An Interview with Manuel F. Varela and Ann F. Varela: Susumu Tonegawa—Somatic Cells, DNA Recombination, and Antibody Diversity

Feb 9, 2021 by

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Susumu Tonegawa

My parents were firm believers that education is the best asset that parents can give to their children.”

—Susumu Tonegawa

It doesn’t matter whether it is chemistry or immunology or neuroscience: I just research what I find interesting.”

—Susumu Tonegawa

Michael F. Shaughnessy

1) Susumu Tonegawa—exciting name—are there any interesting details about where he was born, when, and early events?

Susumu Tonegawa is best known for discovering that the diversity of antibodies was due to somatic gene rearrangement. In 1986, Tonegawa became the first Japanese scientist in world history to win the medicine or physiology Nobel Prize. He was born in Nagoya, Japan, on September 5, 1939. Tonegawa has an older brother and a younger brother and sister. Tonegawa’s mother, Miyoko, was a homemaker. His father, Tsutoma, worked in Japan for a textile company as an engineer, which meant he was obligated to move every so often to a new factory location in various rural towns in the southern part of Japan. Tonegawa and his siblings enjoyed the country living, but once he and his older brother were young adolescents, they were sent to Tokyo for more formal education. Tonegawa and his brother were now the wards of his Uncle. They attended Hibiya High School, where his interest in chemistry was developed.

Tonegawa took an entrance exam for the Department of Chemistry of the University of Kyoto, Japan; having failed his first attempt, he did not abandon his dream. He was accepted into the University of Kyoko in 1959.

2) His initial work was at the University of Kyoto—in chemistry—am I correct? And then Tonegawa tended to drift from there…

Tonegawa earned a B.S. degree in Chemistry from Kyoto University in 1963. While attending Kyoto University, Tonegawa became interested in operon theory. He recalls how reading papers by François Jacob and Jacques Monod sparked his interest in molecular biology.

As you can recollect, in chapter 26 of our book published in 2018 on “The Inventions and Discoveries of the World’s Most Famous Scientists,” that Jacob and Monod took the Nobel in 1965 for their discovery of the lac operon. Research opportunities in the molecular biology field for undergraduate students at the university were somewhat limited during the 1960s.

Tonegawa’s graduate studies focused on molecular biology under Itaru Watanabe at the Institute for Virus Research at Kyoto University. After a short time, Watanabe suggested that Tonegawa should apply for graduate study in the United States. A PubMed search of Tonegawa’s publications with Watanabe did not find any publications during these early years at the University of Kyoto.

3) Apparently, there is a great diversity of various antibodies produced by the immune system—Why is this important to understand?

Antibodies protect the body from dangerous microbes and cancer. The immune system produces antibodies in response to entities that are not a part of the body. From an immune system’s perspective, anything that is not already intrinsically part of the body is considered foreign. Many of these so-called non-self-substances, called antigens, can potentially (but not always) be detrimental to the body, perhaps causing illness or even death in some rare instances. Every single antigen must be recognized and somehow eliminated. The antibody’s role is to specifically encounter every specific antigen in the body and eliminate the invader, lest they cause harm or death.

Once an antibody molecule recognizes and binds to its specific foreign antigen, the potentially dangerous non-self-entity is to be purged from the body or destroyed, preventing it from causing disease and death. Antibody molecules protect us from antigens in several ways. One mechanism is called neutralization. Here, the antibody binds to the foreign antigen and prevents it from getting to our cells and tissues, where it might otherwise do damage. A second protective mode is called agglutination, which causes the antigen-antibody complexes to form giant conglomerations, which prevent the antigen’s pathological functioning. Another mechanism is biochemical in biological nature. Here, the antibody oxidizes the antigen, thereby destroying it in the process.

Lastly, the antibody purges and eliminates the antigens by recruiting other biological forces, such as the complement cascade system, inflammation, phagocytosis, and natural killer cells. Each of these innate systems functions to help the antibody destroy any potentially harmful or deadly foreign antigen.

