Part 4 – The biochemist
In 1908, a biochemist ensconced at the Rockefeller Institute for Medical Research in New York worked away at deciphering the chemistry of nucleic acids, and had just published a paper describing the linear construction of these nuclear compounds. Phoebus Aaron Theodor Levene and his student J. A. Mandel described a linear complex with a phosphoric acid, a carbohydrate (sugar), and a base forming a subunit they called a mononucleotide, with two or more mononucleotides bound together to form what they called a polyphosphoric acid or polynucleotide. The illustration of their best guess of how this polynucleotide compound was linked together is shown below, and is taken from their paper. Although this model was not quite correct, it was a major advance in the understanding of nucleic acids at the time, and Levene would continue to refine and improve his model in the ensuing decades.
Prior to this work by Levene, the laboratory of Albrecht Kossel (who would win a Nobel Prize in Medicine in 1910) had identified the bases called guanine, adenine, thymine, uracil, and cytosine, as key components of nucleic acids. Today we know these bases by their abbreviations as the alphabet of our genetic code: G, A, T and C (for DNA) and G, A, U, and C (for RNA). The bases form the rungs of the helical ladder as we normally visualize DNA today thanks to the crystallographic structure determination of Rosalind Franklin, Francis Crick, and James Watson. Also identified by Kossel were some form of unknown carbohydrate suspected to be a pentose (a five-carbon sugar), as well as large amounts of phosphoric acid as first identified by Friedrich Miescher and published in 1871 (and discussed in a previous blog here). But there was no understanding of how these components were related or connected to each other, and no understanding of their role in biology.
Levene had worked briefly in Kossel’s lab in the late 1890s while recuperating from tuberculosis, before finally landing his lifetime appointment at the Rockefeller Institute in 1905. Levene was hired by Dr. Simon Flexner, the first director of the institute who pulled together what would become an all-star team including Hideyo Noguchi, S. J. Meltzer, Jacques Loeb, Eugene Opie, Rufus Cole, Peyton Rous, and Levene.
Phoebus Levene was born on February 25, 1869 in Sagor Russia, the second of eight children born to Solom and Etta Levene. 1869 was the year that Ulysses S. Grant was inaugurated as the 18th President of the United States; that Dmitri Mendeleev introduced the periodic table of the elements to the Russian Chemical Society; the American Museum of Natural History opened in New York City; a number of states passed anti-Ku Klux Klan laws; the Transcontinental Railroad was completed with the Golden Spike driven on May 10; that Thomas Edison won his first US patent; and the last clipper ship the Cutty Sark was launched from Dumbarton, Scotland. Gregor Mendel’s seminal paper on plant hybridization was published a few years prior in 1866, and Darwin’s On the Origin of Species was published a decade before in 1859.
The family moved to St. Petersburg when Levene was two years-old, and he grew up and was educated there, obtaining his medical degree at the Imperial Military Medical Academy at a time when very few Jews were admitted. It was at the Academy that Levene came under the tutelage of some outstanding professors including Alexander Borodin (from whom he learned chemistry), Ivan Pavlov (who taught physiology and would later gain fame from his experiments on conditioning with dogs), and Alexander Dianin (who taught organic chemistry and gave Levene unrestricted access to his laboratory). This was likely the period in which Levene’s interest in biochemistry grew to displace his training in medicine.
In 1891, part way through his medical studies, Levene’s family decided to move to New York to escape the growing anti-Semitism in Russia. Levene accompanied his family to America, immediately returned to Russia to complete his studies, and in March, 1892 he turned around yet again to land in New York where he gained his medical license. While practicing medicine Levene also spent time in the chemistry labs of Prof. John Curtis at Columbia University, enrolled in the Columbia School of Mines to further his chemistry knowledge, and summered in Berne, Germany in the laboratory of E. Drechsel. In 1896 Levene left his medical practice when he was appointed as an Associate of Physiological Chemistry at the New York State Hospital’s Pathological Institute. This was also the year Levene was struck down by tuberculosis.
Levene was hired in 1905 as a promising young biochemist, and by 1907 was put in charge of the Division of Chemistry, a position he held until his death in 1940. Once established at the Rockefeller Institute, Levene began to work in earnest on the problem of nucleic acid structure. In 1909 Levene and Walter Jacobs published a paper identifying ribose as one of the sugars in nucleic acids (today we know ribose is the sugar in the backbone of RNA or ribonucleic acid). Ribose had been synthesized two decades previously in the laboratory of Emil Fischer (for whom Levene worked briefly in the early 1900s), but saw very little subsequent work until Levene’s discovery of natural ribose.
Also in 1909, Levene published a paper titled On the Biochemistry of Nucleic Acids in which he reviewed the state of the art in nucleic acid research. Within this work, Levene proposed the naming convention that we use to this day: the nitrogenous base combined with the ribose sugar is called a nucleoside; the combination of a nucleoside and a phosphate group is called a nucleotide.
Although identifying ribose as a sugar in nucleic acids was a major accomplishment, Levene also made an error by generalizing that fact and claiming that ribose was present in all nucleic acids. This error became evident as accumulating data suggested that nucleic acids from different sources appeared to contain different sugars.
