Portal de Periódicos da CAPES

The “scientific catastrophe” in nucleic acids research that boosted molecular biology

2019; Elsevier BV; Volume: 294; Issue: 7 Linguagem: Inglês

10.1074/jbc.cl119.007397

1083-351X

Eugenio Frixione, Lourdes Ruiz-Zamarripa,

RNA and protein synthesis mechanisms

The distinctive profile of the double-helix DNA molecule is today, along with Rutherford's depiction of the atom as a miniature planetary system, a worldwide-recognized symbol of twentieth-century science. The complex story of how DNA's tertiary structure was determined is also well-known. Surprisingly, however, far less is commonly known about how the structural subunits of the nucleic acids—i.e. nucleotides, nucleosides, and the specific carbohydrates that distinguish DNA and RNA—were first identified and their connectivity ascertained. This comparative oblivion seems due, at least in part, to the conceptual association of those key findings with an erroneous model of nucleic acid structure, which postulated that these macromolecules would consist of repeating sets of four nucleotides. This model came to be known as the "tetranucleotide hypothesis" and prevailed as the dominant paradigm through almost 4 decades of arduous research in the field. When debunked—with researchers referring by then to this hypothesis as an "effort to force nature into a straitjacket of puerile approximations" (1Chargaff, E., and Davison, J. N., (1955) The Nucleic Acids—Chemistry and Biology, p. 368, Academic Press, New YorkGoogle Scholar), a "scientific catastrophe" (2Glass B. A century of biochemical genetics.Proc. Am. Phil. Soc. 1965; 109: 227-236Google Scholar), and an "absurd" instance of oversimplification (3Chargaff E. What really is DNA?—Remarks on the changing aspects of a scientific concept.Prog. Nucleic Acid Res. Mol. Biol. 1968; 8 (4874234): 297-33310.1016/S0079-6603(08)60549-8Crossref PubMed Scopus (8) Google Scholar)—the whole idea receded into the sidelines of the literature along with its insightful analyses of how the nucleic acids are built in the first place. Early historians have provided more nuanced records (4Olby R. The Path to the Double Helix. Macmillan Press, London1973Google Scholar, 5Portugal F.H. Cohen J.S. A Century of DNA—A History of the Discovery of the Structure and Functions of the Genetic Substance. MIT Press, Cambridge, MA1977Google Scholar6Fruton J.S. P. A. Levene and 2-deoxy-d-ribose.Trends Biochem. Sci. 1979; 4: 49-5010.1016/0968-0004(79)90351-7Abstract Full Text PDF Scopus (2) Google Scholar), but later accounts still ignore or greatly deplore the tetranucleotide hypothesis (7Booth H. Hey M.J. DNA before Watson and Crick—the pioneering studies of J. M. Gulland and D. O. Jordan at Nottingham.J. Chem. Educ. 1996; 73: 928-93110.1021/ed073p928Crossref Scopus (3) Google Scholar, 8Hausmann, R., (2002) To Grasp the Essence of Life—A History of Molecular Biology, pp. 42–83, Kluwer Academic Publishers, Dordrecht, The NetherlandsGoogle Scholar) or mention it just as needed without due references (9Morange M. A History of Molecular Biology. Harvard University Press, Cambridge, MA1998Google Scholar, 10Cobb M. Life's Greatest Secret—The Race to Crack the Genetic Code. Basic Books, New York2015Google Scholar), and even when giving favorable views they come to the usual conclusion that it became a distraction or an obstacle to further advance in the field (11Hunter G.K. Phoebus Levene and the tetranucleotide structure of nucleic acids.Ambix. 1999; 46: 73-10310.1179/amb.1999.46.2.73Crossref Google Scholar, 12Hargittal I. The tetranucleotide hypothesis: a centennial.Struct. Chem. 2009; 20: 753-75610.1007/s11224-009-9497-xCrossref Scopus (12) Google Scholar). Nevertheless, as shown below, the data contained in the tetranucleotide hypothesis—leading up to the first accurate description of DNA polymeric structure (13Levene P.A. Tipson R.S. The ring structure of thymidine.J. Biol. Chem. 1935; 109 (623–630)Abstract Full Text PDF Google Scholar), here recognized as a Classic paper—immediately paved the way toward the basic framework of molecular biology as we know it today. The apparent first observation of a substance later identified as nucleic acid was made by Justus Liebig, who in 1847 reported the presence of an acidic material in a filtrate obtained from beef muscle, which he therefore named "inosinic acid" (from the Greek word inos, fiber, and more specifically those of muscle) (Ref. 14Levene P.A. Bass L.W. Nucleic Acids. Chemical Catalog Company, New York1931Google Scholar, p. 187). Over 20 years later, Friedrich Miescher discovered in nuclei of leukocytes a phosphorus-rich substance remarkably resistant to protein digestion, which he labeled as "nuclein" (15Dahm R. Discovering DNA: Friedrich Miescher and the early years of nucleic acid research.Hum. Genet. 