Dr. Manual Varela: Who was the only Briton to have won two Nobel Prizes?

Oct 2, 2017 by

An Interview with Dr. Manual Varela: Who was the only Briton to have won two Nobel Prizes?

Michael F. Shaughnessy –

1) Dr. Varela, we first have to mourn the passing of Frederick Sanger who recently passed. Obviously he was born in Great Britain, but what do we know about his early life and childhood?

A laureate of two Nobel Prizes, Dr. Frederick (Fred) Sanger was born on the 13th of August in the year 1918 to parents Frederick Sanger and Cicely Crewdson Sanger in the small country village of Rendcomb, located in the county of Gloucestershire, the Cotswold district, in England.  Sanger’s father was an established physician in the small community, and his Quaker mother was a homemaker who kept a diary detailing events concerning mainly about the household and her family with the three children, young Fred being the middle child. Fred had an older sibling, Theodore (b. 1917) and a younger sister, Mary (b. 1923).

The Sanger children had a governess, and the family environment is reported to have been a positive, warm, and friendly household. In her diary, Cicely recorded insight into the behavioral temperament of young Sanger. Sadly, however, she did not have the opportunity to record anything beyond his undergraduate years because she passed away in 1938. His father had died even earlier, in 1937.

In 1927, nine-year old Sanger was enrolled in a Quaker preparatory boarding school called The Downs, located in Malvern.  Although the experience is reported as being a traumatic one for Sanger, as he was bullied, he was, nevertheless, to earn outstanding grades while enrolled at The Downs.

Soon afterwards, in 1932, 14-year old Sanger was enrolled in another newly established boarding public school (in England public schools are actually private schools as a fee is assessed) called Bryanston School, in Dorset, England. Although Sanger failed to secure a lucrative scholarship to attend, he was nonetheless quite happy to be a student at this particular institution where he flourished, especially in the subjects of biology and chemistry.  In fact, Bryanston was such a favorite school that many years later (in 2007) Sanger was to establish a new building in his name, the Sanger Centre for Science and Mathematics, at the school.

In 1936, Sanger attended St. John’s College, Cambridge, his father’s alma mater, majoring in the Natural Sciences and emphasizing in Biochemistry. During his college years, he became a conscientious objector and first met his future wife, Joan Howe.

Initially hoping to ultimately obtain an M.D. degree (perhaps it was more a hope entertained by his father, rather than by Sanger) the death of both of his parents while still in college made Sanger feel that his chances for advancement in medicine were made to be somewhat compromised.  Thus, it is during this time-period that Sanger chose instead to pursue scientific research as a full-fledged career. In 1939, he took his B.A. degree from St. John’s College.

2) His first Nobel Prize was for the sequence of protein. Why is this important in the big scheme of things? When was that awarded, and what did we learn from this?

Sanger earned his first of two Nobel Prizes in 1958, in the area of Chemistry, for his investigative work with the important hormone called insulin.  He was the sole recipient of the 1958 award in the category.

Sanger was the very first scientific investigator in the world to elucidate the entire sequences of the amino acids along the two protein chains that make up insulin. Solving its protein sequence was such an important finding because knowledge of the nature of the amino acids along the insulin protein chains, or of any protein, almost immediately allows an investigator to learn about how the protein chains fold into their three-dimensional structures.

Along these lines, knowledge of the overall 3-D shapes of biologically and biochemically important molecules lends tremendous insights into the nature of their mechanistic functions. All of these great advancements in protein biochemistry started with Sanger’s study of insulin.

Sanger studied a series of biochemical reactions to help him elucidate the nature of the amino acid sequence of both insulin protein chains, called alpha (or A) chain and beta (or B) chain.

3) Apparently, twenty-two years later, the Nobel Committee awarded him the 1980 prize in Chemistry once again for finding a way to determine the ordered sequence of DNA molecules. I suspect this has something to do with the twisted ladder effect? Or am I off on this?

On the contrary, you are not off on your assessment of Sanger’s important work with the double helical DNA molecule. Sanger’s studies on DNA earned him his virtually unprecedented second Nobel Prize, also in Chemistry, in 1980. As of this writing, only four other individuals in history have garnered 2 Nobel Prizes, and Sanger is apparently the only person ever to have gotten two Nobel Prizes in the Chemistry category.