As the numbers and types of microbial pathogens and cancers can range into the hundreds (if not thousands) of antigens, the diversity of antibodies must be correspondingly massive. In theory, however, any substance that is not part of the original body can constitute a potential danger to that body. Thus, the number of potentially dangerous substances now is increased into the billions! Antigenic diversity is enormous from an immunological perspective. Thus, a diverse set of antibodies is necessary to meet the challenge of all of the entities outside the body that may be dangerous to it.

The body’s immune system has developed a series of ingenious mechanisms to meet this challenge and produce an astonishingly diverse array of antibodies. Susumu Tonegawa’s work produced the first known biological mechanism for generating antibody diversity. In so doing, he shook the foundations of biology to its core. He found that the antibody production took the form of two or more independent and distantly located genes that came together to form one new intact gene to express an antibody molecule. Specifically, Tonegawa discovered that the antibody protein’s light chain was encoded by at least two different gene segments, which came together to encode the intact light chain molecule.

With different gene segments recombining in somatic cells to form one protein chain, Tonegawa demonstrated a new mechanism for generating antibody diversity. The breakthrough was profound. The work showed the presence of introns for genes that encoded the antibody. It was unclear whether and to what extent intron and exons were present in genes of the genome.

Furthermore, because each of the body’s immune cells partook in somatic gene rearrangement, Tonegawa made it known that every single B-lymphocyte was genetically unique for a given antigen. Tonegawa’s work had profound implications for the process known as clonal selection. The theory had been proposed by Nobel Laureate Frank McFarlane Burnet, who had proposed that lymphocytes, such as B-cells and T-cells, each had a unique antigenic specificity! Every lymphocytic cell was unique. Thus, a given lymphocyte could be selected by recognizing an invading antigen and selected to become a clone of hundreds or thousands of identical cells, each with specificity against the antigen.

Other mechanisms for generation antibody diversity are known. Based on Tonegawa’s gene recombination discovery, it was discovered that other gene segments could recombine, as well. For instance, gene sections called V, to denote variable, D, for diversity, and J, for joining, all recombined to generate a wealth of diversity to form new antibody variants. Another mechanism for enhancing antibody diversity involves adding nucleotides to antibody-encoding genes. These added nucleotides come in the form of palindromes (called “P addition”) and non-templated (“N-addition”) sequences.

The body is equipped with multiple diversity-generation mechanisms. An additional system involves changing the various locations between gene junctions, a process called junctional flexibility. Somatic hypermutation, another diversity-producing mechanism, entails massive mutational alteration of specific gene regions that encode antibodies.

While this is not necessarily a comprehensive listing of all known antibody diversity-creating mechanisms, it is anticipated that many more ways have yet to be found.

4) Tonegawa traveled to the United States to continue his studies—attending the University of California, San Diego in 1963. What do we know about his early years while there?

By August of 1963, Tonegawa was accepted as a Ph.D. graduate student at the University of California, San Diego. He would now study bacteriophages under Masaki Hayashi in the Department of Biology. In Hayashi’s laboratory, Tonegawa would publish his first known paper in September of 1966 in Biochimica et Biophysica Acta. They studied the phage called ΦX-I74 (Φ is Greek and pronounced Phi). Tonegawa and Hayashi prepared DNA from the phage, added purified DNA-dependent RNA polymerase enzyme, and measured RNA synthesis by detecting ATP and UTP’s radioactive incorporation. Tonegawa and Hayashi found that newly made single-stranded viral RNA formed a hybrid with the single-stranded template DNA from the phage genome. Furthermore, they determined the direction of RNA synthesis as new chains formed alongside the DNA template.

In another project, Tonegawa and Hayashi examined bacteriophage λ (Greek for lambda), a double-stranded DNA virus discovered in 1950 by Esther Lederberg. Tonegawa and Hayashi focused their attention on the phage λ gene called b2. They measured the expression of the b2 RNA synthesized by transcription. They found that the b2 gene was expressed in the early and later stages during the phage λ infection of Escherichia coli. Tonegawa and Hayashi also discovered that b2 RNA synthesis was controlled by the N gene’s product, which functions early on during the phage lytic cycle. Overall, the project was a significant contribution to our knowledge of transcription during phage λ infection. The work was published in the prestigious PNAS journal in late 1968.

