Herbert Boyer: Who was he, and what did he have to do with Molecular Biology? An Interview with Manuel Varela and Ann Varela.

Oct 11, 2020 by

Herbert Boyer

Michael F. Shaughnessy –

1) Herb Boyer—born in Derry, Pennsylvania (not Maine), apparently played football and had some early interests in becoming a doctor. What happened?

Herbert Wayne Boyer was born on the tenth of July in 1936, in Derry, Pennsylvania. Young Boyer was not very interested in academic pursuits while in high school. Boyer was interested in football, basketball, and baseball. Fortunately, Boyer’s high school coach was instrumental in his decision to become a medical doctor, but after taking biology and chemistry in college, Boyer set out to become a researcher. He was determined not to work for the railroad as his father and grandfathers did. His mother had a high school diploma.

Boyer earned his bachelor’s degree in 1958 from Saint Vincent College in Latrobe, Pennsylvania, in biology and chemistry. Boyer married Marigrace Hensler in 1959. In 1963, Boyer took his Ph.D. from the University of Pittsburgh. For three years, Boyer’s post-graduate work was in progress at Yale University in the laboratories of Professors Edward Adelberg and Bruce Carlton.

The University of California, San Francisco, is where he became an assistant professor and a Biochemistry Professor from 1966 to 1991. He learned that genes from bacteria and eukaryotes could be joined. Collaborating with Stanley Cohen, Boyer validated the possibility of producing recombinant DNA in bacteria in 1973. They began their investigation by combining a gene for frog ribosomal RNA with a bacterial plasmid. Next, the combination was then mixed with a strain of Escherichia coli for expression. Based on this procedure, Boyer helped found Genentech in 1976 in collaboration with Robert Swanson.

Boyer was also involved with the Howard Hughes Medical Institute from 1976-1983 as an investigator. In 1977, Boyer’s laboratory and colleagues Keiichi Itakura and Arthur Riggs described the first-ever synthesis and expression of a peptide-coding gene at City of Hope National Medical Center. He produced synthetic insulin in August 1978, by using his new transgenic genetically modified bacteria. Later on, in 1979, he developed a growth hormone. Boyer founded Genentech with entrepreneur Robert A. Swanson in 1976. Boyer served as Vice President of Genentech from 1976 until his retirement in 1991. Genentech’s methodology to the first synthesis of insulin was more accepted than Walter Gilbert’s Biogen approach, which used whole genes from natural sources. Boyer built his gene from its specific nucleotides.

2) Boyer found some methodology to prompt bacteria into producing foreign proteins. The method was linked to “jump-starting” the entire field of genetic engineering. How does all this work?

In 1976, Boyer would help establish one of history’s first biotechnology firms, Genentech, to achieve mass protein production using bacteria cultures. Biotechnology’s purpose was to exploit biological systems for mass production, in a factory-like manner, of products useful to biomedical research and medicine. One of the first foreign proteins to be produced by Boyer’s biotech company included the so-called restriction endonucleases. The corporation would later be geared for producing synthetic insulin to treat human diabetics or the production of therapeutic growth hormones.

The technology was molecular-based, and it focused on producing desired proteins that were difficult to purify by conventional biochemical means. Traditional biochemical techniques involved brute force methods that required massive quantities of animal tissue or human cadaveric material. These biochemical methods were sometimes fraught with failure to develop suitable purification protocols or produced minute quantities that may have been inadequate for use. In many other cases, the desired protein proved impossible to extract from animal or human tissues. Boyer’s molecular biological approach circumvented many of these types of problems encountered during the biochemical extraction of desired proteins.

