An Interview with Manuel F. Varela and Ann F. Varela—Discussing Marshall Nirenberg—And the Genetic Code!

Dec 6, 2020 by

Marshall Nirenberg

He had an idea every two or three minutes…

–Dr. Phillip Leder

One individual alone creates only a note or so that blends with those produced by others.”

–Marshall Warren Nirenberg

The U2 incident started the cold war; the U3 incident started the code war.”

–Rollin Hotchkiss

Michael F. Shaughnessy

1) Marshall Nirenberg—born in New York city—but contracted rheumatic fever—so his family moved to Florida. Just for the record—when was he born, and what do we know about his early years in Florida?

Marshall Warren Nirenberg was born into the homestead of Harry Edward, a shirt maker, and Minerva (Bykowky) Nirenberg in New York City on April 10, 1927. When Nirenberg was around 12 years old, the family moved to Orlando, Florida, expecting that the subtropical climate would benefit their son, who had recovered from rheumatic fever.

Nirenberg was intrigued with biology as a youth; he was fascinated with bird watching and exploring Florida’s marshlands’ varied ecology. Another advantage of his living environment was the instruction provided by local experts such as biochemists and museum curators at neighboring World War II training sites.

Nirenberg was a 1945 graduate of Orlando High. He developed an interest in biochemistry during his college experience. Nirenberg attended the University of Florida at Gainesville and earned his B.S. in zoology and chemistry in 1948. Then, Nirenberg took his M.S. in zoology, studying the ecology and taxonomy of caddisflies in 1952.

At the University of Michigan, Ann Arbor, Nirenberg took a Ph.D. degree in Biochemistry in 1957. After attainment of the doctorate, Nirenberg conducted postdoctoral work at the NIH.

2) His work at Gainesville, Florida, was unparalleled—what do we know about Nirenberg’s accomplishments at the University of Gainesville?

Nirenberg earned a stellar performance on an entrance exam as a high school senior. The honor permitted early admission to the University of Florida during his second semester of high school in 1945. Interestingly, Nirenberg’s elder sister Joan attended university out-of-state because the University of Florida did not accept women until 1947 or African American applicants till 1958.

Nirenberg seemed to impress neither his high school nor university professors as being gifted. He made average grades in college courses. He studied subjects like biology, chemistry, sociology, and infantry. While a student, Nirenberg taught as a teaching assistant for biology and comparative anatomy courses. Although his GPA was a mediocre 2.32, he took his undergraduate degree in just three years.

As a graduate student in the zoology program at the University of Florida, Nirenberg flourished. To help pay for graduate school, Nirenberg became a biochemistry research associate working with radioactive substances in a laboratory committed to the study of nutrition. Dr. George K. Davis ran the nutrition laboratory. The lab facility was occupied by another assistant named Ray Shirley. Nirenberg participated in a project dealing with tracing radioactive compounds through bird tissues as eggs were produced. The work was published in a biochemical journal, and it was Nirenberg’s first foray into the scientific literature. Nirenberg’s work experience in the nutrition biochemistry lab would become an influential history-changing event.

For his M.S. graduate thesis project, Nirenberg chose to study under Professor Lewis Berner. The latter was a professor in the biology department. In Berner’s laboratory, Nirenberg studied the taxonomical classification of caddisflies, which are moth-like insects. Nirenberg collected tens of thousands of caddisfly specimens and examined hundreds collected by others. He found that while the caddisflies were not good food sources for fish, the flies were nevertheless good water quality indicators. The flies survived poorly in polluted water. Nirenberg took only one and a half years to acquire an M.S. in zoology with the highest academic honors. Most remarkable about Nirenberg’s stint at the University of Florida at Gainesville was his experience learning biochemistry, even though his thesis was primarily ecological in scope.

3) Nirenberg’s doctoral work was in Biochemistry from the University of Michigan in Ann Arbor in 1957. He studied “hexose uptake in tumor cells” supervised by James F. Hogg. So why is this relevant, and how does it relate to his later work?

