Meanwhile, even as Miescher's name fell into obscurity by the twentieth century, other scientists continued to investigate the chemical nature of the molecule formerly known as nuclein. One of these other scientists was Russian biochemist Phoebus Levene. A physician turned chemist, Levene was a prolific researcher, publishing more than 700 papers on the chemistry of biological molecules over the course of his career. Levene is credited with many firsts. For instance, he was the first to discover the order of the three major components of a single nucleotide (phosphate-sugar-base); the first to discover the carbohydrate component of RNA (ribose); the first to discover the carbohydrate component of DNA (deoxyribose); and the first to correctly identify the way RNA and DNA molecules are put together.
During the early years of Levene's career, neither Levene nor any other scientist of the time knew how the individual nucleotide components of DNA were arranged in space; discovery of the sugar-phosphate backbone of the DNA molecule was still years away. The large number of molecular groups made available for binding by each nucleotide component meant that there were numerous alternate ways that the components could combine. Several scientists put forth suggestions for how this might occur, but it was Levene's "polynucleotide" model that proved to be the correct one. Based upon years of work using hydrolysis to break down and analyze yeast nucleic acids, Levene proposed that nucleic acids were composed of a series of nucleotides, and that each nucleotide was in turn composed of just one of four nitrogen-containing bases, a sugar molecule, and a phosphate group. Levene made his initial proposal in 1919, discrediting other suggestions that had been put forth about the structure of nucleic acids. In Levene's own words, "New facts and new evidence may cause its alteration, but there is no doubt as to the polynucleotide structure of the yeast nucleic acid" (1919).
Indeed, many new facts and much new evidence soon emerged and caused alterations to Levene's proposal. One key discovery during this period involved the way in which nucleotides are ordered. Levene proposed what he called a tetranucleotide structure, in which the nucleotides were always linked in the same order (i.e., G-C-T-A-G-C-T-A and so on). However, scientists eventually realized that Levene's proposed tetranucleotide structure was overly simplistic and that the order of nucleotides along a stretch of DNA (or RNA) is, in fact, highly variable. Despite this realization, Levene's proposed polynucleotide structure was accurate in many regards. For example, we now know that DNA is in fact composed of a series of nucleotides and that each nucleotide has three components: a phosphate group; either a ribose (in the case of RNA) or a deoxyribose (in the case of DNA) sugar; and a single nitrogen-containing base. We also know that there are two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), each with two fused rings, and the pyrimidines (cytosine [C], thymine [T], and uracil [U]), each with a single ring. Furthermore, it is now widely accepted that RNA contains only A, G, C, and U (no T), whereas DNA contains only A, G, C, and T (no U) (Figure 1).
Figure 1: The chemical structure of a nucleotide.
A single nucleotide is made up of three components: a nitrogen-containing base, a five-carbon sugar, and a phosphate group. The nitrogenous base is either a purine or a pyrimidine. The five-carbon sugar is either a ribose (in RNA) or a deoxyribose (in DNA) molecule.
The discovery of the structure of DNA was reported 50 years ago this month. But the saga began many years before, says Susan Aldridge
On 25 April 1953, a paper appeared in Nature that was to transform the life sciences - from biochemistry and agriculture, to medicine and genetics. James Watson and Francis Crick, then at Cambridge University, reported the discovery of the structure of DNA (deoxyribonucleic acid) - the molecule that genes are made of.
Crick and Watson used model building to reveal the now famous double helix of DNA, but the X-ray crystallographic data of Rosalind Franklin and Maurice Wilkins at King’s College, London, were crucial to the discovery. The breakthrough also owed much to advances in biochemical techniques, microscopy, chemical analysis and theories of chemical bonding that had developed from the mid-19th century. The true significance of the DNA structure was underlined around the same time by the final settlement of a decades-long controversy over whether DNA or protein was the ’life molecule’.
