Severo Ochoa discovered a method for synthesizing the biological molecule RNA, establishing that this process, key to the transcription of genetic material, can occur outside the living cell and thereby admit of manipulation by experimental scientists and genetic engineers.

Transmission of parental characteristics to offspring in humans and other organisms has been assumed over the ages. Nevertheless, not until the mid-eighteenth century was a comprehensive theory put forth to outline the general biological parameters of heredity. These ideas, established by the insightful Austrian monk Gregor Johann Mendel Mendel, Gregor Johann , are now known as classical, or Mendelian, genetics Genetics . By the early twentieth century, Mendel’s ideas had been linked to cell theory (the idea that cells compose the fundamental units of life) by observations that cellular entities known as chromosomes carry Mendel’s hereditary units. The hereditary units had been named genes, and the work of Thomas Hunt Morgan Morgan, Thomas Hunt and his colleagues at Columbia University rooted the new science of genetics firmly within the context of hereditary units being located as well-organized arrays of genes on chromosomes. Ribonucleic acid
Biochemistry;genetic material
[kw]Ochoa Creates Synthetic RNA (1955)
[kw]Synthetic RNA, Ochoa Creates (1955)
[kw]RNA, Ochoa Creates Synthetic (1955)
Ribonucleic acid
Biochemistry;genetic material
[g]North America;1955: Ochoa Creates Synthetic RNA[04700]
[g]United States;1955: Ochoa Creates Synthetic RNA[04700]
[c]Genetics;1955: Ochoa Creates Synthetic RNA[04700]
[c]Science and technology;1955: Ochoa Creates Synthetic RNA[04700]
Ochoa, Severo
Grunberg-Manago, Marianne
Nirenberg, Marshall W.
Lengyel, Peter

Severo Ochoa.

(The Nobel Foundation)

In the early decades of the twentieth century, genetics had not been experimentally united with biochemistry. This merging soon occurred, however, with work in the mold Neurospora crassa. Neurospora crassa This Nobel award-winning work by Edward Lawrie Tatum Tatum, Edward Lawrie and George Wells Beadle Beadle, George Wells showed that genes control production of proteins, which are major functional molecules in cells. Yet, no one knew the chemical composition of genes and chromosomes, or, rather, the molecules of heredity. Oswald T. Avery Avery, Oswald T. and his colleagues at New York’s Rockefeller Institute determined experimentally that the molecular basis of heredity, or the transforming principle, as they called it, was a large polymer known as deoxyribonucleic acid Deoxyribonucleic acid (DNA). DNA had been recognized as a cellular component for some seventy years; but until 1944, its functional importance, if any, was unrecognized.

Avery’s discovery triggered a furious worldwide search for the particular structural characteristics of DNA that allow for the known biological characteristics of genes. One of the most famous studies in the history of science solved this problem in 1953. James D. Watson, Francis Crick Crick, Francis , and Maurice Wilkins postulated that DNA exists as a double helix. That is, two long strands twist about each other in a predictable pattern, with each single strand held to the other by weak, reversible linkages known as hydrogen bonds. About this time, researchers recognized also that a closely related molecule to DNA, ribonucleic acid (RNA), plays an important role in transcribing the genetic information as well as in other biological functions.

Severo Ochoa was born in Spain as the science of genetics was developing. He received his medical degree from the University of Madrid in 1928, idolizing the great Spanish histologist Santiago Ramón y Cajal and finding himself deeply immersed in experimental biology by the end of his medical studies. The 1930’s found him studying and researching in Madrid, Germany, and England, until a combination of professional and political factors caused Ochoa to move to St. Louis and the dynamic biochemistry studies occurring at Washington University’s School of Medicine. He landed in an environment focused upon the central biochemical issues of that time—that is, those surrounding questions of how cells process energy from organic molecules like the sugar glucose to provide usable biological energy in the form of adenosine triphosphate Adenosine triphosphate (ATP). Ochoa made many significant discoveries in biochemical energetics, especially in studies with vitamins, with some of the reactions of what is now often called the Krebs cycle, and in oxidative phosphorylation, the final common pathway that produces most of ATP.