Nirenberg attended the University of Michigan, Ann Arbor, and earned his Ph.D. in Biochemistry in 1957. His thesis studied a permease for hexose transport in ascites tumor cells, with his advisor James Felter Hogg. One could surmise that this sugar transport work mirrored his curiosity in the chemistry of life.

Nirenberg had arrived on the campus of the University of Michigan at the beginning of 1952. He took courses in medical physiology, physiological chemistry, organic chemistry, and physical chemistry. After finishing his graduate coursework and passing his preliminary and oral examinations, Nirenberg entered Hogg’s research laboratory for his thesis project. Initially, the biochemistry department chair Howard Bishop Lewis had assumed that Nirenberg would conduct his thesis work in Lewis’s lab. A disappointed Nirenberg was too polite to refuse Lewis and agreed to work under Lewis. However, soon after Nirenberg met with the chair, Lewis suffered a stroke and never fully recovered, being replaced by a new chair. Considering the unique circumstances, Nirenberg was free to work under Hogg.

Nirenberg’s first Ph.D. thesis project was a failure. His second attempt was an astonishing success. Nirenberg chose to use tumor cells to study how one sugar prevented the cancer cells from using a different sugar.

The word hexose is a technical term for a type of sugar. The prefix “hex-” means “six,” meaning that the molecule has six carbons atoms, and the suffix “-ose” stands for sugar. Thus, hexoses are six-carbon sugars. The sugars were transported into a special kind of cancer cell called Ehrlich ascites tumor cells, and Nirenberg studied them. These cancer cells took up hexose sugars by a transport system so they could grow readily. Nirenberg and Hogg found that the addition of a sugar inhibitor called 2-deoxy-D-glucose prevented the transport of glucose or fructose into the tumor cells and thus prevented glycolysis. They found that the sugar inhibitor was converted to an intermediate called 2-deoxy-D-glucose-6-phosphate by an enzyme called hexokinase. The intermediary was then responsible for glycolysis inhibition. The sugar was affecting two glycolytic enzymes. One was called phosphohexose isomerase, and the other was phosphofructokinase. The inhibition of phosphohexose isomerase prevents the conversion of glucose-6-phosphate to fructose-6-phosphate.

Similarly, inhibiting phosphofructokinase prevents the phosphorylation of fructose-6-phosphate and from producing fructose-1,6-diphosphate. Preventing the activities of either of these enzymes in the tumor cells prevented glycolysis. In any case, the tumor cells would have difficulty growing in the presence of the glycolytic inhibitor if they could not transport and metabolize glucose or fructose. The new study was published in June of 1958.

4) Postdoctoral work—was at the National Institutes of Health in 1957. At that time, Nirenberg began to study the various steps that relate to RNA, DNA, and proteins. Why was this work meaningful?

Nirenberg’s findings started the effort to crack the secret code of DNA. He immediately began working at the National Institutes of Health (NIH) in Bethesda, Maryland, upon completing his Ph.D. requirements. Shortly after that, his research was focused on DNA, RNA, and protein.

In 1958, Nirenberg researched how DNA directed the expression of proteins. He was curious about the role RNA had in these processes. Along with Heinrich J. Matthaei, see Figure 65, at NIH, they produced RNA composed exclusively of uracil. The breakthrough came when Matthaei added “poly-U”—RNA comprised solely of uracil bases—to the mix. After short-term incubation at average body-heat temperature, radioactive measurements suggested the synthesis of long protein-like molecules. These molecules were made entirely of the amino acid phenylalanine. Nirenberg and Matthaei determined that the RNA sequence “UUU” regulates phenylalanine’s addition to any growing protein chain. In some circles, the UUU sequence was known as the “U3 codon,” a reference to the 1960U2 spy plane incident, which sent the world into the Cold War.