Tonegawa earned a Ph.D. in molecular biology from the University of California, San Diego, in 1969 and remained in Hayashi’s laboratory as a postdoctoral fellow for a year. In one last project conducted by Tonegawa in Hayashi’s laboratory at San Diego, they paid attention to the assembly of phage ΦX-I74. In the laboratory, they permitted infection of bacteria with the ΦX-I74 phage, and before the complete infection could occur, they broke open the host bacteria and revealed its internal cellular contents. These infected cells’ contents had pieces of incompletely assembled phage products in the form of misshapen proteins. Tonegawa and Hayashi named these precursor viral products 6S, 9S, and 12S. The 6S particle was a five-piece component of the phage’s G protein, and the 9S particle was a five-piece section of the phage F protein.

Tonegawa and Hayashi reported their findings in the Journal of Molecular Biology in March of 1970. Later work conducted in Hayashi’s lab after Tonegawa had left showed that the 6S and 9S particles assembled to form the 12S protein, a partially assembled shell of the ΦX-I74 phage capsid.

5) Postdoctoral work at SALK—not too shabby—what did Tonegawa investigate there?

In 1969, Tonegawa accepted a postdoctoral fellowship at the Salk Institutes in San Diego in Renato Dulbecco’s laboratory. You can recall that the life and scientific achievements of Professor Dulbecco was featured in Chapter 7 of our 2020 book “An Overview of Biomedical Scientists and their Discoveries.” At the Salk, Tonegawa and Dulbecco studied tumor-causing viruses, such as polyomavirus and simian virus 40 (SV-40). Tonegawa was focused on examining the RNA molecules produced in normal infected cells transformed into tumors by the viruses.

Unfortunately, before he could get off the ground with the cancer work, U.S. policy dictated that Tonegawa had to leave the country because of the limited visa time allowed for foreign workers in the country. Tonegawa and Dulbecco would publish no work as a result. With Dulbecco’s intervention and an offer by astute scientific investigators, the future Nobel Laureate Tonegawa would find better refuge with an offer abroad by Professor Neils Kaj Jerne, a prominent immunologist.

6) The Basel Institute of Immunology—What did he study or research during his time there?

With his U.S. visa about to expire, he applied to work at the Basel Institute for Immunology in Switzerland. Fortunately, he was accepted as a member of the Basel Institute for Immunology in Switzerland from 1971 to 1981, even though he had little immunology experience. While at the Institute, Tonegawa applied the newly devised recombinant DNA methods of molecular biology to immunology and began to undertake one of the most critical unsolved immunological questions of his time regarding how antibody diversity is generated. It is now understood that this diversity is generated by somatic recombination of the inherited gene segments and somatic mutation. The latter process is well known. See Figure 91. For example, plants are known to undergo mutation in the genetic material of somatic cells.

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Figure 91. A red tulip that has undergone a somatic mutation is shown. The outcome is that half of a petal is yellow (left front petal). Such genetic and phenotypic consequences are considered to be due to a somatic mutation.

Part of the Nobel Prize-winning work involved addressing a fundamental question in molecular biology and immunology. The question was how antibodies had such a massive diversity in recognizing and binding the various billions of antigens while simultaneously keeping the same antibody functions of antigen destruction and elimination for all such antigens. It was a vexing question, and Tonegawa, working with Nobumichi Hozumi and others at the Basel Institute in Switzerland, would solve it!

For our discussion, we briefly consider one classic experiment. Tonegawa and Hozumi showed that different genes came together to encode intact antibody molecules. One genetic part, called the V gene (V stood for variable), produced the antibody’s diversity requirement. The second part, called the C gene (C stands for constant), encoded the antibody’s constant region.

One of their experiments was cleverly conducted as follows. First, Tonegawa and Hozumi prepared DNA from mice embryos, representing somatic cells that did not produce antibodies but harbored all of the genes for antibodies in separated locations in the genome. Next, Tonegawa and Hozumi digested the somatic DNA with restriction enzymes to break the somatic DNA into pieces. They had used BamHI to cut apart the somatic DNA and generate fragments.