In 1942, Ochoa moved to New York University, where he continued his interests in oxidative phosphorylation by studying the bacterium Azobacter vinelandii, which was known to be extremely active metabolically. With postdoctoral fellow Marianne Grunberg-Manago, he studied enzymatic reactions capable of incorporating inorganic phosphate (a compound consisting of one atom of phosphorus and four atoms of oxygen) into adenosine diphosphate Adenosine diphosphate (ADP) to form ATP. One particularly interesting reaction was followed by monitoring the amount of radioactive phosphate reacting with ADP.

Following separation of the reaction products, Ochoa and Grunberg-Manago discovered that the main product was not ATP, but a much larger molecule. Chemical characterization demonstrated that this product was a polymer of adenosine monophosphate (AMP). When other nucleocide diphosphates, such as inosine diphosphate (IDP), were used in the reaction, the corresponding polymer of inosine monophosphate (IMP) was formed. Thus, in each case, a polymer—a long string of building-block units—was formed. The polymers formed were synthetic RNAs, and the enzyme responsible for the conversion became known as polynucleotide phosphorylase. This finding was made in 1955. Once early skepticism was resolved, biochemists received the news with great enthusiasm, because no technique outside the cell had yet been discovered in which a nucleic acid similar to RNA could be synthesized.

Ochoa, Peter Lengyel, and Marshall W. Nirenberg at the National Institute of Health took advantage of this breakthrough to synthesize different RNAs useful in cracking the genetic code. Crick had postulated that the flow of information in biological systems is from DNA to RNA to protein. In other words, genetic information contained in DNA structure is transcribed into complementary RNA structures, which, in turn, are translated into protein structure by specifying the particular amino acids to be incorporated into the protein.

Protein synthesis, an extremely complex process, involves bringing a type of RNA, known as message, together with amino acids and huge cellular organelles, known as ribosomes. Investigators did not know the nature of the nucleic acid alphabet—for example, how many single units of the RNA polymer code were needed for each amino acid, and the order that the units must be in to stand for a word in the nucleic acid language. In 1961, Nirenberg demonstrated that the polymer of synthetic RNA with multiple units of uracil (poly U) coded for a protein containing the amino acid phenylalanine only. Each three units (U’s) gave one phenylalanine. Therefore, genetic words each contain three letters. UUU translates into phenylalanine. Poly A, the first polymer discovered with polynucleotide phosphorylase, was coded for a protein containing multiple lysines. That is, AAA translates into the amino acid lysine.

The words containing combinations of letters such as AUG were not as easily studied, but Nirenberg, Ochoa, and Gobind Khorana Khorana, Gobind of the University of Wisconsin uncovered eventually the exact translation for each amino acid. In RNA, there are four possible letters (A, U, G, and C) and three letters to each word. Accordingly, there are sixty-four possible words. With only twenty amino acids, it became clear that more than one RNA word can translate into a given amino acid. However, no given word stands for any more than one amino acid. For example, UUU specifies phenylalanine only. UUC also means phenylalanine but no other amino acid. A few RNA words do not translate into any amino acid; they are stop signals, telling the ribosome to cease translating RNA.

The questions of which direction an RNA is translated are critical. For example, CAA codes for the amino acid glutamine, but the reverse, AAC, translates to the amino acid asparagine. Such a difference is critical because the exact sequence of a protein determines its activity. To a large extent, this problem had to be solved simultaneously. Ochoa and his colleagues made great strides using polynucleotide phosphorylase to build synthetic RNAs with predictable directionality. By 1964, Khorana had developed more direct methods to analyze these questions, and the field began to move in new directions.

In a 1980 autobiographical sketch in the Annual Review of Biochemistry, Severo Ochoa stated: “I tell this story to justify the title of this essay, because in my life biochemistry has been my only and real hobby.” His research pursuits covered vitamins, the central reactions of intermediary metabolism, RNA and the genetic code, and protein synthesis.

Ochoa’s discovery of polynucleotide phosphorylase, leading to the laboratory synthesis of RNA, was a serendipitous side effect of studies planned in the central area of oxidative phosphorylation. Even though Ochoa’s experimental productivity in a quantitative sense may have been greater in the metabolic areas, however, the production of synthetic RNA will likely have the longest-lasting consequences. This conclusion may seem odd in the light of follow-up studies that have shown polynucleotide phosphorylase to be a minor player in general RNA synthesis. For the most part, it is found in bacteria only, and even there, its function remains controversial: Because polynucleotide phosphorylase catalyzes relatively reversible reactions, it is possible that RNA degradation, the opposite of synthesis, is its real biological niche.