2008; 122 (17901982): 565-58110.1007/s00439-007-0433-0Crossref PubMed Scopus (148) Google Scholar). Finally, in 1889, Richard Altmann succeeded in removing, by pepsin treatment and alkaline hydrolysis, most or all of the protein associated with nucleins obtained from either yeasts or some animal organs, hence referring for the first time to the remaining acidic material as "nucleic acid" (Nucleinsäure) (16Altmann R. Ueber nucleinsäuren.Arch. f. Anat. u. Physiol. Physiol. Abt. 1889; : 524-536Google Scholar). This technical achievement opened the way to investigate nucleic acids apart from their accompanying proteins. Over the following years, Albrecht Kossel, a future Nobel Prize laureate in medicine for his contributions to cell chemistry "including the nucleic substances" (17Albrecht Kossel: Nobel Prize in Physiology and Medicine nomination. 2018; (Accessed December 14, 2018)https://www.nobelprize.org/prizes/medicine/1910/summary/Google Scholar), applied Altmann's method plus other procedures devised by his own research team to determine the main chemical components of the nucleic acids (18Jones M.E. Albrecht Kossel, a biographical sketch.Yale J. Biol. Med. 1953; 26 (13103145): 80-97PubMed Google Scholar). Kossel's laboratory found that hydrolysis of nucleic acids obtained from various sources produced, along with large quantities of phosphoric acid as Miescher had described, specific sets of other molecules depending on the original material used as the source. Such compounds included several purine and pyrimidine bases—guanine, adenine, thymine, uracil, and cytosine—and some kind of carbohydrate suspected to be a pentose (19Hammarsten O. Zur kenntnis der nucleoproteide.Z. Physiol. Chem. 1894; 19: 19-37Google Scholar). By the turn of the century, a variety of nucleic acids with different elemental compositions had been reported, depending on the primary material used, the particular methods applied, and the research groups involved (20Levene P.A. On the biochemistry of nucleic acids.J. Am. Chem. Soc. 1910; 32: 231-24010.1021/ja01920a010Crossref Scopus (3) Google Scholar). Eventually, two main kinds of nucleic acids were distinguished, each kind labeled after the example preferred as an experimental system by the biochemists of the period and therefore better characterized: the yeast nucleic acid, believed to be typical of plants (also called "phytonucleic"), and that obtained from the thymus gland ("thymonucleic", or occasionally "zoonucleic"), which was seen as peculiar to animals. The main differences detected between these two sorts of nucleic acids—actually RNA and DNA—were in composition regarding one of the pyrimidine bases (thymine in thymonucleic versus uracil in yeast) and the sugar in each case (a pentose in yeast versus what was by then supposed to be a hexose in animal tissues). Despite these distinctions, however, "it was found that in nearly all the acids the bases were present in approximately equimolecular proportions, that the number of molecules of phosphoric acid corresponded to that of the bases, and the number of molecules of carbohydrate was equal to that of phosphoric acid" (20Levene P.A. On the biochemistry of nucleic acids.J. Am. Chem. Soc. 1910; 32: 231-24010.1021/ja01920a010Crossref Scopus (3) Google Scholar). There was no hint at the time as to how these various constituents of a nucleic acid would join with one another. Still, Kossel included the nucleic acids, together with proteins and huge carbohydrate molecules like starch and cellulose, in a group of cell substances that would likely be composed of specific building stones (Bausteine), explaining "that these units may be united to form larger structures and that their union takes place according to a determined plan or architectural idea" (21Kossel A. The chemical composition of the cell.Harvey Lectures. 