Biotechnology’s genetic engineering processes include finding the desired genes within the living organisms that harbor them. Using one of Boyer’s discoveries, restriction endonucleases, the target gene will be excised from the organism’s genome of origin. Then, the DNA fragment generated by the restriction digests containing the desired gene will be mixed with a plasmid molecule’s DNA, sometimes called a “cloning vector,” which has also been subjected to restriction endonuclease cleavage. The ends of the two different molecules will have single-stranded DNA regions compatible with each other in their nucleotide base-pairing properties and anneal, meaning to join up. Next, the annealed DNA fragments, one fragment containing the desired gene and the other containing the cloning vector (plasmid), are covalently bonded together by ligase enzymes.

Once the ligation procedure has been completed, the newly formed creation is referred to as a recombinant DNA molecule. The recombinant DNA is utilized to transform a microbial host, usually, Escherichia coli, as was first used in Boyer’s day or other cellular hosts, such as yeast, insects, or cultured monkey cells, or even human cells that are grown in culture. The host cells containing the recombinant DNA molecules will use their cellular machinery to express the desired gene to produce mRNA by transcription and then make protein by translation.

The cultured host cells harboring the recombinant DNA will express their cloned gene products, i.e., the desired protein. Then the biotechnologists can proceed with the extraction of the proteins from the cultured cells. The purified proteins can be checked for purity and function. The protein products can then be packaged, prescribed, and used to conduct biomedical research and serve as chemotherapy for a medical ailment.

3) Early on, Boyer became interested in Escherichia coli. Where did this lead, and why is it important?

In the mid-1960s and early 1970s, Boyer discovered a famous restriction endonuclease enzyme called EcoRIfrom the well-known Escherichia coli bacterium. The discovery would lead to revolutionary advances in molecular biology, genetics, and molecular biology.

The restriction enzymes bind to DNA at sites containing specific nucleotide sequences and then cut the DNA at staggered locations within the restriction sequence. Thus, the restriction enzymes are restricted to cutting DNA at their particular sites on the DNA. The DNA cleavage products have ends containing exposed bases that can anneal to back to each other, or other similar DNA ends. Hence, the restriction enzyme cutting sites are often called “sticky ends.”

Boyer’s discovery was spurred by his interest in analyzing the interactive sites between DNA and proteins that bound it. Such DNA-binding proteins had fascinated Boyer. To study these sorts of DNA-specific proteins, Boyer needed to know more about enzyme proteins that targeted DNA and carried out chemistry on it, perhaps modifying DNA in some way. Then, Boyer had become aware of discoveries that dealt with bacterial antibiotic resistance.

In 1966 Boyer learned from the scientific literature about research on phage infection of certain antibiotic-resistant bacteria by investigators Toshiya Takano, Tsutomu Watanabe, and Toshio Fukasawa, from Keio University, in Tokyo, Japan. The articles stated that these bacteria carried drug resistance factors (called R-factors), which were enzymes for phage-specific DNA and chemical modification. In essence, Takano, Watanabe, and Fukasawa had discovered the phenomenon of plasmid-borne antimicrobial resistance in bacteria. The drug-resistant bacteria held the types of proteins that Boyer needed to study DNA-protein interactions. Hence, Boyer had to get ahold of drug-resistant bacteria. These resistant bacteria had the enzymes that Boyer needed. Boyer decided to look at DNA-binding and DNA-modifying enzymes by examining bacteria with antibiotic resistance.

The only source of such antibiotic-resistant bacteria was the healthcare institution with pathogenic specimens—a hospital—and the nearest such institution associated with the University of California, San Francisco. Fortunately, Boyer had a graduate student in his lab, Robert Yoshimori, who had clinical medical microbiology training. Yoshimori was dispatched to the hospital’s clinical laboratory, where they collected and identified bacterial isolates from patients with infectious diseases. He brought many clinical isolates back to the Boyer laboratory.