The DNA saga began in 1869, when Swiss biochemist Friedrich Miescher isolated a new substance from the nuclei of white blood cells. Researchers were recently aware that cells were the basic unit of life and Miescher was interested in their chemical components. Each morning, he called at the local clinic to pick up dirty bandages, for in the days before antiseptics these were soaked in pus - a good source of white blood cells with their large nuclei. Adding alkali made the cell nuclei burst open, releasing their contents, from which Miescher extracted DNA (which he called nuclein).
Analysis of this nuclein showed that it was an acid, containing phosphorus, so it did not fit into any of the known groups of biological molecules, such as carbohydrates and proteins. Miescher calculated its formula as C29H49O22N9P3 - a gross underestimate, reflecting the fact that DNA is a long, fragile molecule that readily fragments. Miescher must have used one of the fragments for the determination of the formula. Nuclein was rechristened nucleic acid and, despite its chemical novelty, its biological significance was not fully realised for many more decades.
Meanwhile, thanks to developments in microscopy, the cell continued to yield its secrets. In 1879 the German biologist Walther Flemming discovered tiny thread-like structures called chromatin (later known as chromosomes) within the nucleus - so-called because they readily absorbed colour from the new stains used to reveal cellular components. Studies on cell division were to reveal the key role played by chromosomes in inheritance - how they double up before the cell splits, and then divide into two sets, taking a fresh copy into each ’daughter’ cell.
Further analysis suggested that chromosomes contained DNA, which led another German researcher, Oskar Hertwig, to declare that ’nuclein is the substance which is responsible ... for the transmission of hereditary characteristics’. Not everyone agreed - Miescher for one. Chromosomes also contained protein, and biochemists were just beginning to appreciate what large, complex molecules proteins were. The fragility of DNA was to conceal its underlying complexity for many more years.
Ironically, Miescher was possibly the first to put forward the idea of a chemical code handing on biological information from one cell to another but he, like many others after him, believed that only proteins were capable of carrying such a code.
By 1900, it was known that the basic building blocks of DNA were phosphate, a sugar (later shown to be deoxyribose) and four heterocyclic bases - two of which were purines [adenine (A) and guanine (G)] while the other two were pyrimidines [cytosine (C) and thymine (T)].
It was Phoebus Levene, of the Rockefeller Institute, New York, and a former student of the Russian chemist and composer Alexander Borodin, who showed that the components of DNA were linked in the order phosphate- sugar-base. He called each of these units a nucleotide, arguing that the DNA molecule consisted of a string of nucleotide units linked together through the phosphate groups, which are the ’backbone’ of the molecule.
But no-one appreciated the extraordinary length of the DNA molecule until well into the 20th century. We now know that the DNA from one human cell would, if laid end to end, make up a molecule of about 1m in length. Even a simple organism like the bacterium E. coli has a DNA molecule just over 1mm long. Miescher had not realised this, of course, and nor did Levene, who insisted that DNA was a relatively small molecule - probably about 10 nucleotides long.
Levene was also convinced that the amounts of the four bases were the same in all DNA molecules, whatever their origin. So even when Swedish researchers Torbj?rn Caspersson and Einar Hammersten showed, in the 1930s, that DNA was a polymer, most people continued to believe in Levene’s ’tetranucleotide hypothesis’. Even if DNA contained millions of nucleotides, they were thought to be arranged in a monotonous and predictable fashion that could have no meaningful information content. Levene’s contemporary, the great German chemist Emil Fischer, had shown that proteins are made of amino acids, linked together in diverse sequences. It looked more and more as if proteins carried the genetic code, while DNA played a supporting role in the chromosomes.
A breakthrough came from Oswald Avery, Colin McLeod and Maclyn McCarty, a team of medical microbiologists at the Rockefeller Institute in New York. They were trying to identify the nature of the ’transforming principle’ - a substance discovered by English microbiologist, Fred Griffith, in 1928. Griffith had been experimenting with two species of pneumococcus, the bacteria that cause pneumonia (much-feared in the days before antibiotics).
One form - known as the smooth form from its appearance when cultured in Petri dishes - was known to be pathogenic, while the second, ’rough’, form was harmless. To his surprise, Griffith found that mixing live rough bacteria with killed smooth pneumococci could transform the rough pneumococci into a virulent smooth form. Evidently some substance - the transforming principle (genes, in other words) - had passed from the smooth bacteria to the rough bacteria. Using enzymes that broke down specific cell components, Avery and his team showed by a process of elimination that DNA, not protein, was the transforming principle.