1911; 7: 33-50Google Scholar). He was thinking, obviously, of the notion known today as "monomers," and apparently unaware that in the case of nucleic acids such building blocks had been tentatively identified and named just 3 years earlier by a one-time visitor to his own laboratory (see below). Phoebus Levene, a Russian physician who at only 22 emigrated to the United States, got his start in science in New York and as a visitor in Germany at the laboratories of both Kossel and Emil Fischer (22van Slyke D.D. Jacobs W.A. Biographical memoir of Phoebus Aaron Theodor Levene (1869–1940).Biograph. Mem. Natl. Acad. Sci. U.S.A. 1944; 24: 73-126Google Scholar, 23Tipson R.S. Phoebus Aaron Theodor Levene, 1869–1940: Obituary.Adv. Carbohydr. Chem. 1957; 12 (13617111): 1-12PubMed Google Scholar). In 1905, he was appointed a member of the recently created Rockefeller Institute for Medical Research, and then head there of a department that reportedly became "the center of bioorganic chemistry in America" (24Kohler, R. E., (1982) From Medical Chemistry to Biochemistry: The Making of a Biomedical Discipline, p. 269, Harvard University Press, Cambridge, MAGoogle Scholar). Levene's interest in nucleic acids was evident right away. First, he devised a novel method for their preparation (25Levene P.A. On the preparation of nucleic acids.J. Am. Chem. Soc. 1900; 22: 329-33110.1021/ja02044a003Crossref Scopus (4) Google Scholar) and continued from here with a number of findings on the subject, including a new more elaborate and sequential rather than simultaneous hydrolysis technique (26Levene P.A. Hydrolysis of spleen-nucleic acid by dilute mineral acid.Am. J. Physiol. 1904; 12: 213-219Crossref Google Scholar). This procedure involved milder pH levels and avoided the use of alcohol for precipitating the liberated parts, as commonly done by his competitors, thus obtaining intermediate products of hydrolysis with enough purity for a consistent and therefore more reliable quantification of their relative amounts. In 1908, Levene communicated results of work carried out in collaboration with his student J. A. Mandel on "thymo-nucleic acid" (27Levene P.A. Mandel J.A. Über die konstitution der thymo-nucleinsäure.Ber. Deut. Chem. Ges. 1908; 41: 1905-190910.1002/cber.19080410266Crossref Scopus (3) Google Scholar)—i.e. DNA extracted from the thymus, where Kossel had identified the base "thymine." They used the new gradual decomposition method described in the former paper, leading them to the preliminary conclusion that linear complexes "consisting each one of a phosphoric acid, a carbohydrate, and a base … bind in such a way that they form a polyphosphoric acid. The base is probably bound to the sugar moiety in glycosidic form" (Fig. 1; see Ref. 14Levene P.A. Bass L.W. Nucleic Acids. Chemical Catalog Company, New York1931Google Scholar, pp. 266–275, for alternative structures of nucleic acids in the contemporary literature). Furthermore, they suggested that those tripartite modules might build also nucleic acids of a higher order, and coined the German words Mononucleotiden and Polynucleotiden to distinguish two levels of integration, although it was not clear to them at the time just how the latter would be integrated. The following year, Levene and Walter A. Jacobs—an assistant and later associate researcher at the Rockefeller Institute—found this primary nucleotide constitution again in the yeast nucleic acid (i.e. RNA) (28Levene P.A. Jacobs W.A. Über die hefe-nucleinsäure.Ber. Deut. chem. Ges. 1909; 42: 2474-247810.1002/cber.190904202148Crossref Scopus (17) Google Scholar). Here, they also announced to have succeeded in purifying the freed glycosidic components of yeast guanylic acid down to their crystalline form and proposed for these subunits "the general term 'nucleosides'." Moreover, this same work provided evidence that the pentose contained in yeast nucleic acid, so far believed to be l-xylose, was in fact "d-ribose" (i.e. a sugar synthetized almost 20 years earlier in Emil Fischer's laboratory (29Fischer E. Piloty O. Ueber eine neue Pentonsäure und die zweite inactive Tryoxyglutarsäure.Ber. Deut. Chem. Ges. 