Finally, in 1929 Levene published a paper with L. A. Mikeska and T. Mori in which they discovered deoxyribose in animal nucleic acids isolated from thymus glands. Today we know that deoxyribose is the carbohydrate component of the backbone in DNA, with each sugar subunit linked by a phosphate group. The chain of alternating deoxyribose and phosphate groups form the twin helical rails of the ladder, connected by the bases that we see as the rungs. But Levene and the scientific community at the time had no notion of the overall structure of nucleic acids, and struggled to conceptually piece together the building blocks one at a time.
In 1935, Levene and R. S. Tipson published a paper describing their updated model of a nucleic acid they called thymidine (DNA obtained from the thymus glands of cows). We see a significant improvement over the model from 1908, and in particular the phosphate linkage between the deoxyribose sugars is now correct.
The same paper also describes their model for RNA, which does not quite get the phosphate linkage correct: the phosphate is shown connecting the 3’ carbon from one ribose sugar to the 2’ carbon of the next ribose. In reality, the RNA phosphodiester bond connects the same 5’ to 3’ carbons between sugars as in DNA, and this would not be corrected until the 1950s, almost two decades later.
A couple more interesting tidbits in these models... Levene correctly assigned the bases guanine, adenine, cytosine, and thymine to DNA, and the same bases but uracil (instead of thymine) for RNA.
Aside from the incorrect RNA bonds, the chemical compositions of the bases were also slightly off.
But these were minor errors that distract from the major accomplishment of understanding the fundamental nucleic acid structures, what we know today as perhaps the most important molecules in science, DNA and RNA. These minor errors also distract from one of the biggest errors in Levene’s career, which was his “tetranucleotide hypothesis”.
In 1901, Walter Sutton published data from grasshopper meiotic division showing that chromosomes were the physical component of Mendel’s heritable factors (discussed in my blog here). The question then was, what was it in chromosomes, the proteins or the nucleic acids, which carried the information required to pass along hereditary traits? Between 1810 and 1922, most of the amino acids we know to make up proteins were discovered, and since there were almost two dozen known amino acids, like an alphabet, there was sufficient complexity in proteins for them to carry genetic information. Therefore proteins, at that time, were considered the primary candidate as the genetic chromosomal material. This was in contrast to nucleic acids which contained only four bases or nucleotides, and therefore possessed insufficient complexity, it was thought, to encode genetic information.
Today we know that DNA and RNA use a triplet of bases to encode each amino acid, and therefore with various combinations of three nucleotides, there is more than sufficient complexity to carry genetic information.
Unfortunately, Levene at the time subscribed to the idea that nucleotides were too simple to carry hereditary information from one generation to the next, and his tetranucleotide hypothesis stated that there were only four nucleotides per nucleic acid molecule – or alternatively that a longer nucleic acid contained repeating identical motifs of the same four nucleotides – as stated in his 1931 monograph, 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. [p262]…. It must be borne in mind that the true molecular weight of nucleic acids is as yet not known. The tetranucleotide theory is the minimum molecular weight and the nucleic acid may as well be a multiple of it. [p289]
Although Levene wisely kept open the idea that nucleic acids may be much larger than four nucleotides long (which was demonstrated shortly after his quote above), that was overridden by his continued emphasis on the lack of complexity in a molecule composed of only four unique components, represented by quotes such as in this 1917 paper:
…nucleic acids…which occur in all tissues, all organs of all species, constant, invariable in their structure, present wherever life is present. They are indispensable for life, but carry no individuality, no specificity, and it may be just to accept the conclusion of the biologist that they do not determine species specificity, nor are they carriers of the Mendelian characters.
Until his death in September, 1940, Levene and the rest of the scientific field held to this erroneous notion that nucleic acids are too simple to be the carriers of genetic information.
Today, most of us know nothing of Phoebus Levene, as if he had been written out of the history and biology books. The scientific community seemed to react in a kind of fury at having been misled by the tetranucleotide theory, and unfairly wiped away all the other critically important contributions that Levene had made to our understanding of nucleic acids, and to the field of biochemistry.
Just as bad were Darwin’s inability to propose a mechanism for his theory of evolution, Mendel’s lack of a physical explanation for his mathematically derived laws of heredity, Einstein’s inability to embrace the statistical nature of quantum mechanics and fear of “God playing dice”, Isaac Newton’s obsession with alchemy and his stubborn adherence to his own confusing notation for calculus over Liebnitz’s notation which we use today… all these and many more show fundamental errors or failings at least as egregious as Levene’s. Yet Darwin, Mendel, Einstein, Newton, and many others are household names today.
It is time to give the biochemist his due. A toast to Phoebus Aaron Theodor Levene!
I hope you enjoyed the latest installment in this series on the discovery of DNA. I'm pretty psyched that so many historical papers are available online and have made the effort to hyperlink quite a few of them as references for key points. Feel free to click on the blue underlined texts and let me know if any of the links are broken.
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