Sanger was able to determine the nucleotide base sequences of DNA by again putting his knowledge of biochemistry to work. He used bacterial enzymes like, for instance, DNA polymerase which he obtained from the bacterium called Escherichia coli, and other chemicals to break up DNA into nested pieces.  Each of these DNA pieces differed chemically by one base-pair in their lengths.

Next, he collected his various pieces of DNA and placed them in so-called electrophoretic gels, ran an electrical current through the DNA-laden gels and then read the radioactively labeled bands on the gels on exposed X-ray film using a light box. From this rather labor-intensive work he was able to determine the entire genome sequence of a bacteriophage virus called ΦX174 (the Greek symbol Φ is pronounced phi).

Application of Sanger’s method for determining the nucleotide base sequences along their twisted DNA chains was a remarkable and profound discovery.

4) Proteins seem to have a very exact, defined, specific sequence or composition. How was Sanger involved in this and why is it important?

Protein chemists, biochemists, and molecular biologists often like to proclaim that although DNA is indeed universally considered to be the “molecule of life,” it is the proteins, however, which “do everything.” Proteins are molecules that partake in the performance of those sort of processes involved in all living systems. Each protein has a specific function or set of functions, and these functions are carried out by the particular shapes, or molecular configurations, inherent within each of the proteins.

These protein structures, in turn, are dictated by the characteristic sequence of amino acids within them. It can be said that proteins are actually chains of varying lengths which are composed of amino acids that are tied together by peptide bonds. The amino acid chains of polypeptides in turn will fold up around each other to form molecular shapes that are virtually limitless in their structures formed but are certainly specific to each and every distinct protein.

Thus, the particular sequences of amino acids along protein chains can be said to determine the precise molecular natures of protein structures. Therefore, if one knows the molecular shape of a protein one might then know the biological function of those proteins.

Sanger’s involvement with this protein chemistry field comes into play as he was the very first scientific investigator to determine the sequence of amino acids for a protein, namely, that of insulin.

Let’s briefly consider Sanger’s Nobel Prize winning work with protein sequence determination of amino acid residues along the insulin hormone protein.

First, Sanger broke up each of the two insulin A and B chains from each other and then broke apart each of these A and B polypeptides into smaller fragments. He had denatured the two chains from each other by adding chemicals to undo the disulfide bonds connecting the A and B polypeptides to separate them from each other and then, using a technique called chromatography, he isolated each of the separated chains.

Then, he developed a ‘labeling’ molecule that would light up or fluoresce when induced and that would bind to the one amino acid that was located at one of the ends of a separated insulin protein chain, whether it was the A or the B chain. The labelling molecule was called 1-fluoro-2-,4-dinitrobenzene (FDNB).

Next, he used very strong acids to disrupt each of the two insulin protein chains, and the amino acids would come apart from each other releasing them including the end-labeled amino acid. He did this disruption for each of the A and B chains separately.

Fourth, Sanger invoked a special biochemical process called the ‘Edman degradation’ which had been developed by the biochemist Dr. Pehr Edman. The process also end-labels protein chains, like Sanger’s FDNB, but instead when the protein chain degrades, only the end-labeled amino acid comes off, not all of them like in Sanger’s strong acid treatment.

Next, Sanger would conduct a series of chemical and biochemical reactions meant to determine the identity of the amino acid that came off of the individual chain during the earlier third step.

Lastly, Sanger then purified and re-used the insulin protein that was left over from the third step with its missing (though identified) amino acid, but essentially intact otherwise, to start another round of the Edman degradation. Then he would simply start over again, label the protein, remove the end-labeled amino acid with the Edman step, and identify the next amino acid along the chain till he was finished with identifying all of the amino acids in the protein.

Now, with insulin consisting of two separate polypeptide chains, the A and the B chains, and each of the polypeptide chains being unique, sequence-wise, it was a very labor intensive process to identify all amino acids in the protein. Insulin’s A chain has 21 amino acids, and the B chain has 30 amino acids.

It took years to complete the entire protein sequence identification for both insulin chains. Much later, the cumbersome protein sequencing technology became automated, the new advanced machines being given the name ‘sequenators.’

The B chain gave away its amino acid sequence secret first, and its sequence was published in 1951, with co-author, Hans Tuppy. The A chain gave away its sequence secret a couple of years later in 1953. No one had ever accomplished such a remarkable task before in scientific and world history.