1891; 24: 4214-4225Crossref Scopus (15) Google Scholar) but scarcely characterized thereafter). Perhaps overconfident after these brilliant successes, in a subsequent paper that year Levene and Jacobs tentatively argued, based upon various considerations, that "Since the pentoses of guanylic acid and of yeast nucleic acid are identical with [that of] inosinic acid, the nature of [the] pentose in this nucleic acid may now be regarded also as d-ribose" (30Levene P.A. Jacobs W.A. Über die pentose in den nucleinsäure.Ber. Deut. Chem. Ges. 1909; 42: 3247-3251Crossref Scopus (8) Google Scholar). This hasty ruling, which simply ignored a critical hydroxyl group in the sugar of inosinic acid (i.e. DNA), was seemingly confirmed a few weeks later as they claimed that in yeast nucleic acid, "the pentose proved to be identical to that of inosine or guanosine, namely to d-ribose," according to results obtained by hydrolysis with picrate (31Levene P.A. Jacobs W.A. Über hefe-nucleinsäure.Ber. Deut. Chem. Ges. 1909; 43: 2703-2706Crossref Scopus (2) Google Scholar). Correcting this major error took Levene and his associates many more years of hard work (see below). The assumption that d-ribose would be the common sugar in all nucleic acids soon became problematic, as the chemical properties of the sugars in each of the two acids proved to be markedly different. Specifically, whereas yeast nucleic acid (RNA) would readily degrade when heated with ammonia, thymus nucleic acid (DNA) "remained apparently unchanged" under these alkaline conditions (32Levene P.A. Jacobs W.A. On the structure of thymus nucleic acid.J. Biol. Chem. 1912; 12 (411–420)Abstract Full Text PDF Google Scholar). At higher temperatures or neutral pH, free purines and pyrimidines were released and no intact nucleosides were obtained, so the authors attributed their results to some instability of the sugar residue. Then World War I intruded, heavily affecting scientific activity and communication. Meanwhile, the identity of the sugar moiety in thymus nucleic acid remained a nagging central doubt. All attempts to obtain this carbohydrate following hydrolysis under various conditions had so far been unsuccessful. In contrast to the yeast nucleic acid, which regularly produced d-ribose, the thymus nucleic acid hydrolysates contained only levulinic acid, a common product of cellulose degradation. Hence, given the above mentioned general consensus that concerning nucleic acids the yeast was representative of plants whereas the thymus stood for animals, the view that the sugar in thymus nucleic acid was likely a hexose became prevalent, as stated in an influential treatise published in 1914 by Walter Jones, a former Kossel student: "Plant nucleic acids contain a pentose group, and all animal nucleic acids a hexose group in their molecule" (33Jones W. Nucleic Acids—Their Chemical Properties and Physiological Conduct. Longmans, Green and Co., London1914Crossref Google Scholar). Clearing up this issue had to wait for a novel experimental approach made possible by a unique chain of incidents. As it happened, in 1923 Levene was urgently contacted by the Russian neuroscientist and Nobel Prize laureate Ivan Pavlov, who had once been his physiology teacher while studying medicine at St. Petersburg. The now elderly professor was at the moment in New York on his way to Paris, and in quite a difficult situation after being robbed of both his money and passport at Grand Central Station. Levene promptly managed to obtain financial help from the Rockefeller Institute and also assisted Pavlov in getting a new visa. This unexpected encounter after so many years allowed both scientists to talk at length about their respective research. Levene became highly interested in the technical details about Pavlov's world-famous demonstration of a conditioned reflex, by which gastric juice was secreted in a dog's stomach at the sound of a bell or other specific sensory stimulus previously associated with food offer. The fresh secretion was collected for analysis through an implanted fistula, straight from the animal's stomach. Levene surely recalled an experiment he had carried out with Jacobs years earlier, using "a dog with an intestinal fistula which permitted feeding the dog on nucleic