In a second phase of the Nobel work, Tonegawa and Hozumi isolated DNA from an antibody-producing cell, a B-lymphocyte-like mouse tumor cell called MOPC 321, which had presumably harbored rearranged gene segments to produce an antibody’s light chain protein. The idea was that if somatic recombination of DNA occurred to make antibodies, then an antibody-producing cell should have rearranged DNA that differs from the genomic DNA of the non-immune somatic cell. Tonegawa and Hozumi digested this DNA from the antibody-making tumor cells with BamHI restriction enzyme, breaking it into DNA fragments.

The third phase of the famous experiment by Tonegawa and Hozumi involved using gel electrophoresis to separate the DNA fragments from the somatic cell (non-antibody making) and the B-cell-like tumor (makes antibody). The gel electrophoresis separated the DNA pieces into individual bands on the gel matrix.

Next, they performed what is considered to be the first case of a now-famous experimental method, called a Southern Blot. In the historical experiment, Tonegawa and Hozumi added one of two radioactive RNA probes. One RNA probe detected the V gene, and the second RNA probe detected the C gene. The results showed that in the somatic cell DNA, the V and C genes were far apart. Incredibly, Tonegawa and Hozumi then observed that in the antibody-producing cells, the V and C genes were near each other and connected!

The published graph showing the somatic cell versus antibody-making cell’s radioactive peaks provided the first demonstration in the scientific history of somatic cell DNA recombination. These data shook the foundations of biology! Remember, the somatic cell’s DNA was supposed to be unchanged. Tonegawa and Hozumi published the shocking Nobel Prize work in October of 1976 in PNAS.

The idea that somatic cell gene rearrangement was possible was met with skepticism. Moreover, it seemed that Tonegawa and his colleagues had to experimentally repeat the process many times in order to definitively demonstrate the startling phenomenon. Before Dreyer and Bennett, the scientific world strongly believed that the genomic contents of the non-sex cell, the somatic cell, were identical and unchanged in every cell. Eventually, somatic recombination as a bona fide molecular immunological mechanism for immune diversity came to be widely accepted.

7) In 1981—Massachusetts Institute of Technology—What had Tonegawa researched or investigated there?

Tonegawa moved to the U.S. in 1981 at the invitation of Salvador Luria. His appointment was a biology professor at the Center for Cancer Research at the Massachusetts Institute of Technology (M.I.T.). In combination with conducting immunological investigations, Tonegawa studied molecular and cellular characteristics of neurobiology.

His research projects concerned two significant problems. The first was to continue investigating the role of somatic rearrangement in the activation of the rearranged antibody genes. The second was to extend Basel’s research to “the other half” of the immune system, specifically the T cells’ antigen receptor and the histocompatibility of antigenic tissues.

At M.I.T., Tonegawa studied the T-cell receptor (TCR) of the lymphocytes. He examined their TCR structures and genes. He showed that the genes encoding the TCR also underwent somatic recombination. Not to be outdone, in much the same way, Tonegawa studied the genes of the immune system major histocompatibility complex (MHC). The studies by Tonegawa on the TCR and MHC systems were extensive and took a great deal of his time during the 1980s and early 1990s. These were highly significant findings, each one likely worthy of a Nobel in its own right.

8) A Nobel Prize—and a Stamp—What did he get the Nobel Prize for, and who awarded him a stamp?

In 1995, the western African country of Nambia issued a commemorative stamp on behalf of various Nobel Laureates. To honor Tonegawa, he was issued Scott stamp number 1635c.

The path to the Nobel for Susumu Tonegawa started with counting genes. He was testing the so-called Dreyer-Bennet hypothesis. According to William J. Dreyer and J. Claude Bennett, postulated in 1965, variable (V) and constant (C) regions of the antibody proteins were products of two distinct genes, the V and C genes. These genes were postulated to be separated in the genetic material of the sex cells. Such DNA was said to be germline derived. In somatic cells, the DNA moved and combined—its DNA recombined. Dreyer and Bennett hypothesized the so-called “two genes encode one protein” idea. If correct, it predicted one of a few C genes and another from a more extensive set of V genes combined to express one antibody protein. Two genes came together in non-sex cells to form a single protein.

Susumu Tonegawa was working with several colleagues at Basel, A. Bernardini, B. J. Weimann, C. Steinberg, S. Dube, and G. Matthyssens. They set out to test the Dreyer-Bennett hypothesis, and they started by counting genes in the late 1970s.

