An Interview with Ann and Manuel Varela: Paul Berg and Recombinant DNA.

Oct 2, 2020 by

Dr. Paul Berg

Michael F. Shaughnessy –

1) Paul Berg—a key figure in genetics and recombinant DNA—where was he born, and where did he go to school?

Dr. Paul Berg is a 1980 chemistry Nobel Laureate who is well known for developing the splicing techniques used to make recombinant DNA possible. Berg is also credited for finding Francis Crick’s “adaptor” molecule, now known as transfer RNA (tRNA), a molecule that Berg found was specific for the amino acid methionine.

Paul Berg was an American born in Brooklyn, New York, on June 30, 1926. His parents were Russian, Jewish immigrants. Berg’s father, Harry, was a clothing manufacturer, and his mother, Sarah Brodsky, was a homemaker. Berg had two younger brothers. From an early age, Berg was interested in biology. He was inspired to follow a research career after reading Sinclair Lewis’s Arrowsmith and Paul de Kruif’s The Microbe Hunters in junior high school.

Berg passed over a grade in elementary school and started high school at the age of 14. While in high school, Berg participated in the Science Club, sponsored by Sophie Wolfe, who proved to be motivational and inspirational in Berg’s interest in science. He earned his high school diploma in 1943 from Abraham Lincoln High School.

As soon as he turned 17, Berg enlisted in the Navy because he was excited to join the war effort. Although flight school was his main interest in the military, he was ultimately recruited for ship duty. His commission was on submarine chaser ships, where he served until 1946. Some of these missions placed Berg in the Atlantic Ocean and the Caribbean Sea. Fortunately, World War II ended before he saw combat, but Berg assisted with escorting American naval ships in the Pacific Ocean to the United States.

Berg earned a B.S. degree in biochemistry from Penn State University in 1948. He took his Ph.D. in biochemistry from Case Western Reserve University in Cleveland, Ohio, in 1952.

2) Berg’s early work was in Copenhagen, Denmark, and several other places (University of Washington) studying cancer. Did he come up with any new insights during this period?

Once his graduate studies were completed, Berg was accepted as a postdoctoral fellow at the American Cancer Society. This work took him to the Institute of Cytophysiology in Copenhagen, Denmark, and the Washington University School of Medicine from 1952-1954.

As a postdoctoral research scientist studying in the laboratory of Herman Kalckar at Copenhagen, Berg teamed up with another new postdoc named Dr. Wolfgang (nicknamed “Bill”) Joklik, who was fresh from obtaining his Ph.D. at Oxford University. Joklik and Berg became acquainted at an afternoon tea gathering, which Kalckar regularly held for his lab members. Based on an idea they formulated during one of these afternoon teas, they decided to test it in the lab.

The new project required a large quantity of radioactive ATP (adenosine triphosphate), but it was too expensive. Therefore, Berg and Joklik took it upon themselves to make their own in the lab. So, they placed a laboratory rabbit in a tank of water and forced it to swim to exhaustion, almost drowning the animal in the interim! The purpose was to deplete the animal of its ATP, which it would need to produce from scratch. If the animal could use radioactive phosphate, the rabbit would incorporate it into newly make ATP. Therefore, Berg and Joklik injected a large dose of radioactive phosphate, labeled with phosphorous-32 (32P), hoping to obtain rabbit-based 32P-ATP. The effort worked. Berg and Joklik were able to extract a suitable amount of radioactively-labeled ATP to test their new idea.

Unfortunately, the original idea that led Berg and Joklik to make their own radioactive ATP is unclear. Nevertheless, the preliminary work between Joklik and Berg in Kalckar’s lab led to discovering a crucial new enzyme by accident! They named their new enzyme nucleoside diphosphokinase, which they colloquially nicknamed “Nudiki.” The new enzyme catalyzes phosphate transfer from ATP to deoxyribonucleoside diphosphates or ribonucleoside diphosphates. One of the reasons for the enzyme’s relevance is that it seems to be a universal protein in all life types, from bacteria to humans. Another reason that their enzyme was important was that it served to produce building blocks to synthesize proteins, co-enzymes, RNA, and DNA. The two young postdoctoral fellows published their findings in two classic papers, one in Nature, in 1953, and the other in the Journal of Biological Chemistry, in 1954.