5) Key word–electrophoresis- what does this mean and why is it relevant?

The term refers to a laboratory technique that’s very often used to separate biological components from each other in order to study each of the components separately. The electrophoresis process involves making a ‘gel’ often in a rectangular shape, like that of a flat brick, but with the consistency of Jell-O.  In fact, investigators making electrophoretic gels often use the same sort of materials in Jell-O, like gelatin, or agarose, or perhaps more sophisticated materials, like polyacrylamide, depending on the purpose of the experiment and the nature of the biological molecules being studied. The gels will also have little ‘wells’ in them, formed during the gel making process. The wells will be located on one end of the gel brick and used as little buckets to place the biological samples.

Once an electrophoretic gel is made, complete with its wells, the investigator will then load the wells with the molecules in question, like proteins, nucleic acids, etc.

Next, here’s where the ‘electro’ part comes into play. The loaded gel will be placed in a buffer with ions and subjected to an electrical current. The current will force the samples loaded in the wells to move into the insides of the gel, called a gel matrix, and redistribute themselves according to their relative sizes and charges.

The smaller molecules will move faster and farther along the gel than their larger counterparts.  The more negatively-charged molecules will move ‘faster’ and farther along the gel than their positively-charged relatives, which stay somewhat behind. Sanger was known to have used electrophoresis when he developed his Sanger method for DNA sequencing.

6) The “Sanger method” supposedly allowed very long stretches of DNA to be accurately and rapidly sequenced. Why is this important in the big scheme of things?

The Sanger method of DNA sequencing has also been referred to as the ‘Sanger dideoxy’ or ‘chain termination’ method. This Nobel-winning work involved the following steps.

First, Sanger added the basic ingredients, such as the DNA to be sequenced, a short DNA primer to start DNA synthesis, a DNA-making enzyme called DNA polymerase, an excess amount of the nucleotide bases, G, A, T, and C, plus a tiny amount of Sanger’s special DNA chain terminator molecules called dideoxy-G, dideoxy-A, dideoxy-T and dideoxy-C. These chain terminators, usually labeled with radiation or a florescent probe, will prevent the continued synthesis of new DNA chains from that point onward.

During the DNA-making reaction, sometimes the machinery added incorporates a regular base into the growing DNA chain, or it incorporates a chain terminator, instead. The reaction proceeds in which new DNA is made but because of the chain terminators, the end-product is a series of new DNA chains of varying lengths, each usually differing by only one nucleotide, if performed correctly.

The mixture of new DNA of different lengths was placed by Sanger into wells of an electrophoretic gel and subjected to an electrical current to separate the fragments according to their lengths, from larger to smaller. The gels are placed on photographic film to expose the DNA, and the nucleotide bases are ‘read’ along the length of the chains, thus revealing the DNA base sequences.

The DNA sequences are stored and archived in computer databases. The DNA sequencing process is important for a variety of reasons. The knowledge of DNA sequences, for instance, facilitated the cloning of genes and for computer determination of their corresponding protein sequences. This alleviated the need for cumbersome protein purification and protein sequencing efforts, saving a tremendous amount of time and money.

Also important was the ability of investigators to study sequence variants of DNA genes and proteins, such as in genetic diseases, or their evolution, or for garnering more efficient industrial efforts to make new products, such as medicines.

7) He studied lysine and insulin. What discoveries or contributions did he make relative to these two things?

While an undergraduate student, and to Sanger’s great surprise, he excelled in Biochemistry, earning the highest honors possible and, thus, qualifying him for advanced studies in graduate school.  Sanger’s work with lysine began in graduate school.

Thus, in 1940, Sanger was granted admission to graduate school at Cambridge, entering the laboratory of Prof. Norman (Bill) Pirie, housed in the institution’s Biochemistry department. In 1936, Prof. Pirie had discovered that a virus, called the tomato bushy stunt virus, was amenable to crystallization. In Pirie’s laboratory, Sanger was given the thesis project involving the purification of an edible protein from grass.  However, very shortly after starting on the new thesis project, Pirie left the institution for another academic post, leaving Sanger to find another thesis advisor at Cambridge.