File:J. Heinreich Matthaei and Marshall Nirenberg (29819142523).jpg

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Figure 65. Marshall Nirenberg and Heinrich Matthaei worked together to decipher the genetic code. The research earned Nirenberg the Nobel Prize in Physiology or Medicine in 1968.

Nirenberg’s NIH work was deemed a highly significant finding because it addressed the DNA’s genetic code. The genetic code warehoused in DNA is transcribed into a codon message as RNA to be translated to dictate the amino sequence along the protein chain. The UUU triplet sequence of the codon message specified the amino acid phenylalanine. Hence, the RNA message’s poly-U chain faithfully produced a string of phenylalanine molecules in a row.

The ramifications of the discovery would be profound. First, Nirenberg’s discovery led to the code-cracking of other repeat triplet codons like AAA, GGG, and CCC on mRNA molecules. The AAA codon on mRNA specified the amino acid lysine. The GGG and CCC codons likewise coded for the amino acids called glycine and proline, respectively. Later, other alternating sequence variations of RNA molecules like poly-UC, poly-CG, and poly-AG, led to discovering the codons for different amino acids. After all of the sequence iterations were thoroughly examined, all possible triplet codons had been made known. Some 64 codons were ultimately discovered to encode 20 amino acids and three so-called “stop” codons, UAA, UAG, and UGA, coded for ends of proteins. The ultimate solving of the genetic code was one of the significant scientific discoveries of the 20th century.

The knowledge of the genetic code could be put to use in significant ways. One example led to DNA sequencing technology development, finding the order of nucleotide bases along a DNA chain. If one could read the DNA sequence, then the RNA and the protein sequences could be made known, as well. The genetic code could also be used to learn about the nature of protein structures in mutated variants. Thus, the function of specific evolutionarily conserved amino acid sequences could be analyzed readily by reading the genetic code and directly translating it into their final protein sequences. Another significant advance dealt with the ability to direct the formation of protein structures and use them to correlate with their functions.

What is more, the code would be universal. All living beings, from bacteria to humans, would use the same codon language of DNA and RNA to produce identical amino acid sequences. It was a breathtaking discovery.

In its essence, the genetic code opened up the floodgates of molecular biology. In modern times, the genetic code would be exploited to determine the human genome map. With the knowledge of the secret code, molecular biologists discovered new disease diagnosis methods. They provided a seemingly promising avenue for genome editing.

The working knowledge held in the genetic code could be harnessed with modern computers’ power to open up vistas with bioinformatics. The genetic code could be used directly by using computers to study the function of individual biomolecules. The genetic code held in DNA can be compared between specific sequences, genes, proteins, individual organisms, or between groups of organisms. The evolutionary tree of life would be constructed to learn where various species of living beings fall into the scheme of things on Earth and where the multiple billions of species are headed.

5) Fast forward to August 1961, and the International Congress of Biochemistry in Moscow. What happened?

During the Congress in Russia, Nirenberg presented data that stunned the scientific world. He told the investigators present that UUU on mRNA encoded phenylalanine! Shockingly, Nirenberg stated that when cellular machinery was fed RNA called poly-U, a single protein was produced that consisted of one type of amino acid! The poly-U RNA was a long string of uracils, i.e., UUUUUUUUUUUUUUUU. Nirenberg announced the artificial formation of a protein with a long line of phenylalanine residues, i.e., poly-Phe! See Figure 66.

File:Nirenberg's experiment.png

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Figure 66. A scheme for Marshall W. Nirenberg’s experiment that elucidated the genetic code’s first “letter.” He used poly-uracil (shown in the top row) as a template for in vitro protein synthesis; he obtained a polypeptide composed only of phenylalanine (bottom row).

Nirenberg’s data, presented at the Congress, was the first time in scientific history that the long-awaited deciphering of the genetic code had been cracked open! It was a stunning revelation! The world’s leading molecular biologists realized that the genetic code would soon be solved.