Berg also studied cancer research with Arthur Kornberg in 1954 in the Department of Microbiology at the School of Medicine connected to Washington University in St. Louis, Missouri. Berg and Kornberg studied a problem concerning the synthesis of acetyl-substituted Co-enzyme A (acetyl-CoA), a necessary intermediary product in the central metabolic process that breaks down food to produce usable energy. See Figure 13.

Berg conducted a new project after he arrived at St. Louis in Kornberg’s laboratory. Berg set out to purify the “enzyme-CoA” complex. The famous Fritz Lipmann and his colleague Feodor Lynen had speculated in 1953 that such a molecule was an intermediate in acetyl-CoA production. Thus, the enzyme-CoA complex was thought to exist in living cells, such as those of yeast. The putative enzyme would be named acetyl-CoA synthetase, and Berg hoped to isolate it when connected to CoA.

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Figure 13. Co-enzyme A structure.

An interesting side story arises out of this period. Kornberg had invited Dr. Edward Korn to join them in his lab, but Korn turned down the offer. In later years, Kornberg would lament that had Korn accepted the offer and collaborated with Berg; they could have published a paper together, written by “Korn, Berg, and Kornberg!”

In any case, Berg set out to study and purify the so-called enzyme-bound nucleotidyl complexes that might exist in living cells. Almost immediately after starting the project, Berg obtained a sample of purified protein from cultured yeast cells. To Berg’s surprise, the enzyme prep failed to perform the biochemical step predicted by Lipmann and Lynen. Therefore, Berg began artificially adding substances that he hoped might trigger the enzyme prep to conduct the desired biochemical reaction. He found that pure acetate was required. The result predicted that an intermediate called acetyl adenylate was involved in the process. If the prediction was accurate, it further predicted that inorganic pyrophosphate (PPi) would be released. Therefore, Berg worked to isolate the requisite acetyl adenylate, and he used pure biochemical to activate his enzyme preparation. The exploration worked.

Berg found that the enzyme plus the acetyl adenylate readily converted ATP and acetate to make the PPi, and eventually, acetyl-CoA was produced! Thus, Berg discovered that the synthesis occurred in a two-step biochemical process, rather than the complicated three-step reactions predicted by Lipmann and Lynen. The new contribution to biochemistry by Berg was as follows. First, ATP plus acetate made acetyl adenylate and PPi. Then CoA plus the acetyl adenylate (from step one) produced the desired acetyl-CoA, releasing AMP.

Berg published the new work as two articles in the Journal of Biological Chemistry, both in 1956. Berg had discovered the important enzyme, now known as acetyl CoA synthetase. Berg would later relate that this work was one of the most gratifying discoveries he ever made, even more than the discovery to earn him the Nobel.

Berg built on the success by discovering the acetyl CoA synthetase, which invoked the exchange between PPi and ATP. Therefore, he and his new postdoctoral fellow Fred Bergman concentrated his studies on other biochemical processes that used the same sort of factor exchanges. Soon they discovered that amino acid metabolism involved the ATP and PPi exchange mechanisms. Berg deduced that the amino acid-based reactions likewise involved an intermediate enzyme bound to aminoacyl-adenylate, similar to what he had seen with his successful discovery of the acetyl-CoA synthetase. Furthermore, because CoA served as a sort of acceptor molecule in the synthesis of Acetyl-CoA, then, as Berg brilliantly reasoned, perhaps amino acid biochemistry likewise involved such an acceptor-like molecule. That is, Berg hypothesized the presence of a so-called aminoacyl-accepting molecule. Thus, Berg began the tasks of purifying each of the enzymes involved in amino acid biochemistry. They focused on the metabolism of methionine first. To the surprise of Bergman and Berg, the purified acceptor molecule turned out to be RNA!

