An Interview with Manuel and Ann Varela—about Richard J. Roberts—From Derby England to Split Genes and the Nobel Prize

Dec 17, 2020 by

There is need for more science in politics and less politics in science.”

—Richard J. Roberts

Invest in basic research so you can make the discoveries that everybody else will want to use. You don’t have to do everything. You just find some little niche area, something that you’re interested in here, or something that there’s an individual here in this country with a very good idea of how to do it that no one else is doing.”

—Richard J. Roberts

I’m a great believer in youth; I think the nice thing about young people doing science is that they ask the sort of questions that I would no longer ask. Because if I were to ask some of these questions, people would look at me and say, “Well, how come you don’t know the answer?” And then they’d think I was stupid. And so what happens is when you get older, you tend not to ask the stupid questions that sometimes lead to new insights.”

—Richard J. Roberts

Michael Shaughnessy

  1. It is often fascinating to read about people who rise from somewhat humble beginnings to the Nobel Prize. Richard J. Roberts was born in Derby, England—but when exactly was he born, and what were his early school experiences?

Richard John Roberts is a 1993 Nobel Laureate in physiology or medicine for his discovery of the molecular phenomenon called “split genes.”

Roberts shared the Nobel accolade with Phillip Allen Sharp, who discovered the gene-splicing mechanism in adenoviruses. On September 6, 1943, Richard Roberts was born in Derby, England, to John and Edna (Allsop) Roberts. He was an only child. Roberts’ father was an automotive mechanic, and his mother was a homemaker.

By the time he was four, the Roberts family had moved to Bath, England. There, young Richard attended the City of Bath Boys School. Roberts considered the headmaster to be a mentor of sorts because he encouraged him to excel in mathematics and provided him with crosswords and logic-based puzzles to solve. Roberts was a curious lad and had diverse interests, such as detective work and chemistry. His father assisted him with building a rudimentary chemistry lab in the family’s home basement. The interest in chemistry stemmed from being gifted a chemistry set. He also did quite well in school with chemistry and math; however, physics was problematic. An extra year of school to repeat physics would be needed to complete the University entrance requirements.

Roberts attended the University of Sheffield in England and earned his Bachelor of Science degree with honors in Chemistry in 1965. Roberts was the Ph.D. student of Dr. David Ollis, Professor of Organic Chemistry.

2) Roberts’ doctoral dissertation involved “Phytochemical studies of neoflavonoids and isoflavonoids.” First, what do these things mean, and why are they important?

In 1968, Roberts completed his Ph.D. in organic chemistry from the University of Sheffield, England. His thesis, completed in 1969, was titled Phytochemical studies involving neoflavanoids and isoflavonoids. Neoflavonoids found in pieces of heartwood from a Brazilian tree provided Roberts with many interesting new compounds.

Roberts studied these compounds because they were novel intermediates in the biosynthesis of a class of plant-based phytochemicals called flavonoids. These chemicals are found in fruits and vegetables that many organisms consume in their diets, including the diet of humans. Thus, these phytochemicals are essential dietary constituents in natural foods.

In particular, the flavonoids serve important protective roles against the detrimental effects of oxidants. Plants use flavonoids for protection against bacterial pathogens, which can produce oxidative agents. Hence, flavonoid compounds are necessary antioxidants that counteract the oxidative effects of agents produced by plant pathogens.

Plants that release flavonoids affect particular microbes like bacteria or fungi that reside in the plant roots. The flavonoids will signal, for example, nitrogen-fixing bacteria of the Rhizobium genus to produce proteins called “Nod,” which are nodulation factors. The Nod proteins will signal the root hairs of the plant to form nodule havens for the microbes. The microbes help the plant fix nitrogen molecules extracted from the air to form ammonia, which supplies nitrogen to the plant to make protein.