Consequently, the new graduate advisor whom Sanger chose to work with was a newly appointed faculty member by the name of Albert Neuberger. Many years later, Sanger would recount how it was Prof. Neuberger himself who taught him (Sanger) how to properly conduct scientific research and, importantly, not to be afraid of studying new things and to not despair too terribly if an experiment did not work out—just try another experimental route.

In Neuberger’s laboratory Sanger set out to study the degradative metabolism of an important and ‘essential’ amino acid called lysine, in laboratory rats.  Sanger also partook in so-called war-work pertaining to World War II, namely, the analysis of nitrogen and protein composition in different strains of potatoes.

Although Sanger was unable to definitively elucidate the entire degradative biochemical pathway for the metabolism of lysine, he was nevertheless able to advance the work sufficiently enough to publish five papers on the topic and in his potato work to finish his Ph.D. thesis project, and make a very good starting point for other investigators to complete the lysine breakdown pathway. Sanger defended his Ph.D. thesis project in 1943 and took his doctorate in Biochemistry from the University of Cambridge.

Sanger’s insulin work involved elucidating the entire amino acid sequences of the two polypeptide chains, called A and B, that make up the hormone insulin. He used labor-intensive and harsh chemical techniques in order to do so. He had worked with the insulin molecule from bovine, or cows. The novel and Nobel Prize worthy insulin sequence work ultimately led to elucidation of the sequence for human insulin. It then led to the cloning of the human gene encoding insulin; and it further led to the determination of its three-dimensional protein structure by Prof. Dorothy Crowfoot Hodgkin, the 1964 Nobel Laureate in Chemistry.

8) Insulin, is obviously tied to diabetes- a major medical problem- but what exactly is lysine, what does it do and why is it important?

Lysine is essential in the sense that humans and other animals cannot make this particular amino acid and must, therefore, acquire it from the diet. Good sources of this nutrient include foods rich in protein like meats, fish, legumes, poultry, etc. This amino acid is used by the body to make larger protein molecules, which we know are vitally important for life’s processes.

Much biochemical work has been placed on elucidating its metabolism.  The biosynthetic pathway for making lysine has been well defined.  Likewise the catabolic pathway for degrading the amino acid into its smaller chemical parts has been definitively determined. Both aspects of its metabolism are regularly included in biochemistry and physiology textbooks. The topics are routinely taught to students of healthcare sciences, like medicine, dentistry, and in the biomedical sciences.

It’s been postulated that lysine is important for immune system modulation and as an anti-anxiety supplement. It has been used as an external dietary supplement, a characteristic featured in a popular television show, ‘The Big Bang Theory’ about science and computer ‘nerds’ in which one of the main characters touts the virtues of lysine. Its use is also important in agriculture for feeding of farm animals. Industrial production of lysine involves the fermentative process using microbes and various sugars as a starting point for lysine synthesis. Recombinant DNA technology, made possible by Sanger’s DNA work, has been used to boost the production process for lysine.

It is interesting to note that in Michael Crichton’s book ‘Jurassic Park’ the fictional book in which dinosaurs are brought back to life using ancient DNA fossilized in amber samples of mosquito blood meals, the cloned dinosaur DNA was genetically engineered to be defective in their ability to make lysine. This made the cloned dinosaurs dependent upon the fictional scientists for their source of dinosaur dietary lysine.

9) He apparently was called “the father of the genomic era”. Can you explain this for us?

Because of Sanger’s DNA sequencing invention, it allowed the elucidation of the molecular nature of both genes and proteins. Furthermore, improvements in the efficiency of Sanger’s work was greatly facilitated by converting from the slow and cumbersome gel electrophoresis to the more advanced and efficient capillary approach.

New technological improvements, such as the capillary mode, allowed investigators to sequence DNA with greater efficiency and more rapidly than ever anticipated. It became possible to map genes and to sequence the DNA of entire genomes of organisms, thus, instituting the new field dedicated to the study of whole genomes from living organisms.  The new discipline is referred to as genomics. Sanger’s DNA work made the new and burgeoning genomics field acutely possible.

Prior to the improvements in gene sequencing technology in the 1980s, it had been a laborious task to sequence even one gene.  Entire Ph.D. theses might have been devoted solely to the cloning and sequencing of a particular gene of biological or biomedical importance.