In 1960, the enzyme responsible for most of the RNA synthesis in cells, RNA polymerase, was discovered by the University of Chicago’s Samuel Weiss and Jerard Hurwitz at New York University. In a related area, the enzyme catalyzing DNA synthesis, DNA polymerase, was discovered by Arthur Kornberg Kornberg, Arthur of Stanford University, who worked with Ochoa in the late 1940’s. Ochoa and Kornberg shared the Nobel Prize in Physiology or Medicine Nobel Prize in Physiology or Medicine;Arthur Kornberg[Kornberg]
Nobel Prize in Physiology or Medicine;Severo Ochoa[Ochoa] in 1959.

The germinal nature of Ochoa’s elicitation of RNA synthesis from this obscure enzyme lies in methodology. Synthetic RNAs provided the key to understanding the genetic code. The genetic code is universal; it operates in all organisms, simple or complex. It is used by viruses, which are near life, yet still not alive. Spelling out the genetic code was one of the top discoveries of the twentieth century. Nearly all work in molecular biology depends on this knowledge. Further, availability of synthetic RNAs provided hybridization tools for molecular geneticists.

Hybridization Hybridization, genetic
Genetics;hybridization is a technique in which RNA is allowed to bind in a complementary fashion to DNA under investigation. The greater the similarity between the RNA and DNA, the greater the amount of binding. The differential binding allows for seeking, finding, and ultimately isolating a target DNA from a large diverse pool of DNA, in short, finding a needle in a haystack. Hybridization approaches are indispensable aids in experimental molecular genetics as well as in applied sciences, such as forensics.

Historians and philosophers of science debate the relative merits of methodological breakthrough versus conceptual revolution, of convergent thinking as opposed to the divergent, and luck compared to the rationally planned. Ochoa’s work with synthetic RNA was not planned but led eventually to rigorous step-by-step experimental unraveling of the genetic code. It diverged from the expected but provided the tool to converge upon a universal principle. It made available a technique that ultimately revolutionized biochemists’ ideas about life and its evolution. Ochoa’s life and work embodied a creative synergy of these qualities, resulting in world-class science. Ribonucleic acid
Biochemistry;genetic material

• Grunberg-Manago, Marianne, and Severo Ochoa. “Enzymatic Synthesis and Breakdown of Polynucleotides; Polynucleotide Phosphorylase.” Journal of the American Chemical Society 77 (June 5, 1955): 3165-3166. This article in one of the world’s leading chemical journals is a short, coherent account of the key investigation with synthetic RNA. Presented in a concise and straightforward manner.
• Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. 4th ed. New York: W. H. Freeman, 2005. Useful for those readers who wish some knowledge of the chemistry involved in Ochoa’s RNA discovery. It is very readable and provides historical perspective as well as other context for this area of biochemistry.
• Ochoa, Severo. “The Pursuit of a Hobby.” Annual Review of Biochemistry 49 (1980): 1-30. This highly personal reflection provides insight into the man, his formative years in Spain and his life in New York, and his work from developmental experiences with other biochemists to his independent forays into metabolism and molecular biology.
• 
  Recombinant DNA: Readings from “Scientific American.” Introductions by David Freifelder. San Francisco: W. H. Freeman, 1978. This collection of articles from Scientific American provides one of the best opportunities for popular reading that borders on the semitechnical. A reader seeking an introduction to molecular biology will be treated to the best possible writing, fine illustrations, and historical perspective from cell biology through gene engineering.
• Watson, J. D., et al. Molecular Biology of the Gene. 5th ed. San Francisco: Pearson/Benjamin Cummings, 2004. This highly successful multiedition book by nucleic acid pioneer James Watson is for the technical reader. Yet, it goes to great lengths to bring novices to equal footing with brilliant descriptions of fundamental biochemical tenets, in conjunction with historical perspective.

Avery, MacLeod, and McCarty Determine That DNA Carries Hereditary Information

Bevis Describes Amniocentesis as a Method to Check Fetal Genetic Traits

Watson and Crick Announce the Double-Helix Model for DNA

Horsfall Detects the Link Between Cancer and Altered DNA

Nirenberg Cracks the Genetic Code

Kornberg and Colleagues Synthesize Biologically Active DNA