When Nirenberg presented his striking data at the Moscow Congress, it was held in a small classroom with the slides suffering from the poor-quality projection technology. At first, Nirenberg was disappointed that no one in the room would understand the astonishing disclosure. He had divulged that the genetic code’s cracking would soon be at hand, starting with UUU and phenylalanine. Then, a young scientist named Matthew Meselson (see Chapter 14) ran up to Nirenberg afterward. Meselson expressed his great relief that a clue to the secret of life had just been revealed. Meselson informed Nirenberg that they had to find the famous Nobel Laureate Francis Crick quickly and tell him.

Francis Crick realized the DNA code bomb that had just been dropped in Moscow! He quickly organized another time the next day so that Nirenberg could give his talk again! This time, the attendees of the makeshift plenary session would number well into the hundreds. One of these Congress attendees of Nirenberg’s second speech was none other than the astute Severo Ochoa, who would later share the Nobel with Nirenberg.

6) We have to mention a time between approximately 1961 and 1962, and this zeitgeist is known as the “coding race.” What exactly was the coding race? I understand some outstanding scientists (Severo Ochoa) were also involved in this truly collaborative effort!

The coding race can best be described as a push by several laboratories to be the first to crack the genetic code and completely solve it. Elucidation of the secret DNA code of life was a Nobel Prize-worthy endeavor. Many prominent molecular biologists of the day were on about it. For instance, the genetic code puzzle interested the likes of Nobel Laureate Francis Crick and his collaborators, Har Gobind Khorana and his laboratory, and Severo Ochoa in New York. The life and science of Professor Ochoa are featured in Chapter 19 of our 2020 book titled “Biochemistry and Biochemists: Who Were They and What Did They Discover?

Nirenberg decoded other triplets in the genetic code, a total of thirty-five by 1963, and over sixty by 1966. At the University of Wisconsin, Khorana modified Nirenberg’s experimental system and verified and extended his work. Khorana had used synthetic ribonucleotides to make various base sequence combinations. Using a cell-free translation system, they deduced the codon specificity for other amino acids and start and stop instructions to finish up the missing pieces of the genetic code. Ultimately, the Nobel Prize in medicine or physiology would go to Nirenberg and Khorana in 1968 to solve the elusive puzzle of the genetic code. Professor Khorana is featured in Chapter 34 of our book “The Inventions and Discoveries of the World’s Most Famous Scientists.

In the interval between 1961 and 1962, however, the code was unsolved. During this time, Severo Ochoa was already a Nobel Laureate who received the 1959 accolade. Ochoa had discovered the polynucleotide phosphorylase enzyme, which permitted one to synthesize RNA artificially. Ochoa was in the audience at the 1961 Moscow Congress speech from Nirenberg. Ochoa learned that Nirenberg had discovered the UUU codon and poly-U artificially made phenylalanine and poly-Phe, respectively. Ochoa was reported to have run out of Nirenberg’s plenary session and telegraph his New York University laboratory to start doing the logical next set of experiments!

During the Cold War’s zenith in the early 1960s, the new scientific competition had been transformed into a raging “Code War.” The ramifications would be turbulent and its effects long-lasting.

A graduate student in the Ochoa laboratory named Peter Lengyel did the requisite experiment, as instructed by Ochoa during his long-distance phone call from Moscow to New York. Lengyel first repeated the Nirenberg experiment and confirmed the findings. He prepared poly-A and similarly observed the production of poly-Phe like Nirenberg reported in Moscow. Back home in his New York laboratory, Ochoa, Lengyel, and Joseph Speyer had thought about using random mixtures of so-called copolynucleotides to solve the code. The random copolynucleotide method consisted of mixing two or more different nucleotides to produce new sequences, which could be examined for the types of amino acids they specified. They prepared poly-(U, C) and poly-(U, A) chains of RNA. Lengyel and Ochoa quickly elucidated additional codons for new amino acids. The copolynucleotide mixing process produced novel codon sequences, like UUC, UCU, CUU, etc., plus UUA, UAU, AUU, etc. They found that their cell-free systems made proteins with various new amino acids. Ochoa’s groups found codons that specified phenylalanine, leucine, serine, tyrosine, and isoleucine. See the genetic code diagram in Figure 67. Ochoa Lengyel and Speyer published their findings in October of 1961 in the Proceedings of the National Academy of Sciences.