Furthermore, flavonoids play central roles as vitamins, such as vitamins C or E. Since humans cannot make these and other vitamins in their bodies, the chemicals must be obtained in their diet. In addition to their helpful roles as antioxidants, vitamins serve as cofactors for the body’s various enzymes. Hence, the flavonoids help provide the cell with its necessary conditions for life. Thus, these phytochemicals can be viewed as being indispensable for life.

3) Roberts first did postdoctoral training at Harvard. What did he work on there?

Roberts’ postdoctoral research was done at Harvard University in the research laboratory of Jack L. Strominger. He spent a month in Frederick Sanger’s lab in Cambridge, Massachusetts, learning a new sequencing method of tRNA, which was involved in bacterial cell wall biosynthesis. Roberts’ goal had been to determine the sequence of the tRNA molecule. Roberts felt that the radioactive method of Sanger was preferable for sequencing. Once he returned to his lab at Harvard, Roberts taught his colleagues and local scientists the new radioactive sequencing approach. The tRNA sequence analysis work was also important because it involved bacteria of the Staphylococcus genus.

Another exciting project conducted in Strominger’s Harvard laboratory involved their discovery of a new type of tRNA. Working with Thomas Stewart in the lab, they studied the bacterium Staphylococcus epidermidis, a typical human skin inhabitant. From these resident bacteria, Roberts and colleagues isolated a tRNA molecule specific for the amino acid glycine. Their new tRNA molecule had several noteworthy features.

Their first exciting feature was that tRNA had a single modified ribonucleotide base, called 4-thiouridine. Another attractive property of their novel tRNA was that it participated in synthesizing a bacterial cell wall component called peptidoglycan. The peptidoglycan molecules function to protect the bacteria and help the Staphylococcus epidermidis and Sporosarcina ureae bacteria form their characteristic circular shapes. Roberts and his colleagues added their purified tRNA to a tube containing ATP and the amino acids glutamate and glycine. The bacteria then formed a so-called “interpeptide bridge” between various repeating chemical units of the peptidoglycan. A third exciting feature involving their novel tRNA species was that it did not make protein through the standard translation process using the bacterial protein-making machinery such as the ribosomes.

A related project conducted by Roberts at Harvard concerned a structural comparison between regular protein-making tRNA and his new tRNA species with its modified base and its refusal to make protein. He studied the molecular structures of both species of the tRNA molecules. One curious feature of his novel tRNA molecule was that the anticodon loop shared the glycine-specific bases typical of a regular glycine. Nevertheless, the novel tRNA refused to incorporate glycine into a growing polypeptide chain. However, Roberts reasoned that the novel tRNA species had likely evolved from the regular glycine-specific tRNA molecules. The idea indicated that bacteria could adapt their cellular machinery for other purposes, like making protective cell wall peptidoglycan.

4) He then moved to Cold Spring Harbor Laboratory—first, what went on at this laboratory?

In 1972, James Watson offered Roberts a position, which he accepted, at the Cold Spring Harbor Laboratory in New York. The job included an excellent salary and set-up money to supply his lab. The topic of research was sequencing the DNA of the simian virus 40 (SV40) virus genome. Roberts accepted the position and began studying a group of enzymes called restriction endonucleases. The first enzyme he examined, called endonuclease R from Escherichia coli bacteria, had been discovered by Dan Nathans. The endonuclease R enzyme broke down the adenoviral genome into specific DNA fragments at particular base sequences. Eventually, the endonuclease R enzyme was renamed EcoRI, for Escherichia coli restriction enzyme I. See Figure 77.

File:PDB 1ckq EBI.jpg

Figure 77. Molecular ribbon structure of EcoRI restriction enzyme, an endonuclease protein bound to DNA. EBI registered with code 1ckq.