From 214 of these clinical isolates, a total of 33 so-called “R-factors” were recovered by Boyer and Yoshimori. One of these clinical isolates, a bacterium of the Escherichia coli species, had originated from a female patient with a urinary tract infection. The Escherichia coli strain proved to be resistant to multiple antibiotics, making it almost untreatable. They found that the pathogen contained a plasmid DNA molecule harboring a new R-factor, called a Type I restriction endonuclease. This new restriction endonuclease would later be call EcoRI because it had been found in an Escherichia coli plasmid, and it was of the Type I designation. A second factor was found, Type II, and Boyer and Yoshimori called it EcoRII restriction endonuclease.

Additional R-factors, Types II and III, would be found by Hamilton (Ham) Smith at Johns Hopkins Medical School. From clinical pathogens of Hemophilus influenzae of the “d serotype,” restriction endonucleases would be purified and called HindII and HindIII. Smith and his colleagues Dan Nathans and Werner Arber would garner a Nobel Prize in 1978 for their discovery of the HindII and HindIII restriction enzymes.

Boyer would discover that the EcoRI restriction enzyme cleaved the covalent bond holding guanine and adenine together in the specific G-AATTC sequence along a double-stranded molecule of DNA. Likewise, the complement sequence of GAATTC, denoted as CTTAAG, on the other strand would be cleaved at the A-G bond, e.g., CTTAA-G. See Figure 19 for the restriction sequence recognized by the EcoRI DNA-binding protein. The arrows indicate the cut sites of EcoRI endonuclease.

Figure 19. DNA sequence for EcoRI restriction enzyme cleavage sites.

The digest of a double-stranded DNA molecule by the EcoRI restriction enzyme would be a staggered cleavage. The staggered DNA cut would generate single-stranded regions with exposed bases that could readily anneal with other DNA pieces that had complementary base sequences. The discovery of these and other restriction enzymes would, in short order, revolutionize the history of molecular biology.

4) Stanley Norman Cohen and plasmids—First, who was Stanley N. Cohen? Why are plasmids important, and even more importantly, what are they?

As you will learn in the next chapter of this book, Stanley N. Cohen, an expert on plasmid DNA molecules, would work with Herbert Boyer to artificially construct recombinant DNA molecules, clone them into a plasmid vector, and express the recombinant proteins in bacteria. Cohen and Boyer would set the stage for the emergence of molecular cloning of genes. The gene-cloning method ushered innovation to biology and biochemistry. The technique improved immunology, physiology, cell biology, genetics, biomedicine, and biotechnology.

Plasmids are short stretches of double-stranded DNA molecules. Some plasmids harbor so-called resistance factors or other genetic elements that encode proteins that confer useful survival properties for microorganisms that have them. The genes carried on naturally occurring plasmids might encode antimicrobial resistance, fermentative properties, or even virulence factors for mediating pathogenesis and disease.

In late 1972, Boyer and Cohen would meet for the first time at a scientific conference in Hawaii, and their meeting would change the course of history. The meeting was devoted to plasmid biology research. Some details are unclear regarding the timing of certain events that transpired during the session. However, the consensus is that Boyer and Cohen suddenly realized that DNA molecules from different species could be recombined to create a novel chimera!

All of the clues that led to their historic realization seem to have presented themselves at the conference. One clue was Cohen’s methodology of plasmid extraction from bacteria and his possession of plasmids with reportable resistance to antibiotics, such as tetracycline or penicillin. Another clue was the idea that an antibiotic gene might be transferred to another plasmid. Boyer and Cohen wondered if such a gene transfer was possible. A third clue was the notion that bacterial resistance genes might serve as “reporters,” signaling their genetic presence by conferring antibiotic resistance upon bacteria that harbored them. Another clue was the discovery of Boyer’s restriction enzymes might be used to cut and anneal two DNA molecules together. The annealed molecules could be ligated using a phage T4 DNA ligase enzyme. One last clue was the observation that one restriction site on DNA could linearize a circular plasmid, and a foreign piece of DNA with two restriction ends on each side could be inserted into the one restriction site on the plasmid.

In theory, Boyer and Cohen realized that they could recombine DNA from two separate plasmids from different bacteria and propagate the hybrid DNA without the need for phages. They needed the right plasmids, though.

