. At the conference, Chargaff gave Wilkins samples of DNA.
After attending a conference in Stockholm, Wilkins returned to London in September. He met with Rosalind. Wilkins found that Rosalind had made an important discovery. She found that there were two types of DNA, depending of the level of humidity. At humidity levels of 75%, DNA existed in the form of A-DNA, but at the higher level of 92%, DNA existed in a new pattern called B-DNA. At this time, a strained relationship existed between Wilkins and Franklin. In order to reduce friction in the working relationship Wilkins suggested that he should study the DNA that Chargaff had given him; and that Rosalind should continue working with the DNA obtained from Signer. He also agreed to use the old Raymax X-ray set that Gosling and he had used before, while Rosalind was given exclusive use of the new fine-focus X-ray tube (Wilkins, 2003, p 157).”
In October 1951 a laboratory colloquium was presented at King’s to summarize the DNA X-ray work that had been done there. The meeting was attended by James Watson. Wilkins went through the X-ray evidence that DNA from a wide range of species gave a basically similar ‘cross-ways’ X-ray pattern indicating the same helical structure (Wilkins, 2003, p 163).” “Stokes described his Bessel function description of diffraction from a helical structure. This technique allowed one to calculate X-ray diffraction from a helical structure. It provided support for the idea that the B-DNA structure discovered by Franklin was helical. Rosalind then summarized the recent progress of her work on DNA structure. She presented clearly reasons why the phosphate groups should be on the outside of the molecule, and the importance of understanding the role of water in DNA structures A and B.”
Watson and Crick Determine the Molecular Structure of DNA
Watson and Crick approached the problem of finding the molecular structure of DNA by building a model. They were influenced by the work of the American Biochemist, Linus Pauling, who had successfully employed this approach to elucidate the structure of large protein molecules. Pauling described the structure of polypeptide chains as an α helix. The amino acids in a helix are arranged in a right-handed helical structure. Watson and Crick believed that they would be able to determine the structure of DNA in the same way. According to Watson, “All we had to do was to construct a set of molecular models and begin to play—with luck, the structure would be a helix (Watson, 1968 pp. 50-51).” Meanwhile, Rosalind Franklin was approaching the problem by using a laborious and time consuming mathematical technique called Paterson Function Analysis.
Two weeks after the colloquium at King’s, Francis Crick telephoned Wilkins to say that he and Jim Watson had built a model of DNA and invited him to Cambridge to see it. Wilkins, along with Rosalind, Gosling, Bill Seeds, and Bruce Fraser traveled to Cambridge (Wilkins, 2003, P 171). “We were surprised to find the model disappointing. According to our thinking it was completely inside out, with the helical regularity in its three chains being established by the phosphate groups held together along the helix axis. But magnesium ions (which we had never heard of in DNA) were needed to hold the phosphates together. On the outside of the helix, the bases seemed to flop about without being stacked on top of each other, or stabilized in any way (Wilkins, 2003, p 172).”
After learning of the incorrect model built by Watson and Crick, the MRC directors, Randall and Bragg, decided that Watson and Crick should stop working on DNA (Wilkins, 2003, p175). Watson and Crick then handed over to the group at King’s the set of metal jigs that they had designed carefully so that the workshop mechanics could make accurate parts for constructing DNA models (Wilkins, 2003, p175).” “Francis and Jim were disappointed that we did not use the jigs for making DNA models: Rosalind scorned the equipment, sticking to her view that model-building was what one did after finding the structure by proper X-ray procedures.”
In January, 1953 Watson and Crick received news that Linus Pauling had constructed a model of DNA. Two copies of the manuscript were being sent to the lab, one to Lawrence Bragg, the other to Peter Pauling, Linus’ son, who was visiting the lab. Watson and Crick found that the model was a three chain helix with the sugar-phosphate backbone in the center. Watson the realized that “the phosphate groups on Linus’ model were not ionized, but that each group contained a bound hydrogen atom and so had no net charge (Watson, 1968, p102). The model was not correct.
One day in January 1953, Raymond Gosling met Maurice Wilkins in the corridor and handed him an excellent B pattern photograph that Rosalind and he had taken. Wilkins described it as being much clearer and sharper than the first clear B pattern the Rosalind had produced in October 1951, and pointed out that the new pattern showed the helix X-shape more clearly than ever before. A few days later James Watson made a visit to King’s College and Wilkins showed him the photograph. In his book, “The Double Helix”, Watson described his reaction to seeing the photograph: “The instant I saw the picture my mouth fell open and my pulse began to race. The pattern was unbelievably simpler than those obtained previously (‘A” form). Moreover, the black cross of reflections which dominated the picture could arise only from a helical structure (Watson, 1968 pp. 167-169).” Much controversy has erupted as to whether it was appropriate for Wilkins to have shown Watson the picture without Rosalind Franklin’s knowledge.
Watson explained to Bragg what he had learned about the B pattern of DNA. He also expressed his concern that Linus Pauling would beat them to the structure of DNA. He convinced Bragg to allow them to begin model building again.
At this time Watson had been trying to understand how the nitrogenous bases joined together. He had been using diagrams from a book on nucleic acids by Davidson. The book depicted the nitrogenous bases in their enol form. At that time the American crystallographer Jerry Donohue was visiting the lab. Jerry contended that the enol forms shown in the book were incorrect and that the bases were actually in the keto form. Watson cut out accurate representations of the bases on stiff cardboard. He had been shifting the bases in and out of various pairing possibilities. He describes the insight that came next: “Suddenly I became aware that an adenine-thymine pair held together by two hydrogen bonds was identical in shape to a guanine-cytosine pair held together by at least two hydrogen bonds (Watson, 1968, p123).” This became the base pairing rule. It explained Chargaff’s rule that the amount of adenine in DNA equaled the amount of thymine and the amount of cytosine equaled the amount of guanine. The discovery also suggested a copying mechanism for DNA.
Francis then arrived. “A few minutes later he spotted the fact that the two glycosidic bonds (joining base and sugar) of each base pair were systematically related by a diad axis perpendicular to the helical axis. Thus, both pairs could be flip-flopped over and still have their glycosidic bonds facing in the same direction. This had the important consequence that a given chain could contain both purines and pyrimidines. At the same time, it strongly suggested that the backbones of the two chains must run in opposite directions (Watson, 1968, p 125).”
Watson and Crick then set to work building a model of DNA. They used metal jigs they had ordered from the machine shop. The DNA model had two strands – the famous Double Helix. The nitrogenous bases were stacked in pairs in the center. The bases were joined by hydrogen bonds. Adenine was joined to thymine and guanine was joined to cytosine. The sugars and phosphates alternated along the sides of the molecule.
After it was clear that their model accurately portrayed the structure of deoxyribonucleic acid, Francis Crick went into the Eagle pub on February 13, 1953, “telling anyone within earshot that we had found the secret of life (Watson, 1968 pp. xv-xvi).”
Maurice Wilkins and Rosalind found that their X-ray data strongly supported the double helix. Watson and Crick reported their results in the journal Nature on April 25, 1953. Two other papers on DNA also appeared in that issue, one by Wilkins, Stokes, and Wilson, and the other by Franklin and Gosling.
Study Guide History of DNA The Genetic Material draft 3 Corrections made 11/24/18
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