Unbeknownst to Berg, however, Francis Crick had previously formulated the notion of an “adaptor” molecule to serve as a conduit between amino acids and RNA, which later turned out to be transfer-RNA. Berg had discovered a tRNA specific for an amino acid!

The discoveries involving acetyl-CoA synthesis and the tRNA adaptor molecule would solidify Berg’s career. From 1955 to 1959, Berg was an assistant professor of microbiology at Washington University until his departure for California to follow Kornberg.

3) Stanford University was lucky enough to have Berg teach biochemistry from 1959—to the year 2000—no easy feat. What did he accomplish during his time there?

In the latter part of 1959, Berg relocated to Stanford University, along with Kornberg and most of the Washington University department, to teach biochemistry and was appointed chair of the biochemistry department from 1969 to 1974. In his Stanford lab, Berg began to focus his studies on molecular biology, specifically mammalian cells: how genes act and proteins are made.

At Stanford, Berg would now build on his successes with the biochemistry of amino acids. They would inspire an interest in the synthesis of proteins. His early work involved purifying enzymes that played roles in producing messenger RNA (mRNA) and the mechanism by which aminoacyl-RNA molecules participated in making proteins. He also concentrated on searching for the enzymes called aminoacyl tRNA synthetases that produced amino acid-specific tRNA molecules. He further purified one of the first enzymes connected to their dedicated aminoacyl adenylates!

Postdoctoral fellow Dr. Anne Norris Baldwin had performed the work in Berg’s laboratory. Baldwin had isolated a tRNA synthetase bound to the amino acid isoleucine. Baldwin and Berg went on to show that ATP and PPi were involved in the biochemistry. Baldwin further demonstrated that particular tRNA molecules were dedicated to specific amino acids. A remarkable series of studies proved useful to the study of protein synthesis and its proofreading mechanisms to ensure sequence fidelity. Berg was on the brink of exploiting these new advances to solve the genetic code and was prepared to make a big announcement that his laboratory was set to do so. Then another announcement was made at a scientific conference held in Russia that Marshall Nirenberg at NIH was already well on the way to the genetic code solution. It was a disappointment to Berg.

Berg also collaborated with another renowned biochemist and tennis partner, Professor Charles Yanofsky. In the Biological Sciences department at Stanford, Yanofsky was interested in suppressing enzymes that participated in the amino acid tryptophan’s metabolism. Yanofsky had discovered bacterial mutants that overcame this genetic suppression. The collaboration was serenely productive, and the laboratories of Berg and Yanofsky published several significant scientific articles together.

Berg became Willson professor in 1970 and director of the Beckman Center for Molecular and Genetic Medicine in 1985. Berg remained at Stanford University teaching biochemistry until 2000.

4) Gene splicing and modern genetic engineering. What were his contributions?

In 1972, Paul Berg was the first human being artificially to combine DNA from a bacterium with a virus’s DNA! He had recombined two foreign DNA pieces together, from entirely different origins, creating a so-called hybrid DNA molecule, known today as a recombinant.

Berg had been inspired in 1965 to conduct this historic scientific experiment, recombining different DNA molecules, by a colleague named Dale Kaiser. Kaiser had pointed to similarities between lysogeny of a bacterium by phage virus and that of mammalian cells transformed by a cancer-causing virus called SV40. The term SV40 stood for Simian vacuolating virus 40 or simply Simian virus 40. Genetic recombination, as a natural phenomenon, had been known in the field for a long time. However, joining two DNA pieces from completely different organisms in the laboratory was another situation entirely. It had never been done before. Berg would be the first.

The original purpose for Berg was to find evidence of the same sort of lysogenic-type relationship between phages and bacteria in the cancer-causing SV40 virus and eukaryotic host cells. He hoped to find just such a hybrid between a virus and a mammal cell that occurred naturally. After Berg conducted a quick theoretical calculation, he realized that the chances of finding such a naturally occurring viral-mammalian hybrid were exceptionally rare. Success would be elusive, if not impossible.