Physicists had also contributed to this debate - for instance, Erwin Schr?dinger put forward the concept of the ’aperiodic crystal’ in his influential book What is life?. Simple crystals such as sodium chloride cannot carry genetic information because their ions are arranged in a periodic pattern. What Schr?dinger was proposing was that the ’blueprint’ of life would be found in a compound whose components were arranged in a long irregular sequence, which carried information in the form of a genetic code, embedded within its chemical structure. Proteins had been the obvious candidate for the aperiodic crystal, with the amino acid sequence providing the code. Now, with Avery’s discoveries, the spotlight fell on DNA as an alternative choice for the genetic material.
Research to determine the structure of DNA took on an added urgency (although final confirmation of its central role was still to come, from experiments carried out by Alfred Hershey and Martha Chase in the US in the early 1950s). The Austrian chemist, Erwin Chargaff - for one - was deeply impressed by Avery’s work. He wrote: ’I saw before me in dark contours the beginning of a grammar of biology. Avery gave us the first text of a new language, or rather he showed us where to look for it. I resolved to search for this text.’ Chargaff pioneered the paper chromatography of nucleic acids, using this to determine how much of each of the component nucleotides was contained in a DNA sample. He rapidly demolished Levene’s tetranucleotide hypothesis. Each species differed in the amount of A, C, G and T - but within the species, the proportions of each are identical, no matter which tissue the DNA is extracted from. It was just what might be expected for a molecule that is the biological signature for the species.
Even more significant was Chargaff’s further discovery that the proportion of A in any DNA molecule was always equal to the proportion of T and, likewise, the amount of G and C always corresponded - a rule that became known as Chargaff’s ratios. Although Chargaff himself appears to have made little direct use of his findings, the idea of base-pairing (A with T, C with G) was to be a crucial step in piecing together the three-dimensional structure of DNA.
The final phase of solving the puzzle of the DNA structure relied on X-ray crystallography. The use of X-rays to solve the structures of large biological molecules began with Dorothy Hodgkin’s work on penicillin, lysosyme, and vitamin B12, and Max Perutz’s work on haemoglobin from the 1930s. By 1938, William Astbury, a student of William Bragg (who, with son Lawrence, had invented the technique in 1913) had X-ray pictures of DNA, but they were hard to interpret.
The late 1940s saw three separate groups working intensively on the DNA structure. At King’s College, London, Maurice Wilkins was intrigued by the long fibres that DNA forms when it is pulled out of watery solutions with a glass rod, wondering if this meant there was some regularity to its structure. He produced more X-ray pictures, using makeshift apparatus the like of which is hard to imagine nowadays. In 1951, Wilkins was joined by Rosalind Franklin, a British physical chemist who already had an international reputation for her work on the X-ray crystallography of coals. She set about building a dedicated X-ray lab at King’s and was soon producing the best images ever of DNA. These led her to the idea that maybe the DNA molecule was coiled into a helical shape.
Linus Pauling, the US chemist, and author of The nature of the chemical bond, began to think along similar lines. After all, Pauling had already discovered helical motifs in protein structures. Around this time, Francis Crick - with a background in maths and physics, and the younger James Watson, with expertise in the molecular biology of phage (viruses that infect bacteria, then used as a laboratory tool for genetic studies), joined forces at the Cavendish Laboratory in Cambridge, intent on cracking the DNA structure themselves, using a model building approach.
They had the idea that the structure of DNA had to allow the molecule to copy itself during cell division, so that an exact replica of its code - which, again, was embedded in the structure - could pass into each new cell. A visit to the Cavendish by Chargaff in 1952 prompted the further thought that perhaps the sequence of bases might represent the genes in a chemical code. Meanwhile, Pauling published a paper on the DNA structure, but it contained a major error (he put the phosphate groups on the inside). The entry of this scientific giant into the race spurred Crick and Watson to greater efforts, while Wilkins and Franklin were not really getting on well and were making little progress with DNA.