The plasmid candidates would soon become world-famous. Each plasmid was found in different bacteria. One of these plasmids was called R6-5. The “R” referred to the fact that the DNA carried an antibiotic resistance reporter factor. In bacterial hosts, the R6-5 plasmid conferred resistance to the antibiotics called kanamycin and chloramphenicol but not tetracycline.

The other plasmid previously referred to as “Tc6-5,” was later renamed to pSC101. The “p” label means that the DNA molecule is a plasmid. The “SC” in the term pSC101stood for Stanley Cohen, its discoverer, and the number “101” refers to the laboratory designation of the DNA sample. Intriguingly, Boyer at UCSF and Cohen at Stanford would commute to each other’s respective laboratories by car on Highway 101. The pSC101 plasmid DNA carried a tetracycline resistance gene. Thus, any cell that contained this plasmid within it would survive exposure to the antibiotic. However, the beauty of the pSC101 plasmid was it possessed only one EcoRI restriction enzyme digestion site! The plasmid could, therefore, accept foreign DNA if it harbored complementary restriction ends. The proposed gene cloning method was a promising idea.

The molecular cloning work began in earnest. Boyer, Cohen, and co-workers Anne C.Y. Chang and Robert Helling purified the two plasmids after propagating them in bacterial culture. Plasmid handling was Cohen’s contribution to the study. They also isolated the necessary restriction endonucleases. Supplying these DNA sequence-restricted modifying enzymes was the contribution of Boyer.

Next, the investigators “restriction digested” the two different DNA plasmids, using the purified EcoRI endonuclease. They separated the resulting DNA “restriction fragments” using gel electrophoresis. The EcoRI digest of pSC101 generated only one DNA fragment, confirming that the plasmid had only one EcoRI restriction site. The R6-5 plasmid, on the other hand, generated eight DNA fragments with EcoRI digestion. Next, the investigators combined the various restriction DNA fragments from R6-5 with the one restriction fragment of pSC101 caused by the EcoRI digestions. Then, they ligated the multiple mixtures of the DNA fragments using a DNA ligase enzyme. Next, the investigators transformed host bacteria. In one experiment, they plated the transformed bacteria with the recombined DNA molecules on agar plates containing tetracycline and kanamycin. Only truly recombined DNA molecules would permit the bacteria transformants to grow on both antibiotics. The transformants’ growth with the two antimicrobials indicated that DNA recombination had occurred—two different genes would have come together!

The researchers headed by Boyer and Cohen found several recombinant candidates. One such clone that was derived from their doubly antibiotic-resistant transformants was called pSC105. It turned out to be the first artificially made recombinant DNA molecule in which DNA from entirely different plasmids, each from entirely different bacteria, had become stitched together from scratch. Restriction analysis revealed that the original pSC101 cloning vector had taken on a foreign DNA fragment from plasmid R6-5, making a larger pSC105 recombinant. The new molecular size of pSC105 reflected the dimensions of each smaller DNA fragments.

Cohen, Boyer, and their lab workers published the history-changing findings in the National Academy of Sciences Proceedings journal in November of 1973. The work would have profound ramifications for cloning DNA harboring novel genes and, in particular, for cloning genes by combing DNA from different species.

5) Somewhere along the way, Boyer ascertained that some bacteria genes could be linked with eukaryote genes. 

After Cohen and Boyer’s successful experiments towards putting together disparate pieces of plasmid DNA, they further realized that they could, in theory, combine a foreign DNA, say from a eukaryotic organism to a bacterium! That is, a recombinantDNA molecule could be made artificially and propagated in a bacterium, and a eukaryotic gene could be cloned. Genes from eukaryotic organisms could be cloned! For the first time in history, such an experiment would generate a genetic chimera between completely different organisms, say between a bacterium and a frog!