acid and collecting the nucleic acid impregnated with enzyme through the fistula" (see the experimental section of Ref. 34Levene P.A. London E.S. The structure of thymonucleic acid.J. Biol. Chem. 1929; 83 (793–802)Abstract Full Text PDF Google Scholar). They were then attempting once again to obtain free nucleosides from thymus nucleic acid, but the test had produced only an impure substance unsuitable for definitive chemical identification. Now, however, Pavlov's experimental setup suggested to Levene that he could try to digest the nucleic acid with fresh gastric juice in a test tube. The following year, Levene visited Pavlov's laboratory in Russia to perform this study, but these attempts were again unsuccessful, "undoubtedly for the reason that the juice was very poor in enzymes" according to Levene. Nevertheless, it was agreed that an expert member of Pavlov's research team would go to the Rockefeller Institute to carry out additional trials. That visit was delayed until 1928, but this time the results were quite interesting. In order to work with an empty and clean gastro-intestinal segment, Russian professor Efim S. London prepared dogs with two fistulas, one for passing nucleic acids into the stomach and the other for collecting the products of digestion from the upper intestine, after the process had occurred within the animal in a fairly natural way. Early the next year, they published a two-page note announcing the isolation and subsequent hydrolysis of a guaninedesoxypentoside [sic] from thymus nucleic acid (35Levene P.A. London E.S. Guaninedesoxypentoside from thymus nucleic acid.J. Biol. Chem. 1929; 81 (711–712)Abstract Full Text PDF Google Scholar), and shortly after they reported, at a meeting of the National Academy of Sciences, the successful isolation of nucleosides from "thymonucleic acid" (i.e. DNA) as well as their sugar component which, "contrary to expectation, is not a hexose but a desoxypentose" (36Levene P.A. London E.S. The structure of thymonucleic acid.Science. 1929; 69: 556Crossref Scopus (1) Google Scholar). By the time this meeting was held, Levene and London had been able to show the sugar moiety to be also a deoxypentose in three other nucleosides of this same acid (see the experimental section of Ref. 34Levene P.A. London E.S. The structure of thymonucleic acid.J. Biol. Chem. 1929; 83 (793–802)Abstract Full Text PDF Google Scholar). The finding by Levene's team of a possible third sugar ring (37Levene P.A. Mori T. Ribodesose and xylodesose and their bearing on the structure of thyminose.J. Biol. Chem. 1929; 83 (803–816)Abstract Full Text PDF Google Scholar) presented one additional puzzle, but further inspection revealed some errors in this study. Therefore, soon they were able to declare: "Hence, the carbohydrate of thymonucleic acid is d-2-ribodesose" (38Levene P.A. Mikeska L.A. Mori T. On the carbohydrate of thymonucleic acid.J. Biol. Chem. 1930; 85 (785–787)Abstract Full Text PDF Google Scholar) (i.e. 2-deoxy-d-ribose). And then they used this assignment to anchor their—and our—thinking about the fundamental nature of nucleic acid polymers, saying (Ref. 14Levene P.A. Bass L.W. Nucleic Acids. Chemical Catalog Company, New York1931Google Scholar, pp. 261–262): Thus, comparing the components of each nucleic acid, it is seen that they differ from each other in the structure of one pyrimidine base and in that of the carbohydrates. The striking differences in the chemical and also in the physical properties of the acids are determined principally by the carbohydrate which enters into their structures. […] It is therefore logical to classify nucleic acids according to their component carbohydrate into ribonucleic acids and ribodesose [i.e. deoxyribose] nucleic acids. In each group the nucleic acid can then be classified by the number of nucleotides contained in it into mononucleotides and polynucleotides. Among the latter, tetra-, penta-, and hexanucleotides have been described. It must be stated, however, that whereas the existence of tetranucleotides is established beyond doubt, that of the higher order is a question in need of further investigation. Next, Levene and co-workers concentrated on the detailed characterization of the junctions of the phosphate groups with their