SEB Bulletin July 2006 - Thymus desoxypentose nucleic acid...sixty years on
Sixty years ago this July the SEB held the first of an annual series of symposia. The subject of this symposium was nucleic acid and the proceedings of this meeting provide a remarkable insight into the early days of DNA research, a full seven years before Watson and Crick proposed the double helix structure and just one year after the end of the Second World War. Contributed papers range from those that address the structure and synthesis of nucleotides to those describing nucleic acid metabolism in different sub-cellular compartments and in various cell types. However, with the benefit of hindsight, it is the papers that discuss the structure of the nucleic acids that are the most fascinating. Two forms of nucleic acid were recognised, that in which the carbohydrate was pentose and that in which it was desoxypentose (RNA and DNA). The nucleic acid prepared from yeast was considered as characteristic of the first type whilst that obtained from thymus tissue was predominantly composed of the latter. Thus “yeast ribonucleic acid” and “thymus desoxypentose nucleic acid” are used as synonyms for RNA and DNA respectively.
The paper “X-ray analysis of nucleic acids” presented by W. Astbury, then working in Leeds, builds on work that he had initiated some years earlier but which had been interrupted by the war, an event alluded to in the understated opening paragraph “…there has been little opportunity to go on with the nucleic acids until the last few months…” From the X-ray diffraction images he obtained (Figure 1A), it was clear that DNA had a crystalline structure that repeated along its axis. Using a combination of X-ray diffraction image analysis and model building, Astbury proposed that the nucleotides were joined via 3’ and 5’ linkages and were stacked “like a pile of plates” at intervals of 3.34 Å perpendicular to the long axis. An alternative arrangement in which the nucleotides were “…disposed spiral wise along the long axis of the molecule” was considered but not favoured due to the requirement to closely pack neighbouring DNA molecules in order to achieve the relatively high density (1.63 gcm-3 ) observed for dried DNA. Whilst DNA gave images indicating a regular crystalline structure, the chemically very similar yeast nucleic acid (RNA) showed little evidence of crystallinity (Figure 1B). Viewing these diffraction images now, it is clear that that obtained for DNA is very similar to the higher resolution images later obtained by Raymond Gosling and Maurice Wilkins for the low humidity “A” form which so excited Jim Watson when shown at the Naples meeting in 1951 (Watson 1968).
In the preceding paper, “The macromolecular behaviour of nucleic acids”, by Masson Gulland and Jordan (University College, Nottingham), dissociation curves for the forward and back titration of calf thymus DNA with acid and alkali were reported. These authors concluded that the groups titrated between pH 8.0 and 12.0 were the enolic hydroxyl groups of guanine and thymine whilst those titrating between 2.5 and 6.3 were the amino groups of guanine adenine and cytosine (at this time the wrong tautomeric forms of the bases were widely used). Due to the complete identity of the back titration curves, these authors reached the remarkable conclusion that hydrogen bonds exist between the hydroxyl and amino groups of bases “..in the same or adjacent covalent chains”. It was also observed that the high viscosity of a DNA solution was maintained between pH 5.6 and 10.9 but fell sharply above and below these values and it was considered “significant” that these limits of pH were coincident with those interpreted as causing the breakage of hydrogen bonds between bases. “…the fall in viscosity is closely associated with the liberation of the amino and hydroxyl groups which are blocked in the original nucleic acid, and the titration evidence, as has been pointed out above, strongly suggests that the blocking of these groups is caused by a form of linkage between them.” Breaking of these hydrogen bonds resulted in disaggregation into smaller molecular units of covalently linked polynucleotides – the DNA was now single stranded. However, if a DNA solution treated with alkali at pH 12.5 was allowed to stand at pH 7.0 for 96 hours, the viscosity approached that of the original solution – interpreted as some form of re-polymerisation as the bonds reformed.
Avery and co-workers had three years earlier demonstrated the ability of purified DNA from a Type III Pneumococcus strain to convert an avirulent Type II strain to the virulent Type III (Avery et al 1944). This experiment is now credited as being the first demonstration of DNA’s role as the carrier of heritable genetic information and is referenced in three of the manuscripts contributed to the Symposium proceedings. Despite this, it is not always clear that the importance of DNA is fully appreciated. Thus, Edgar and Ellen Stedman (University of Edinburgh) writing on “The function of deoxyribose-nucleic acid in the cell nucleus” state that “The material of which the chromosomes is composed must clearly fulfil two requirements: it must be capable of accounting in a broad manner for the hereditary functions of the chromosomes, and it must possess …. basophilic properties. The first of these requirements can be satisfied only by one known type of compound, a protein.”
Elsewhere, Astbury interprets the crystalline, repeating structure evident from his X-ray diffraction work to indicate that the order of the four nucleotides could not be random “It seems improbable, too, to judge by the degree of perfection of the X-ray fibre diagram, that these four different kinds of nucleotides are distributed simply at random; rather they must follow one another in some definite order – at least, in the more crystalline regions of the structure that give rise to the regular diffraction pattern.” In a second paper, “The structures of nucleic acids”, given at the Symposium, Masson Gulland is more positive about the potential of nucleic acids; “There is at present no indisputable chemical evidence that the nucleotides are arranged in anything other than a random manner in the polynucleotides. It must be realized that the existence of tetranucleotide units, repeated throughout the molecule, would limit the potential number of isomers and hence diminish the possibilities of biological specificity. Thus the immense numbers of variations presented by a polynucleotide in which the nucleotides occur in random sequence is reduced, in the case of an unbranched polynucleotide, to a single possible structure, varying only in its length, if the same, uniform tetranucleotide unit occurs throughout”. John Masson Gulland was to die the following year in a railway accident aged 49, Bill Astbury continued a distinguished carer at Leeds until his death in 1961.
Much has been written about the competition and conflicts between the groups at Kings and Cambridge in the lead up to the discovery of the double helix, but it is perhaps fitting, sixty years after this Symposium, to remember some of the other scientists who contributed to this field in the years preceding this discovery.
References
Astbury WT. (1947) X-ray analysis of nucleic acids. Symposia of the Society for Experimental Biology 1 66-76.
Avery OT, MacLeod CM, McCarty M. (1944) Studies on the chemical nature of the substance inducing transformation of the Pneumococcal types. J. Exp. Med
79 137-156.
Masson Gulland J. (1947) The structures of nucleic acids. Symposia of the Society for Experimental Biology 1 1-14.
Masson Gulland J, Jordan DO. (1947) The macromolecular behaviour of nucleic acids. Symposia of the Society for Experimental Biology 1 56-65.
Stedman E, Stedman E. (1947) The function of deoxyribose-nucleic acid in the cell nucleus. Symposia of the Society for Experimental Biology 1 232-251.
Watson JD. (1968) The Double Helix: A Personal Account of the Discovery of the Structure of DNA. (Athenaeum, New York).
Dr. Rupert Fray is a lecturer in Plant Sciences at Nottingham University. In addition to a personal interest in the history of science, his Group’s primary research areas are in mRNA metabolism and also in bacterial quorum sensing in relation to plant-microbe interactions.