Thus, Berg wondered instead if he could construct in the laboratory a DNA hybrid himself. He would have to start with simple living systems, with organisms that were not as complicated as mammalian cells. He turned to bacteria, and the bacterium of choice was the famous Escherichia coli. The bacteriophage lambda (λ phage) would be the infecting virus of choice. Berg set out to determine whether SV40 genes could be expressed in bacteria and whether bacterial genes could be harbored successfully in a mammalian cell.

Berg used a plasmid DNA molecule called “λdvgal,” which harbored λ phage genome plus Escherichia coli genes encoding proteins that permitted galactose metabolism. Berg, working with postdoc fellow David Jackson and Robert Symons, a visiting scientist, set out to create the first recombinant DNA molecule, a hybrid between SV40 and λ phage DNA harboring bacterial galactose-utilizing genes, a construct they would call “SV40λdvgal.”

The first stage in becoming the basis for Berg’s Nobel Prize was to cut the plasmid (λdvgal) DNA and the SV40 DNA molecules. Both types of DNA (phage-bacterial plasmid and mammalian virus DNA) were circular. Thus, they cut both DNA molecules with a restriction endonuclease called EcoRI, which cleaves DNA at certain sites restricted to a specific DNA sequence. Luckily for Berg and his colleagues, there would be only one such restriction site on each of their two different DNA molecules.

Unbeknown to Berg and his laboratory workers, the ends of the DNA molecules generated by the EcoRI enzymes could readily anneal (come together) to each other with minimal coaxing. They had nevertheless coaxed the DNA ends to connect by producing complementary ends on purpose. They used an enzyme called λ exonuclease to cleave a few bases off their two DNA molecules’ ends. Then the investigators added short strings adenines to one of the DNA molecules (the SV40 DNA) and complementary strings of thymines to the other (i.e., plasmid λdvgal DNA).

Next, Berg and colleagues mixed the two linearized and base-modified DNA molecules, the SV40 and λdvgal plasmid DNAs. The mixture of the two different molecules was expected to anneal to each other, as they should have been complementary—they were—and they annealed quite readily!

Next, Berg and co-workers added purified DNA polymerase plus a mixture of all four deoxynucleotide bases (G, A, T, and C), to fill in any gaps left behind by the previous biochemical manipulations of the DNA. The DNA polymerase and bases permitted DNA synthesis to occur; the process would complete the gap-filling process.

Lastly, Berg and his laboratory incubated the cut, modified, mixed, annealed, and filled DNA hybrids with a phage enzyme called DNA ligase. The enzyme permitted the hybrid DNA to make a covalent phosphodiester bond between completely different DNA molecules! Thus, they had, for the first time in scientific history, successfully recombined DNA from two species of organisms, a mammalian-based virus DNA and a phage-bacterium-based DNA! They published the work in 1972. Figure 14 denotes the cloning process using plasmid vectors and reporter genes.

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Figure 14. Recombinant DNA Molecule.

It was a remarkable but controversial discovery. Berg and his group were immediately criticized for having spliced together two foreign pieces of DNA. No one knew what a new combination would result in that could be destructive to human health. After all, they had combined a cancer-causing viral genome to a bacterium that could reside in the human gut. Perhaps they had created new cancer-causing contagion! It was a serious concern. Michael Crichton’s new 1969 novel The Andromeda Strain had spoken of a deadly fictional alien microbe that wreaked havoc on the human population.

In the ensuing years, Berg and others would be embroiled in the recombinant DNA controversy. After many scientific conferences, culminating in the famous Asilomar Conference, which prominent molecular biologists of the day, Berg included, formulated specific guidelines for the laboratory containment of any new recombinant DNA molecules and organisms which might harbor them. The controversy eventually diminished with time, and Berg’s contributions to the scientific community towards igniting the field of DNA technology would be recognized with the Nobel in 1980 for chemistry, alongside Walter Gilbert and Fred Sanger.