respective bases in the two nucleic acids. In a paper co-authored with R. Stuart Tipson a few years later, they argued as "evident that in desoxy-ribose nucleic acid the positions of the phosphoric acid radicles are carbon atoms (3) and (5) of the desoxy-ribose," whereas in the "ribose nucleic acid … the phosphoryl residues are attached at positions (2) and (3)" (13Levene P.A. Tipson R.S. The ring structure of thymidine.J. Biol. Chem. 1935; 109 (623–630)Abstract Full Text PDF Google Scholar) (Figs. 2A and 3A here). And upon these distinctions, they explained the contrasting responses of the two nucleic acids to alkaline and acidic conditions.Figure 3Structure of RNA. A, Fig. II in Ref. 13Levene P.A. Tipson R.S. The ring structure of thymidine.J. Biol. Chem. 1935; 109 (623–630)Abstract Full Text PDF Google Scholar (reproduced here with permission). This research was originally published in the Journal of Biological Chemistry. Levene, P. A., and Tipson, R. S. The ring structure of thymidine. J. Biol. Chem. 1935; 109:623–630. © the American Society for Biochemistry and Molecular Biology. Four ribose rings, each coupled to one of the four specific bases, are linked in series through phosphate groups, thus constituting a tetranucleotide. The phosphate groups join carbons 3′ and (incorrectly) 2′ of the successive riboses, but condensed formulae of the bases do not show the position of their linkage to carbon 1 of the corresponding ribose. Dotted lines indicate the limits of each nucleotide. This figure is the first approximately correct depiction of the RNA structure. B, current model. Comparative graphic drawn according to the general design and labeling of Fig. II in Levene and Tipson (13Levene P.A. Tipson R.S. The ring structure of thymidine.J. Biol. Chem. 1935; 109 (623–630)Abstract Full Text PDF Google Scholar). The only differences between both interpretations, apart from the correct connectivity between carbons 3′ and 5′ of adjoining nucleotides, are the elemental compositions of the nucleobases, which are here updated. Dotted lines indicate the limits between adjacent nucleotides.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It took almost 20 more years to realize, through extended digestion with ribonucleases and other novel procedures, that in fact the internucleotide phosphodiester linkages in RNA are attached at carbons 3′ and 5′ of the pentose, just as in DNA (39Whitfeld P.R. Markham R. The natural configuration of the purine nucleotides in ribonucleic acids: chemical hydrolysis of the dinucleoside phosphates.Nature. 1953; 171 (13072524): 1151-115210.1038/1711151a0Crossref PubMed Scopus (34) Google Scholar, 40Brown D.M. Heppel L.A. Hilmoe R.J. Nucleotides. Part XXIV. The action of some nucleases on simple esters of mononucleotides.J. Chem. Soc. 1954; : 40-46Crossref Google Scholar). Other than this, however, Figures I and II in Levene and Tipson's paper of 1935 (13Levene P.A. Tipson R.S. The ring structure of thymidine.J. Biol. Chem. 1935; 109 (623–630)Abstract Full Text PDF Google Scholar) showed for the first time the correct—for DNA—and nearly correct—for RNA—chemical structures of the two nucleic acids. And this triumph of modern science opened the map toward a whole new age in biology. As described above, the idea that nucleic acids could be constituted by nucleotides linked up in a series was first conceived by Levene and Mandel in 1908 (27Levene P.A. Mandel J.A. Über die konstitution der thymo-nucleinsäure.Ber. Deut. Chem. Ges. 1908; 41: 1905-190910.1002/cber.19080410266Crossref Scopus (3) Google Scholar), and it was in that work that they also mentioned the possibility that a tetranucleotide or perhaps a pentanucleotide might be involved. This supposition ultimately led to the notion that nucleic acids would consist of several repeats of a set of four nucleotides, with each of the latter corresponding to one of the respective bases. The actual origin of such a view, which became known in the literature as the "tetranucleotide hypothesis," can be traced back to Kossel and Neumann's 1893 speculation that "It is highly probable that there are four nucleic acids, each of which contains only one of the nucleobases" (41Kossel A. Neumann A. Ueber das Thymin, ein Spaltungsprodukt der