Roberts and colleagues found a second endonuclease enzyme, later called HhaI, from the bacterium called Haemophilus haemolyticus. The HhaI restriction enzyme could cut the genomic DNA molecules of SV40 and bacteriophage lambda, also known as phage λ. A third restriction enzyme was discovered from the bacterium called Haemophilus aegyptius. The so-called HaeIII could digest the genomic DNA of adenoviruses and phage λ. Of particular note was the restriction enzyme called BamHI, also discovered by Roberts. The BamHI endonuclease had been purified from the bacterium called Bacillus amyloliquefaciens strain H. The BamHI enzyme cleaved DNA from phage λ, adenoviruses, and bacteriophage Φ (phi). See Figure 78.

File:PDB 2bam EBI.jpg

Figure 78. Molecular ribbon structure of BamHI restriction endonuclease enzyme protein bound to DNA. EBI code 2bam.

Not long after, Roberts and his lab had a whole collection of restriction enzymes. In general, these endonucleases shared two intriguing properties. First, the enzymes seemed to be sequence-specific. That is, each restriction endonuclease could bind to a specific DNA base sequence and break the DNA at that sequence site.

That is, the enzyme was restricted to that sequence site. Second, the enzymes seemed to target foreign DNA molecules that were not derived from the bacteria that made the enzyme. Such alien DNA molecules often were of the viral type. Roberts came to realize that if there were more of these sequence-restricted enzymes, he could use them to break DNA into manageable sizes and utilize them for sequencing.

The restriction endonucleases would also be used for gene cloning. The restriction fragments could be annealed and ligated to form new recombinant DNA molecules.

Nowadays, more than 300 unique restriction enzymes have been characterized, along with several hundred isoschizomers (enzymes that recognize the same sequences as some of those). Still, by examining the DNA of the sequenced bacterial genomes, we know that many more exist. Scientists think it is likely that many thousands could be present in nature.

5) His discovery of RNA splicing—why was this important?

The result of the RNA splicing discovery has been described as an alteration of the fundamental ways in which molecular biologists view the world! The RNA splicing process involved splitting the gene into distinctive segments and splicing them together while leaving out specific sections in the mRNA message. The gene segments that were left out were called “introns” because the excluded regions were merely “intervening” within the gene. The DNA segments that included the messenger transcript and expressed into protein were named “exons.” The exons were “expressed.” Introns were left out of the gene message, and exons were left in the coding-message for expression into protein. See Figure 79.

File:Introductory figure for transcript and splicingV2.png

Figure 79. Transcription, RNA-splicing, and translation during expression of a eukaryotic gene.

The RNA splicing mechanism is significant for several fundamental reasons. First, the RNA splicing process provided a molecular-based means for organisms to undergo evolution. Living beings could alter the gene’s continuous nature to produce a new gene variant. Several introns and exon combinations could be performed, creating in effect several gene variants. The various RNA-spliced products could involve different intron-exon splicing events.

Furthermore, the resulting number of exon variants could be translated into distinct proteins, each with a new biological function. Hence, the RNA splicing system provided a means for generating a tremendous amount of gene diversity from a more limited genome content. See Figure 80. This diversity-generating system aligned nicely with the relatively fewer number of genes in the human genome.

File:Split Gene Theory figure 3.png

Figure 80. How was a gene selected from the random primordial DNA sequence?

A gene sequence on DNA can harbor a variety of stop codons. Splitting the gene up by RNA splicing can permit new genes, each with different stop codons. Thus, the alternative splicing mechanism can enable the production of the desired protein, depending on the cell’s needs.

Another significant consequence of RNA splicing was the opportunity for molecular biologists to investigate cancer and genetic diseases. Altered gene sequence combinations produced by specific RNA splicing events could lead to carcinogenesis, notably when oncoviruses transformed healthy cells. When genetic recombination processes occur during meiosis, newly formed variations could alter or inactivate genes that are critical for the biological functioning of a specialized cell or tissue or metabolic system.