A seminal moment came when Wilkins showed Watson one of Franklin’s photos of the so-called B form of DNA. Previous studies had used the A form, which contains less water and had led to images that were hard to analyse. This picture, by contrast, was beautifully simple and seemed to point clearly to a helical structure for the molecule. As Watson puts it in his famous memoir: ’The instant I saw the picture, my mouth fell open and my heart began to race’.
Model building - using metal plates for the nucleotides and rods for the bonds between them - now began in earnest. But Crick and Watson did not know whether to build their helix with the phosphates inside or outside, and they were unsure how to incorporate Chargaff’s ideas on base pairing.
The final clue came from another visitor to the Cavendish, the American chemist Jerry Donohue, who pointed out how hydrogen bonding allows A to bond to T and C to G. This allows a double helical structure for DNA, where the two strands have the bases on the inside, paired up, and the phosphates on the outside.
The true beauty of the model that Crick and Watson built was that the structure immediately suggested function. As they hinted, in their Nature paper: ’It has not escaped our notice that the specific pairing we have postulated suggests a possible copying mechanism for the genetic material’.
The DNA molecule is self-replicating (as was proved by experiments a few years later) because it can unwind into two single strands. Each base then attracts its complementary base, by hydrogen bonding, so that two new double helices are assembled.
Franklin and Wilkins did not completely miss out on credit for the DNA structure; their own separate papers were published back to back with Crick and Watson’s in the same issue of Nature. Crick, Watson and Wilkins went on to win the Nobel prize for their work in 1962 (Franklin died of cancer at the age of 37 in 1958).
The discovery of the DNA structure was the start of a new era in biology, leading, over the next two decades, to the cracking of the genetic code and the realisation that DNA directs the synthesis of proteins. There were technical advances too, such as DNA sequencing, genetic engineering, and gene cloning. More recently, the complete sequences of many organisms have been solved - including the human genome in June 2000. The next 50 years of the DNA story will be all about realising the practical benefits of Crick and Watson’s discovery for humanity - in industry, medicine, food and agriculture.
Source: Chemistry in Britain
- 2003 Double helix celebrations, Cold Spring Harbor Laboratory, DNA50 website.
- H. F. Judson, The eighth day of creation. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1996.
- J. D. Watson, The double helix. London: Penguin, 1999.
- E. Schr?dinger, What is life?. Cambridge: CUP, 1992.
- D. Chambers, Ann. N Y Acad. Sci., 1995, 758.
- S. Aldridge, The thread of life: the story of genes and genetic engineering. Cambridge: Cambridge University Press, 1998.
- B. Maddox, Rosalind Franklin: The dark lady of DNA. London: Harper Collins, 2002.
- J. D. Watson and F. H. C. Crick, Nature, 1953, 171, 737.
- M. H. F. Wilkins, A. R. Stokes and H. R. Wilson, Nature, 1953, 171, 738.
- R. E. Franklin and R. G. Gosling, Nature, 1953, 171, 740.
A historic paper
In their famous Nature paper announcing the structure of DNA, Crick and Watson come straight to the point. ’We wish to put forward a radically different structure for the salt of deoxyribose nucleic acid.’ It’s often assumed that if the pair were to submit this paper today, they would be required to say ’A radically different structure for the salt of deoxyribose is proposed’. In fact, Nature has always encouraged the use of the active, personal voice, in the interests of clarity and readability. A glance through any recent issue confirms that there is no ban on the words ’we’ or ’our’. But many researchers remain resistant - believing, perhaps, that the passive voice adds authority and objectivity to their work.
Although the DNA paper is short, lively and readable, it did not make a huge impact when it first appeared. While Sydney Brenner (who shared the 2002 Nobel prize for physiology or medicine in recognition of his contribution to molecular biology) immediately judged it a milestone, many others were either indifferent or declared it to be just wrong. Crick and Watson’s work got far greater exposure in 1968 with the publication of Watson’s lively and controversial account of his life in research, which is said to have inspired many young people to a career in science.