Nucleinsäure.Ber. Deut. Chem. Ges. 1893; 26 (Quote translated in Portugal and Cohen, 1977, p. 60): 2753-2756Crossref Scopus (17) Google Scholar). This last statement clearly referred to only one base being present in a particular nucleic acid, understood then as a small molecule, whatever the source. In the meantime, however, evidence had accumulated showing that most of the nucleic acids so far examined from many sources, including wheat, yeast, calf thymus, and fish sperm (42Osborne T.B. Harris I.F. Die nucleinsäure des weizenembryos.Hoppe-Seyler Z. Physiol. Chem. 1902; 36: 85-13310.1515/bchm2.1902.36.2-3.85Crossref Scopus (8) Google Scholar, 43Steudel H. Die zusammensetzung der nucleinsäuren aus thymus und aus heringsmilch.Hoppe-Seyler Z. Physiol. Chem. 1906; 49: 406-40910.1515/bchm2.1906.49.4-6.406Crossref Scopus (7) Google Scholar44Levene P.A. Über die hefenucleinsäure.Biochem. Z. 1909; 17: 120-131Google Scholar), contained roughly equal ratios of the four bases corresponding to their particular type, either yeast-like or thymus-like. Hence, there was wide experimental support to sustain the notion of a four-base regularity in the constitution of nucleic acids, by then already suspected by Levene and others to be chains of subunits. Still, although Levene continued arguing in favor of the basic tetranucleotide model up to the end of his days, he also kept in mind the possibility that the tetranucleotide unit was "the minimum molecular weight and the nucleic acid may as well be a multiple of it" (Ref. 14Levene P.A. Bass L.W. Nucleic Acids. Chemical Catalog Company, New York1931Google Scholar, p. 289; see also last remark in the above-quoted paragraph of p. 262). This suspicion was strengthened when various methods showed that at least the thymus nucleic acid could have a molecular weight of up to about one million (45Signer R. Caspersson T. Hammarsten E. Molecular shape and size of thymonucleic acid.Nature. 1938; 141: 12210.1038/141122a0Crossref Google Scholar), and Levene and his team immediately took this as a fact for their own experimental approaches (46Schmidt G. Levene P.A. The effect of nucleophosphatase on "native" and depolymerized thymonucleic acid.Science. 1938; 88 (17829666): 172-17310.1126/science.88.2277.172Crossref PubMed Scopus (11) Google Scholar). Moreover, Levene and Gerhard Schmidt offered evidence that this novel viewpoint applied also to the yeast nucleic acid, because pancreatin was found to act as "a depolymerizing agent, limited to the dissociation of the tetranucleotides of high molecular weight into those of lower molecular weight," so the "native RNA is a polymer of the tetranucleotide" (47Schmidt G. Levene P.A. Ribonucleodepolymerase (the Jones-Dubos enzyme).J. Biol. Chem. 1938; 126 (423–434)Abstract Full Text PDF Google Scholar). They even pioneered one of the innovative techniques for gauging the relative sizes of large molecules—sedimentation by ultracentrifugation— to examine the effects of enzymatic treatment on DNA of various "degrees of polymerization" (i.e. chain lengths) (48Schmidt G. Pickels E.G. Levene P.A. Enzymatic dephosphorylation of desoxyribonucleic acids of various degrees of polymerization.J. Biol. Chem. 1939; 127 (251–259)Abstract Full Text PDF Google Scholar). These data contributed to the emerging conception of the nucleic acids as large linear macromolecules. At the dawn of the twentieth century, most of the molecular components of the nucleic acids had been already identified and grossly quantified, but only faint guesses had appeared as to their connectivity. The modular basic structure of both nucleic acid types, each module consisting of a nitrogenous base associated with a specific pentose, which is in turn linked through phosphate groups to adjoining similar subunits in a linear series, was first elucidated by Levene and his co-workers starting from 1908. Already by the end of the following year, Jacobs and Levene summarized their early but relevant views in the abstract of a paper presented at a meeting of the American Society of Biological Chemists: "We believe it very likely that the nucleic acids are built up of groups, nucleotids [sic], similar in composition to the inosinic acid, which are joined together as the phosphoric acid radicals in the polyphosphoric