5) Apparently, Berg was also a writer and crafted a book about George Beadle—why is this important?

In 2003, Paul Berg collaborated with his longtime friend and colleague Dr. Maxine Singer to publish a compelling biography about the inimitable George Beadle, entitled, “George Beadle, an Uncommon Farmer: the Emergence of Genetics in the 20th Century.” The book would become a classic, read by many a young molecular biologist, biochemist, or geneticist. Singer and Berg wrote the book because they realized students in their genetics and biochemistry courses were astonishingly ignorant of the scientific pioneers and their experiments. To its readers, the resulting book would become an endearing classic in the scientific literature.

Singer and Berg were deeply impressed with the enormity of Beadle’s work, especially of the ramifications of his work towards the development of genetics in the context of the 20th century. The authors astutely realized that Beadle’s work changed the course of scientific history profoundly. Berg and Singer were also impressed by Beadle’s enormous challenges, both personally and scientifically. They delineated how Beadle overcame such enormous challenges.

It is a deeply inspiring biography, for young and old readers alike. Berg and Singer were convincingly able to convey how deeply gratifying it could be to go into a laboratory and conduct a scientific investigation. Beadle’s example was but one of these prime examples. Scientific investigators who spend a lifetime devoted to similar activities can relate to the same type of rewards inherent in knowing they made the world somehow a little bit better for all.

6) “Dissections and reconstructions of genes and chromosomes” was, I believe, the title of his speech upon receiving the Nobel Prize. Why is Berg’s Nobel Prize lecture important in the larger scheme of things?

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Figure 15. Queen Beatrix receives Nobel laureates: Woman, man, Mildred Levy, Paul Berg, Nicolaas Bloembergen, woman, Christian de Duve, man, Louise Weinberg, Steven Weinberg, Queen Beatrix, man, woman, Manfred Eigen, man, woman.

In an autobiographical article, published in 2008, in the Annual Review of Biochemistry, Berg eloquently described his Nobel experience. He noted the 5:00 a.m. phone call, the jubilant celebrations with Stanford friends, colleagues, students, the Nobel award ceremonies, their Nobel lectures, the post-award parties, and the Stockholm sightseeing afterward. Fortuitously, the text of the Nobel Laureates’ speeches is routinely published for interested readers who cannot make the trip. Figure 15 shows some Nobel Laureates meeting Queen Beatrix.

In Stockholm, Berg’s Nobel Prize lecture, delivered on the evening of December 8, 1980, provided the definitive background leading to the eventual experiments described above. He described his remarkable findings. Berg’s discovery that pieces of DNA molecules from entirely different organisms could be artificially spliced together would form the basis of modern recombinant DNA technology. Berg’s new experiments and findings would revolutionize science and change the world forever.

Using the new technology, the cloning of genes became possible—and routine. Hence, one could now isolate a gene that encodes a specific function, a trait, a process, etc. They could study the biological activities of the gene product in detail. Genetic diseases could be studied for diagnosis, or perhaps therapy, if not for elucidating disease pathology in cell biology and biochemistry terms. One may use early DNA technology to correct defective genes or edit genomes using CRISPR in modern times. One could also focus on entire genomes of disparate organisms and compare them. In short, the scientific contribution of Paul Berg will benefit humanity for eons.

7) Berg made the transition from classical biochemistry to molecular biology, focusing specifically on how genes act and how proteins are made. Why is this transition important in the big scheme of things, or at least in his life?

Indeed, many biochemists (and investigators from all biology fields) made the transition to molecular biology. The molecular biological approach to studying all aspects of living beings’ biology and chemistry is of enormous value for humankind. The phenomenon is called “going molecular,” and it involves cloning new genes from all types of living organisms and perpetuating them in new host cells in culture. The molecular biological approach permits one to focus on genes. One can study how genes are expressed in RNA and protein. One may study more closely how gene structure is related to those of other genes from distinct organisms. A molecular biologist can analyze how gene expression is regulated.