At first, they found only a few numbers of V regions encoding one of the light chains of the antibody, inconsistent with the Dreyer-Bennett hypothesis, which predicted a relatively larger number of V genes. However, Tonegawa would correspondingly refine his previous conclusion using more sophisticated methods and agree that a larger number of V regions could exist, perhaps 30 genes, even for the light chain. In modern times, we know that in the mouse, between 120 and 140 different V genes exist for the light chain that Tonegawa studied, the so-called kappa version of the light chain. See Figure 92a.

File:2220 Four Chain Structure of a Generic Antibody-IgG2 Structures.jpg

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Figure 92. Four Chain Structure of Generic Antibody-IgG Structures.

A typical antibody structure consists of two identical large protein molecules called heavy chains because of their large molecular weights. Disulfide bridges connect the heavy chains. See Figure 92a. Likewise, two identical light chains are composed of lower-molecular weight proteins. The two N-terminal ends of a heavy chain and a light chain constitute the so-called antigen-binding site, and there are two such sites on an antibody. They are sometimes called “Fab” because they are fragments for antigen-binding. These two regions are also called variable because each antibody molecule is different. The remaining domains are constant in their amino acid sequences, and they are denoted as the constant regions. The Fc region is easily crystallizable if cleaved apart from the two Fab portions. The Fc region helps the antibody recruit help for the antibody and stimulates other immune system components. The various domains of the antibody that can appear are “globs,” and, hence, these globs are called “Immunoglobulins” or “Ig” for short. See Figure 92b. There are five immunoglobulin classes, called IgG, IgA, IgM, IgE, and IgE. Each immunoglobulin class has its dedicated structures and functions.

Tonegawa soon focused on mapping the genes encoding the various antibody chains, both heavy and light chains. He also studied the organizations of the various antibody-encoding genes. In 1980, he made the breakthrough of demonstrating gene rearrangement to generate a complete antibody molecule in B-cells, which are precursors to antibody-producing plasma cells, publishing the work during September of 1980 in the journal Science.

In the early 1980s, Tonegawa’s laboratory at Basel characterized the D gene, a DNA segment that was further responsible for generating new varieties of antibody, D for diversity. It was a significant finding, and the work was published in Nature in 1981.

Tonegawa would involve many others in his laboratory conducting new experiments and providing new data repeatedly throughout the 1980s for somatic recombination. Each time he showed another new piece of evidence that demonstrated the validity of the shocking notion of somatic cell DNA recombination.

9) What have I neglected to ask about this molecular biologist?

Tonegawa is an enthusiast of the Boston Red Sox and was chosen to throw out an opening pitch during their 2004 World Series championship season.

Tonegawa was a Howard Hughes Medical Institute investigator from 1988 to 2009. He has been the director of the RIKEN-MIT Center for Neural Circuit Genetics since 2008 and of the RIKEN Brain Science Institute in Japan from 2009 to 2017.

In 1994, after shifting his emphasis on neuroscience, he became the Center for Learning and Memory director at MIT. This center is now the Picower Institute for Learning and Memory. His investigation concentrated on the function of the hippocampus in the processes of memory formation and recall. See Figure 93. Susumu Tonegawa was intrigued by the brain mechanisms fundamental to learning and memory. He and his colleagues have also investigated how memory plays into emotion and how the memory of past experiences is transformed into knowledge supporting new learning.

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Figure 93. Brain highlighting the hippocampus.

In 1987, Tonegawa was presented the Nobel Prize for Physiology or Medicine for his discovery that the genetic mechanisms were causing the pronounced assortment of antibodies produced by the vertebrate immune system. Tonegawa received several prestigious awards throughout his career, including the Louisa Gross Horwitz Prize (1982), the Person of Cultural Merit prize (Bunka Korosha; 1983), conferred by the Japanese government, and the Order of Culture (Bunka Kunsho; 1984). Tonegawa was honored by the Albert and Mary Lasker Award in Basic Research, 1987. Tonegawa was inducted into the National Academy of Sciences as a member in 1986.

For additional information about this remarkable scientist who changed the molecular biology world, visit:

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