Salary History from Grad School in 1966 to 2010 – No, I didn’t get rich.

 

The Garret Ihler Story – My Grad School Mentor

Who was the first to isolate a gene?

It was Garret Ihler in Charlie Thomas’ lab at Harvard in 1968-69 before the advent of recombinant DNA. The paper, appropriately titled “Isolation of Pure lac Operon DNA”, was published in Nature (vol. 224 pages 768-774) in 1969.  This paper, certainly, was fresh in the minds of Dan Nathans and Hamilton Smith a few years later as they developed restriction enzymes to cut and paste defined DNA fragments (Nobel Prize for Medicine in 1978). Ihler’s work started molecular biologists thinking of the benefits of molecular cloning of recombinant genes. Although lac is an E. coli (lactose) operon consisting of three structural genes (encoding the enzymes required for metabolism of lactose), lac is a single transcriptional unit with one promoter and two regulatory domains (for the repressor and operator). Ihler’s paper points out that purification of individual genes would permit investigation of their mechanisms of transcriptional control and expression (the subject of my Ph.D. Thesis). To put this event in perspective, the hottest area of genetics at that time was transcriptional control in the E. coli bacterium with the discovery of sigma factor and investigations into the mechanism by which RNA polymerase transcribed genes. A few years later we would be able to cut genes with restriction enzymes and paste these genes with a ligase into vectors that could be used to transfer genes into cells. Nathans and Smith’s work allowed Paul Berg to put the E. coli gene gpt (encoding guanine phosphoribosyltransferase) next to the SV40 promoter to correct the genetic defect for Lesh-Nyan Syndrome (Nobel Prize in Chemistry, 1980). I benefited from all four of these developments because through their work I had the incentive and was able to clone the human beta-actin gene in December of 1982 and characterize its function in recipient cells using Paul Berg’s vector. Garret Ihler’s work and his mentoring instilled in my mind the goal and benefits of purifying a gene. There is more to the story of Garret Ihler’s accomplishment.

If you look at the 1969 Nature paper, you will find that it has 6 authors – Shapiro, MacHattie, Eron, Ihler, Ippen, and Beckwith. If you look at the acknowledgement at the end of the paper, you will see that Garret Ihler was actually a postdoctoral fellow in Charlie Thomas’ lab and that Thomas’ grant supported the work. In fact, the lac operon was isolated and purified in Charlie Thomas’ lab (noted for DNA replication research) by Garret Ihler.

Picture the group in Beckwith’s lab pondering the prospect of isolating a pure gene but not knowing how. Shortly afterwards Karin Ippen takes a stroll across campus with Ihler describing to him the issue of the day in the Beckwith lab. Picture Ihler enamored with Karin Ippen (the feeling turned out to be mutual). He was also clever especially with lambda transducing phages – these phages with the transduced lac operon from E. coli had been isolated in the mid-1960’s. In fact Ihler could take these phage strains “off the shelf” at a moment’s notice with the lac operon inserted into the lambda genome in opposite directions. Shortly Ihler had thought of the solution for the Beckwith lab with Ippen as his audience. He had the tools at his fingertips – the strains of transduced lac, a reliable exonuclease, and a method of separating and purifying each DNA strand from the lambda double helix. Nevertheless, he offered the idea to Ippen who took it back to the Beckwith lab; subsequently, she was pushed aside by the aggressive members of the group. So Ihler decided to act quickly. He grew up two transducing phages each with the lac operon in opposite orientations, he separated the DNA strands using poly-UG, and then hybridized the two opposite strands. He isolated the “heavy” DNA lambda phage strands (higher poly-UG binding strands) from two different phages each with the opposite lac orientation. When preparations of the two heavy strands were allowed to hybridize only the lac operon sequences could hybridize – the two strands were complimentary only in this part of the phage genome because the flanking lambda DNA sequences of the hybrid DNA were from the same heavy strand of the lambda genome and thus homologous, not complimentary sequences. Ihler nibbled away the lambda DNA “tails” with an exonuclease and precipitated the remaining DNA to yield a pure DNA fragment encoding the lac operon.

Today this seems quite a simple experiment (if you are a molecular biologist), but in the context of the late 1960’s it was a clever experiment with a conceptual pay-off. What happened next poisoned the environment for molecular geneticists especially in the Boston area.