A significant advance in gene cloning became possible with the RNA splicing discovery. Investigators could identify specific genes that were involved in conferring a desirable biological function. The process involved first harvesting RNA from cells undergoing their operations. This functional cellular RNA reflects the active genes that are expressed to operate. Next, a DNA version would be produced using the cellular RNA as a template. This process of making a DNA copy (cDNA) from RNA is called reverse transcription. Once the cDNA is synthesized, it can be inserted into a plasmid vector using one or two of many restriction enzymes, such as those discovered by Roberts and others. The insert DNA and cloning vector DNA are covalently connected using DNA ligase. Then, the recombinant DNA molecules are introduced into host cells, grown culture, and the desired product is extracted. The new creations can be studied more closely, used for diagnosis or chemotherapies, or used in the industrial sector. The technology would be critical for producing new biotechnology products, disease diagnosis, industry, and chemotherapies.

Similarly, a new investigative push is geared towards identifying the genetic program of expressed gene products that make needed living cells, tissues, or even organs. Suppose a patient needs a cell transfusion, a tissue graft, or even a new organ. The new technologies made available because of RNA splicing can be exploited to generate such needed living materials.

6) Richard John Roberts’ Nobel Prize was shared with Philip Allen Sharp. What happened when they were at Cold Spring Harbor, and for what were they recognized?

During a symposium in June of 1977 at Cold Springer Harbor, Richard Roberts and Phil Sharp announced their independently-derived discovery. Roberts and Sharp would win the Nobel Prize in 1993 in the physiology or medicine category for discovering that genes were interrupted. Roberts and Sharp found that such interrupted genes were, thus, discontinuous, a phenomenon that came to be known as the “split genes.”

The CSH announcement that genes were discontinuous had shocked the molecular biology community. The response to the startling discovery was skepticism. Roberts and Sharp had to provide rigorous scientific evidence supporting the new notion.

Roberts began his journey to the Nobel in 1972 at Cold Spring Harbor. Roberts had hired Richard Gelinas, whom Roberts had met while at Harvard, to join his CSH laboratory. Together, they searched the mRNA molecules of adenoviruses for signals indicating initiation and termination of transcription. Their approach had been to first sequence the beginning of a transcription start-site of an adenoviral mRNA molecule, the so-called 5’-end of the transcript. Then Gelinas and Roberts would map the initiation signal on an mRNA fragment generated by a restriction enzyme digest. Lastly, they would build on the finding by examining the mRNA fragment’s so-called upstream region to locate the promoter.

Next, Gelinas and Roberts focused on the capped 5’-ends of all mRNA molecules produced during each stage of the adenoviral replication process. It was known that during the early and late stages of viral replication, various genes were expressed, as evidenced by their different mRNA molecules. To their surprise, however, when Gelinas and Roberts examined the capped 5’-ends of early and late mRNA molecules, the ends all had the same starting nucleotide! Furthermore, they were startled to find that the starting nucleotide, with its cap, was different from the DNA code that the mRNA was based on during the early and late transcriptions.

Gelinas and Roberts reasoned that the viral genes had split during transcription of the adenoviral mRNA production. To demonstrate the viral mRNA’s reflection of a split-up gene nature, Roberts and colleagues Louise Chow and Thomas Broker conducted a definitive experiment. They used an electron microscope to visualize the broken genes directly. They mixed intact DNA templates and the mRNA molecules that were hybridized to each other. What they saw was astonishing. The genes had been split. The mRNA was attached to the DNA at two different regions along the broken gene—the work showed that the gene’s discontinuous nature would be worth a Nobel Prize for Roberts.

Phil Sharp arrived at Cold Spring Harbor as a senior staff scientist in the early 1970s, working under its director James Watson. The experimental approach that Sharp’s group invoked was inspired by a comparative analysis of the RNA present in the cell’s nucleus versus RNA in the cell’s cytoplasm. The nuclear RNA was the presumed precursor type, indicating the pre-spliced nature of the gene. The cytoplasmic RNA, on the other hand, was indicative of the spliced RNA segments. They were also inspired by the observation by David Hogness and Ron Davis that RNA-DNA hybrids were stable entities. The electron microscope could then analyze such stable complexes. Another advance that made Sharp’s work possible was creating DNA fragments using the restriction endonucleases.