The purposes of gene cloning are many. For instance, an investigator may determine the cloned genes’ base sequences, revealing their genetic codes. With such sequence codes in hand, one can deduce the string of amino acid sequences along a protein molecule. Further, one can determine how much amino acid strings fold into a three-dimensional structure, which can lend insight into the proteins’ biochemical natures. With this molecular structural information known, one can study how the proteins function in precise detail.

Using molecular cloning technology, one can generate mutations in the cloned genes, and the resulting mutations can be expressed in the proteins encoded by the altered genes. The effects on the function of the mutants versus the natural codes can be compared. The knowledge base of biochemistry greatly expanded to include many thousands of proteins and their variants.

The pioneering studies of Berg also led to the development of genomics. The close study of genomes and their relationship to each other became possible. Finally, as mentioned above, genome editing technology CRISPR has recently become possible. The genome-editing method may be useful for studying individual genes within the context of an intact organism, or perhaps even for the correction of a defective gene in a person suffering from the detrimental effects of a genetic ailment.

8) Walter Gilbert and Frederick Sanger shared the Nobel Prize with Paul Berg. What exactly did each contribute, or for what were they known?

As mentioned above, Paul Berg shared the Nobel Prize in chemistry in 1980 with Fred Sanger and Wally Gilbert for their scientific contributions to molecular biology and, in particular, for their work with recombinant DNA. Paul Berg’s contribution led to the invention of recombinant DNA technology, to clone genes at the molecular level, by incorporating genes into cloning vectors and producing many gene copies. The gene cloning permitted close molecular analysis of gene structure.

A Harvard faculty, Walter Gilbert earned the Nobel Prize for his invention of the sequencing of DNA. The new chemical-based sequencing method by Gilbert and his collaborator Allan Maxam allowed one to elucidate the nucleotide sequences along the length of DNA chains. The approach was known as the Maxam-Gilbert method of DNA sequencing. While molecular scientists throughout the ensuing years did not uniformly use the Maxam-Gilbert technique, it would prove beneficial towards sequencing key genes and living organisms for the first time in history.

Frederick Sanger, an investigator at the MRC Laboratory of Molecular Biology, took the Nobel for his invention regarding the sequencing of DNA in England. The Sanger method was based on the so-called chain termination method, which proved to be less cumbersome than the Maxam-Gilbert approach. The 1980 Nobel for Sanger was his second—the first Nobel, also in chemistry, went to Sanger in 1958 for his invention towards solving the sequences of amino acids along chains of proteins. While the protein sequence determination technique was rather cumbersome, Sanger’s DNA sequencing technique would ultimately lead to the human genome sequence being determined close to its final form in 2003.

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

Berg published more than 200 scientific articles. He and Maxine Singer wrote several books for non-scientists about genetics. Berg has been widely celebrated with accolades. Among these include Nobel Prize in Chemistry (1980) for his work on protein synthesis and recombinant DNA. He also took the AAAS Award for Scientific Freedom and Responsibility (1982), the National Medal of Science (1983), election to the National Academy of Sciences in 1966, election to the American Philosophical Society in 1983, and election the Royal Society in 1992. Berg was bestowed with four honorary doctorates.

He was an extraordinary teacher, and he received Stanford University School of Medicine’s Henry J. Kaiser award in 1969 and again in 1973. He received the American Chemical Society’s Eli Lilly Prize in biochemistry (1959), the V. D. Mattia Award of the Roche Institute of Molecular Biology (1972), the Albert Lasker Basic Medical Research Award (1980), a precursor to the Nobel. Berg has been a fellow of the American Academy of Arts and Sciences and a Foreign Member of the Japanese Biochemistry Society and the Académie des Sciences, France.

For further information on the extraordinary molecular biologist, the reader may visit:

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