In the laboratory, Boyer, Cohen, and a small team of lab workers set out to clone a frog gene into their cloning vector. This pSC101 plasmid vector had been used earlier to recombine the DNA from different bacteria. First, the investigators harvested DNA from the lab frog called Xenopus laevis. Then they isolated the genetic elements that encoded frog ribosomal RNA molecules. Next, they restriction digested the frog DNA and their cloning vector DNA with EcoRI. As before, they separated the resulting DNA fragments from the frog DNA and purified as many of the DNA fragments as they could. Next, they mixed each of their various frog DNA segments with the pSC101 vector linearized with EcoRI restriction endonuclease.

The mixtures of cloning plasmid DNA and frog DNA were then allowed to anneal, taking advantage of the restriction fragments’ sticky ends that had been generated by EcoRI digestion. Next, the investigators used purified DNA ligase from Escherichia coli to connect the two types of DNA, one from a bacterial plasmid, pSC101, and the other from the Xenopus laevis frog DNA. This ligation step would be the first time in history that animal DNA would be connected to DNA of bacterial origin.

The DNA from a prokaryotic had been combined with that of a eukaryote. A wholly different chimera had been produced in the laboratory. The study by Boyer and colleagues was published in Proceedings of the National Academy of Sciences in May of 1974. The gene encoding a frog rRNA gene had been cloned. It would be the first animal gene to be cloned.

The implications were enormous, as it now became possible to consider the cloning of other animal genes, such as human genes. In time, it became possible to do that precisely. It would become necessary to clone all genes to study their genetic properties and the functions of their encoded gene products, the proteins. The discovery would enhance the studies of all genes known to science.

6) Genentech and human insulin—What did Boyer have to do with this, and why is it important?

Boyer and geneticist Stanley Cohen founded a new scientific field called recombinant DNA technology in the early 1970’s while meeting at a Honolulu delicatessen. After learning about this collaboration, Robert A. Swanson, a 28-year-old biotech industrialist, contacted Boyer and requested a conference. Boyer granted Swanson 10 minutes of his time, but Genentech was to become a reality three hours later. See Figure 20.

File:Genentech HQ buildings 9, 8, 6.JPG

https://commons.wikimedia.org/wiki/File:Genentech_HQ_buildings_9,_8,_6.JPG

Figure 20. Buildings 9, 8, and 6 on the Genentech headquarters campus in South San Francisco, California.

The new biotech firm considered producing insulin. The molecule was needed to provide diabetes treatment, a growing disease in numbers of patients diagnosed with it. Perhaps, Genentech could clone the insulin gene, propagate it in bacteria, harvest it, and make it available commercially. As simple as the idea seemed at the time, it nevertheless proved to be too daunting of a task. The insulin molecule was a large and complicated two-chain protein affair. They needed a simpler test molecule that would provide a proof-of-concept—clone the gene for a potential therapeutic agent and coax microorganisms to propagate the agent in large quantities for biomedical use.

Boyer and Genentech chose somatostatin. It was a molecule that was smaller than insulin. Somatostatin was also more straightforward because it did not require a two-polypeptide chain assembly to be a fully functional molecule. Boyer hired Keiichi Itakura and Art Riggs to begin the molecular gene cloning for the somatostatin gene and its protein expression in bacteria. Their first attempt failed. The somatostatin did not show itself within the bacteria, and it was a devastating blow.

Boyer thought of a brilliant solution. Perhaps they could fuse the somatostatin coding gene to another, more easily acceptable bacterial gene—one that the host bacteria would readily make and bring the Somatostatin gene along with it—a bacterial decoy gene. The idea was to construct a human-bacterial genetic fusion! If the gene-fusion hybrid expressed itself in the bacteria, then the somatostatin molecule could be cleaved off the bacterial decoy protein. The strategy worked! Somatostatin would be produced in sufficient quantities to be purified.