Sharp worked with Sue Berget and Claire Moore, and others at CSH. Sharp and his group prepared hybrids between mRNA for the so-called hexon protein of adenovirus-2 microbe and the DNA fragment generated by a HindIII restriction digest. The result of the production of an RNA-DNA hybrid molecule. Next, the newly formed RNA-DNA complexes were studied under the electron microscope and visualized.

Because the bound RNA displaced the unbound DNA, the electron micrograph pictures revealed the presence of the so-called “R-loops.” The DNA R-loop formation indicated that the unbound DNA sections were introns, the non-expressed intervening segments of the gene. They were also quite surprised when they noticed that at the 5’-end of the hybrid was a single-stranded RNA “tail.” Among the various explanations for the presence of the RNA tails, one intriguing possibility was evident. Sharp had reasoned that the RNA tails corresponded to a different location upstream of the hexon protein-encoding mRNA sequence along the viral DNA genome.

The formation of DNA loops and RNA tails strongly indicated that the viral genes had split up into various pieces and rejoined to produce new variants. Thus, Sharp and his colleagues essentially used the same type of viruses, genomic DNA, and mRNA molecules and, independently of each other, discovered the same split-gene phenomenon as Roberts. Throughout the various reviews of the split-gene discoveries, Sharp and Roberts’ respective works are typically mentioned together, inaccurately implying that they worked together. On the contrary, they arrived at nearly the same sort of discovery that genes could be differentially spliced independently of the other.

7) Next stop—New England Biolabs—what did he investigate or study there?

Roberts joined the biotechnology firm of New England Biolabs (NEB) in 1992 as co-director of research with Ira Schildkraut. Before taking the NEB post at Beverly, Massachusetts, Roberts had already been a consultant and chair of their scientific advisory board for 12 years. During these 12 years, Roberts studied the restriction endonucleases, discovering new ones on a routine basis.

He had also been interested in determining the nucleotide sequences of viral genome fragments. Roberts and colleagues also began to map the genome of the adenovirus-2 strain, a DNA-based virus. They created a so-called restriction map of the virus’s genome. A restriction map highlights the locations on a DNA diagram where specific endonucleases bind and cleave DNA. Further, as mentioned above, when Roberts and his team attempted to find a promoter at the 5’-end of the gene’s initiation site, they discovered the split-gene nature of the adenovirus genome and RNA splicing.

In the early 1990s, Roberts acquired an interest in a group of proteins called methyltransferases. These enzymes can attach methyl groups of chemicals (CH3) to nucleotides on DNA molecules. Some bacteria are good at putting these methyl molecules on DNA base sequences of restriction sites. Methylation of restriction sites protected bacteria from DNA cleavage by their own or rouge restriction enzymes. Other methyltransferases could attach methyl groups to specific DNA sequences. For instance, the enzyme called Dam methyltransferase places a methyl group only on the A residue of a palindromic 5’-GATC-3’ sequence.

At first, Roberts and his group attempted to test the hypothesis that restriction enzymes and the methyltransferases that recognized the same DNA sequences had the same active protein chemistry sites. Unfortunately, they found no similarities in DNA sequences for the methyltransferases and restriction endonucleases. Further, they came up empty when searching for homology between their genes. Nevertheless, they found the genes encoding the methyltransferases shared homologies, suggesting they were evolutionarily related and shared a common ancestor.

Thus, Roberts, his laboratory workers, and investigators from other laboratories conducted systematic analyses for the methyltransferases. They cloned the genes encoding as many methyltransferases as they could find. Next, they determined the genes’ nucleotide sequences. They used the DNA sequence data to deduce the amino acid sequences of the various methyltransferase proteins. Next, they compared the protein sequences for these methyltransferases. They found several highly conserved amino acid sequence motifs, which were present in each of the methyltransferases. Next, they assigned functional roles for each of the distinctive conserved protein sequence motifs shared between the methyltransferases. The Roberts group participated in an elegant series of protein chemistry experiments. They swapped various peptide domains harboring specific conserved motifs to confer new functions upon the newly created hybrid proteins.