File:Codons aminoacids table.png

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Figure 67. The Genetic Code with Amino Acids.

What happened next is a feature of controversy between the Nirenberg and Ochoa camps. According to Nirenberg, Lengyel had interrupted Nirenberg’s seminar at M.I.T. to announce new findings from the Ochoa laboratory. Thus, in Nirenberg’s mind, the Ochoa group had invaded their genetic code territory. However, Ochoa’s group had already been in the genetic code field in Lengyel’s mind before knowing about Nirenberg’s UUU codon data.

The code war had specific positive and negative ramifications for Nirenberg. For example, Nirenburg despaired about the perceived intrusion by Ochoa’s laboratory in the coding race. He needn’t have worried, as Nirenburg and none of Ochoa’s group took the Nobel Prize. Yet, because Nirenburg had to concentrate his efforts in garnering the rest of the genetic code’s codons, he could not pursue his studies on messenger RNA’s potential discovery. The RNA work would have to be placed on the back burner to have others involved in its definitive demonstration by others.

7) His later work focused on neuroscience, neural development, and the homeobox genes—First of all, what is meant by these homeobox genes? What research did he do on neural development, and how did Nirenberg define neuroscience? I realize his definition of neuroscience may be different than what we adhere to currently.

In 1962, Nirenberg headed the Section of Biochemical Genetics at the National Institutes of Health. In the late 1960s, Nirenberg began to study neurobiology. This discipline deals with the nervous system, such as the brain, spinal cord, and peripheral nerves. Nirenberg explored the neurotransmitters called norepinephrine (noradrenaline) and dopamine. These agents were involved in a cancerous brain tissue called neuroblastoma. Working with Dr. Phillip Nelson, Nirenberg learned to grow the brain cancer neurons in the laboratory as artificial cultures. His neuroblastoma study allowed him to create an investigative model. The model system served as the basis for a broad range of neurobiological research. Nirenberg routinely provided cell culture samples of neuronal cancer tissue to other investigators, who in turn made significant strides in many aspects of neurobiology.

Nirenberg’s research program involved several lines of investigation. One avenue involved the formation of synapses between developing neurons. Another push from Nirenberg’s laboratory involved individual mapping of neurons in a developing retina. He endeavored to discover the final locations of neurons in a fully developed brain. Nirenberg had called this goal to determine the “molecular address” of individual neurons of the retina. In modern times, this address is referred to as a neuronal topographic map.

Nirenberg invoked a molecular biological approach to neuroscience. He cloned the gene encoding specific antibodies to recognize and bind various parts of the retina. However, when several attempts to clone particular genes encoding proteins that were resident to retinal cells, he turned to polymerase chain reaction (PCR) technology. The PCR method allowed him to isolate distinct genetic elements. Soon Nirenberg acquired a massive DNA library of these genetic elements.

During this time, in the early-1980s, Nirenberg learned about an exciting discovery about cancer. The formation of cancer involved a gene family called protooncogenes, which stopped tumor suppression when turned off or mutated, leading to tumor formation. Oncogenes could transform a healthy cell into a tumorogenic one. Nirenberg and others soon realized that in brain cancer, oncogenes might be involved in starting tumor growth. Along these lines, Nirenberg reasoned that the homeobox genes, discovered in 1983 by Dr. Walter Gehring, who was at the University of Basel, could serve as gene regulators to turn on or off certain genetic elements. Nirenberg had felt that the homeobox genes could bridge the relationship between molecular biology and neuronal development.