In 1978 Boyer and colleagues could now turn their attention to insulin. Almost immediately, and to their dismay, they heard the rumor that Walter Gilbert’s team at Harvard had already cloned the human insulin gene and was gearing up to proceed with its expression in bacteria. It was another profoundly disappointing situation for the Genentech team. Unfortunately for Gilbert, however, they had somehow cloned a contaminating rat insulin gene, instead of the desired human version.

Gilbert’s failure provided a reprieve and, importantly, badly needed time to play catch up. Employing their proven molecular cloning techniques, including the human-bacteria gene fusion approach, Boyer’s group at Genentech managed in 1978 to successfully clone the human insulin gene and make bacteria produce the two polypeptide chains of the human-based insulin protein. Next, they separated the bacterial decoy protein from the insulin chains and extracted the insulin protein from cultured bacteria vats. With each insulin chain now purified, it was necessary to combine the two polypeptides to assemble the full structural nature of insulin—the two-chain affair. The assembly process worked, and a recombinant insulin molecule was now available for testing.

The biotechnological news that insulin could be made in the laboratory test tube using bacteria and DNA was a significant milestone in scientific history. The discovery would catapult Genentech as a blockbuster firm for the production of needed biomedicines. The new biotech company would become a leading figure in producing additional therapeutic molecules for new generations. The firm would serve as models for the establishment of new biotech companies to spawn worldwide.

7) In addition to being a scientist, Boyer was also a philanthropist—what was his biggest contribution?

In 1990, Boyer and his wife Marigrace contributed $10,000,000 to the Yale School of Medicine, which was the single most significant donation given by an individual donor. In 1991, the Boyer Center for Molecular Medicine was named after the Boyer family. St. Vincent College has renamed the School of Natural Science, Mathematics, and Computing the Herbert W. Boyer School during the 2007 Commencement.

File:Herbert Boyer HD2005 Winthrop Sears Medal.JPG

https://commons.wikimedia.org/wiki/File:Herbert_Boyer_HD2005_Winthrop_Sears_Medal.JPG

Figure 21. Photograph of Herbert Boyer, chemist, and recipient of the 2005 Winthrop-Sears Medal, presented on June 9, 2005, at Heritage Days, Chemical Heritage Foundation, Philadelphia, PA, USA.

It is interesting to note that Boyer participated as an activist in the civil rights movement.

In 1980, Boyer was awarded the Albert Lasker Award for Basic Medical Research and the Golden Plate Award sponsored by the American Academy of Achievement. In 1982 Boyer was given the Industrial Research Institute (IRI) Achievement Award. In Boyer took, in 1989, the National Medal of Technology, and in 1990 President George H. W. Bush awarded Dr. Boyer the National Medal of Science. In 1993 Boyer was given the Helmut Horten Research Award.

In the year 2000, Boyer received the Biotechnology Heritage Award with Robert A. Swanson, from the Chemical Heritage Foundation and the so-called Biotechnology Industry Organization (BIO). In 2004 Boyer shared the Albany Medical Center Prize with Stanley N. Cohen. Also, in 2004 Boyer received the Shaw Prize in the category of Life Sciences and Medicine. In 2005 Boyer took the Winthrop-Sears Medal and, in 2007, the Perkin Medal. As recently as 2009, Boyer became a CSHL Double Helix Medal Honoree. See Figure 21.

8) Apparently, Boyer is still alive and well and teaching. What is going on in his current endeavors? 

Indeed, as of this writing, Dr. Boyer is 84 years of age. He had retired from Genentech as its vice president at the age of 55 years. Boyer is credited with having said, “There is life after science.” It is reported that he has been enjoying retirement fishing and enjoying outdoor activities. He has continued to serve on editorial boards of several molecular biological journals. More recently, Boyer has been operating on board of directors for Scripps Research, a non-profit medical research institute.

Boyer’s scientific contributions to molecular biology, biotechnology, and biomedicine cannot be overstated. The implications for improving our quality of life for countless billions will be felt in all aspects of therapeutics for generations.

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