They also studied the crystal structures of the methyltransferase enzymes. One of the first of these methylating proteins was purified by Sanjay Kumar, an investigator in Roberts’ laboratory. The enzyme was a member of the “m5C-MTase” group, as it biochemically placed a methyl group on carbon number five of cytosine (called “m5C”). The enzyme methylated the second cytosine on a DNA recognition sequence 5’-GCGG-3’ to produce 5’-GmCGG-3’. The “m” signifies the added methyl group. Thus the methyltransferase was explicitly named “M.HhaI” as it was a methylator enzyme from the bacterium Haemophilus huemolyticus. Next, Kumar was able to obtain crystals of the new enzyme in December of 1991. In his 1993 Nobel address, Roberts had referred to the crystallization of m5C-MTase by Kumar as a “wonderful Christmas present.”

The M.HhaI methyltransferase structure was soon elucidated, and Roberts and his colleagues studied its properties. They discovered a crystal structure complexed between their M.HhaI methyltransferase and a cofactor called S-adenosylmethionine (AdoMet). Then, in an astonishing feat of molecular biology, the Roberts laboratory managed to isolate and deduce structures of M.HhaI- AdoMet complexes bound to DNA! Soon they found contact sites between their M.HhaI methyltransferase and DNA. They discovered that DNA bound to M.HhaI methyltransferase at its conserved amino acids sequence motifs! Not to be equally unremarkable, Roberts and his group also found the contact sites between the conserved amino acids of AdoMet and its specific DNA recognition sequence! From there, they worked out the biochemical mechanisms for the methylation of cytosine on DNA. It was all remarkable work!

8) Is Roberts still alive and contributing to science?

As of this writing, Roberts is 77 years old. He is quite active. In 2015, Roberts gave a speech entitled “A Crime against Humanity.” See Figure 81. He spoke of the detrimental effects of anti-genetically modified organisms for food production (anti-GMO) activists. He related the false claims made by anti-GMO activists, which seek to ban genetically modified foods. Such foods were developed to enhance the crop yield and reduce hunger, starvation, and nutritional deficiency diseases in underdeveloped regions. He spoke of the safety of GMOs and their crops as foods. In this book, a link to Roberts’ speech is provided at the end of this chapter.

The safety and efficacy of GMOs and their food have been definitively established, with many years of supporting scientific evidence. Anti-GMO activists have failed to realize that for millennia humanity has already participated in genetically modifying farm crops and animals to improve yield and nutritional status. Such activists also fail to understand that GMOs and their foods are modified with only minor alterations in their genomes. In these cases, perhaps only one gene or one base nucleotide substitution is enough. The genomic changes in GMOs are minor.

On the other hand, for eons, humanity has been doing much the same sorts of genetic modification with traditional plant and farm animal breeding. However, these conventional breeding and farming practices proceed except with major changes in DNA. Such large DNA changed occur with naturally occurring recombination events that transpire during natural meiosis and natural sex cell production. The genomic changes in conventionally cultivated crops and farm food animals are major.

File:Richard Roberts at GYSS 21Jan2016 1.jpg

Figure 81. Sir Richard Roberts was giving a talk, “A Crime against Humanity” at the Global Young Scientists Summit (GYSS@one-north, Singapore, 17-22 January 2016).

9) What have I neglected to ask?

In addition to the 1993 Nobel Prize in medicine or physiology, Roberts received the FRS (1995), EMBO Membership (1995), and Knight Bachelor (2008). Roberts and his wife Jean have four children, Alison, Andrew, Christopher, and Amanda.

For further information regarding this astonishing molecular biologist, visit:

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