Homeobox genes refer to specifically conserved DNA sequences that encode the so-called homeodomains. These homeobox genes are members of the so-called homeotic genes, which direct the body’s specific structures. Many of these homeotic genes are called hox genes, which is an abbreviated form of homeobox. These homeobox genes, hox, encode proteins that contain the homeodomains, which are 60-amino acids long and conserved in many gene regulatory proteins in virtually all living organisms. The protein structure of the homeodomain serves as a DNA-binding motif. The motif has a molecular design called a “helix-turn-helix,” which binds to DNA domains called homeoboxes. See Figure 68. The bottom of the TetR protein harbors two helix-turn-helix motifs capable of securing the homeodomain boxes of DNA.

File:TetR-overview (2).png

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Figure 68. TetR homodimer. Each monomer is found on the left and right. The helix-turn-helix motifs of each monomer are shown on the bottom.

Nirenberg studied homeobox genes from fruit flies of the Drosophila melanogaster species. Working with a postdoctoral fellow, Dr. Yongsok Kim, four new homeobox genes, NK-1, NK-2, NK-3, and NK-4, were discovered in the chromosomes of the fruit fly.

Interestingly, these homeobox genes were found in the human genome! These homeobox genes are thought to play functional roles in the embryonic development of the brain. Furthermore, in the 289 human genes that harbor the homeobox genes, many are now implicated in specific human genetic diseases of the brain, cancer, metabolism, and the immune system.

8) Nirenberg received three major awards—The Nobel Prize in Physiology and Medicine in 1968, the Franklin Medal the same year, and the Louisa Gross Horwitz Prize in 1968—a three-peat indeed! For what was each award given?

The three accolades to Nirenberg that you mentioned in your question all pertain to his role in solving the genetic code. The work had tremendous implications for the mechanism of protein synthesis. See Figure 69. Indeed, Nirenberg was soundly honored throughout his scientific career.

File:Marshall Nirenberg performing experiment.jpg

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Figure 69. Photograph of experiment likely related to protein synthesis and genetic code translation.

Nirenberg holds honorary degrees from several Universities. Additionally, Nirenberg was granted the Molecular Biology Award and had become a member of the National Academy of Sciences in 1962. Nirenberg was given the Paul Lewis Award in Enzyme Chemistry by the American Chemical Society in 1964. Afterward, he was promoted to director of biochemical genetics and kept that title until his retirement.

Nirenberg earned the National Medal of Science, in 1965, the Research Corporation Award, in 1966, the Hildebrand Award, in 1966, the Gairdner Foundation Award of Merit, in 1967, the Prix Charles Leopold Meyer from the French Academy of Sciences, in 1967, and the Albert Lasker Award, in 1968.

Nirenberg was a co-recipient of the Nobel Prize in Physiology or Medicine in 1968 with Robert William Holley and Har Gobind Khorana. In 1968, Nirenberg was awarded the Joseph Priestly Award, the Franklin Medal. Nirenberg shared the Louisa Gross Horwitz Prize and the Lasker Award with H. G. Khorana. Nirenberg was a member of the American Academy of Arts and Sciences and the National Academy of Sciences.

9) Is Nirenberg still alive? Or has he gone to his rest after a long productive career?

File:Marshall Nirenberg 2003.jpg

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Figure 70. Marshall Warren Nirenberg.

Sadly, Nirenberg died of cancer at 82 years on January 15, 2010, in New York City. See Figure 70. Nirenberg had married a chemist, Perola Zaltzman, from the University of Brazil, Rio de Janeiro, in 1961. Four years after her death, Nirenberg married Myrna Weissman in 2005.

Interestingly, Pope Paul VI appointed Nirenberg to the Pontifical Academy of Sciences. And in 2000, Nirenberg was honored on a stamp issued in 2000 by the Palau Islands.

For additional biographical details of this remarkable solver of the genetic code, visit the book link:

https://www.google.com/books/edition/The_Least_Likely_Man/Ro-pBgAAQBAJ?hl=en&gbpv=1
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