With the advent of genomics, it became possible to determine the entire nucleotide base sequence of an intact (i.e., whole or complete) genome.

Furthermore, it was possible to compare genome sequences with other genomes from other individuals or other species. This comparative genomics approach has made possible the study of the evolution of living organisms as they interact with other species or how they behave in their native or novel environments. Such evolutionary information may shed light into what the future has in store for generations of living beings throughout the world.

Comparison of genomes has greatly enhanced the search for targets with novel biological mechanisms and therapies.  For example, in our research laboratory, we determined the entire genome sequence of a non-pathogenic variant of a dangerous bacterium called Vibrio cholerae, the causative agent of cholera—a serious disease in many parts of the world. Using our genome sequence data, we then compared our ‘good’ bacterium with the known genome sequence of the classically dangerous cholera bacterium.

Identifying the differences between the good and not-so-good bacteria allowed us to recognize those genes in the cholera-causing bacterium which might be involved in conferring the serious nature of its effects on their patients. Thus, these new genes may encode cellular targets that we may be able to exploit by reducing their activities in order to minimize their deleterious effects.

Currently, it is possible to take samples of DNA from an environment and ascertain the entire organismal content of that environment by sequencing and learning about the entire make-up of all of the species that reside in that environment.  This has been done recently with the human gut, which is predicted to harbor thousands, if not millions of microbial species. The gut microbiome has been implicated in determining various aspects of human health, such as immunity, behavior, and personality, such as whether one is generally a happy individual.  Much work will be needed to determine the extent to which each of the millions of bacterial species interact with others. In fact, with genomics-based studies, it has been recently postulated that Earth harbors a trillion microbial species. If true, it will require efforts based on genomics to find and identify all of these yet undiscovered species.

10) There are all kinds of stories about him- apparently he was a Quaker, a pacifist, and, I quote “A chap who just messed about in his lab”. What other gems do you know about this iconic scientist?

It is interesting to note that despite his advancing age, he spent a great deal of time literally ‘at the laboratory bench’ doing experiments with his own hands up until the time of his retirement. Many senior scientists are often loathe to work at the bench or are encumbered to do so by having to perform so many ‘required’ administrative duties, or to participate in grant-writing and manuscript-preparation, or serving on numerous committees, etc.

Prof. Sanger was known to be a great scientific mentor, with several of his doctoral and post-doctoral students themselves having earned a Nobel of their own. One of his Nobel mentees participated in structural studies of antibodies while another was involved in studying the molecular edges of chromosomes, called telomeres.

When asked in an interview how he felt about being the recipient of two Nobel Prizes, he replied that it was more difficult to get the first one than it had been to get the second one. The first Nobel made it possible to more easily acquire new laboratory facilities and other research opportunities that made his subsequent research endeavors more readily possible.

It may interest our readers to know that Sanger’s laboratory was not the only one devoted to discovering how to sequence DNA, a procedure that was highly sought after by many molecular biologists of the day. The Harvard laboratory of Walter Gilbert proved to be Sanger’s biggest competitor. Gilbert’s approach was called the Maxam (after Allan Maxam) and Gilbert chemical method. The story is told that once during a Gordon Research Conference, members of Gilbert’s lab presented their sequencing methods and data without referencing the work of Sanger, which they (members of the Gilbert lab) had used.  This slight had rankled Sanger. Nevertheless, Gilbert and Sanger shared the 1980 Nobel in Chemistry for their efforts in inventing the DNA sequencing technology.

When Sanger determined the genome sequence of the ΦX174 phage, he found that there were two overlapping genes encoded within the genome. Thus, Sanger was one of the first investigators to go beyond the ‘one gene, one enzyme’ notion that was prevalent throughout the field of molecular biology.

Another finding by Sanger that was of great importance was his sequencing of mitochondrial DNA.  The mitochondria are sub-cellular organelles that function to produce the cellular energy needed for life and are thought to have arisen from an ancient bacterial infection of an ancient eukaryote. When Sanger sequenced mitochondrial DNA, he found that the genetic code was not entirely universal, as was once thought.

Others who knew Sanger well on a personal basis have observed that he was quiet, humble, humorous, and often partook in self-deprecating behavior. It has been reported that he refused the offer to be knighted by the Queen, remarking only that he had not wanted to be changed by the honor and to be called ‘Sir Fred.’

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