Beckwith took this work and euphorically ran with it. Four co-authors from the Beckwith lab were added with trivial contributions and the paper was published. Beckwith scheduled a news conference basically to take credit for the work much to the embarrassment of Ihler. He used this news conference to belittle the accomplishment and used this media platform to express his personal political agenda which had no connection to Ihler’s work. Surprisingly, the tone of this press conference is preserved in a paper by Beckwith published a year later (Bacteriological Reviews 34:222-227, 1970) where he underplays the role of Ihler – he contributed “a critical idea”. When I read the final section of this paper I could only conclude that Beckwith was deluded by his perception of self-importance. This paper can be found on the Internet for anyone wishing to read it along with Ihler’s recounting of the story at http://mbch.tamu.edu/ihler/  .

Luckily for me, Ihler took a faculty position at the University of Pittsburgh, School of Medicine, and earned his M.D. at the same time. He and Karin married while I was finishing up my Ph.D. He later became an esteemed Professor at Texas A&M noted and especially appreciated by medical students for his stimulating lectures. Karin also developed a productive research program as a Full Professor of Microbiology and Genetics at A&M. Sadly Karin died of breast cancer in 1995 with Garret at her side. Her presence and work has been memorialized through the annual Karin Ippen-Ihler Lecture Series at A&M.

Ihler was probably correct in his belief that Beckwith’s press conference led to misplaced fears and “widespread attempts to regulate cloning and gene transfer”. It did not help that Michael Crichton published “Andromeda Strain” in the same year. Fortunately, all of this concern had fallen by the wayside 13 years later when I was doing this type of work. But now we are experiencing the same hysteria over stem cell research.

Some Trials and Tribulations at Johns Hopkins

I was reminded of the following experiences during my six and a half years at Johns Hopkins by the news in 2006 of fraud by the Korean stem cell scientists who made worldwide news in May of 2005 for their published cloning of human stem cell lines corresponding to afflicted human individuals. Although it seems certain that the Koreans’ work will be disproved and retracted, I prefer to wait for the dust to settle before commenting further on this fiasco. However, in this context I thought that I could discuss my own brushes with overzealous ambition and fraud that occurred during my 30 year career as a bench scientist and researcher. Here is part 1 which relates to events of the 1970’s at Johns Hopkins. In part 2, I follow-up with some more bizarre examples that occurred in the 1980’s while I was at NIH and then at the Pauling Institute.

During my career as a research scientist I met and worked with many great scientists like Kivie Moldave, Garret Ihler, and Dai Nakada in my formative years at the University of Pittsburgh where I received my Ph.D. Later in my career during my postdoctoral years at Johns Hopkins, I collaborated with Aaron Bendich at Sloan Kettering who was an inspiring cell biologist and protégée of Erwin Chargaff at Columbia. As I established myself as a productive molecular biologist, I formed collaborations with high-powered notable scientists like Klaus Weber and Joel Vandekerckhove at that Max-Plank Institute, Rudi Aebersold and Lee Hood at Cal Tech, and Larry Kedes and Peter Gunning at Stanford. I also interacted with Nobel Prize winning scientists like Linus Pauling, Dan Nathans, Hamilton Smith, David Baltimore, and Howard Temin to name a few. I was uniformly impressed by the precision, intelligence, and focus of these scientists. Never did I see any hint of ambitiousness in these people and their discoveries were infinitely correct, substantiated, and proved by others who followed. No doubt these people were ambitious, but intelligence and precision dominated their personas. This kind of character helped these special people to make great discoveries that advanced human civilization in meaningful and measurable ways.

My first brush with overzealous ambition occurred when I returned to the lab from a long and much needed vacation in the spring of 1973 during the Watergate period. I was walking up the steps to the front door of the School of Hygiene and Public Health of Johns Hopkins University. A professor who was leaving the building and approaching me said “Congratulations, your work was written up in Science (Magazine).” I was dumb-founded and immediately went to my lab to try and understand what he was talking about. Sure enough, I found an article by Gina Kolata in Science describing ‘our discovery’ that bacteriophage DNA could replicate in mammalian cells. This was a period of infancy for the field of genetic engineering and there were several labs that were interested in knowing if one could repair defective human genes with bacterial genes transduced by bacteriophages. This was the basis of my collaboration with Aaron Bendich at Sloan-Kettering. However, I was employed and working in the lab of Paul Ts’o at Hopkins on a Postdoctoral Fellowship from the National Cancer Institute.

When I took the postdoctoral position with Ts’o, I knew I was entering into one of the highest funded labs in the country. I quickly learned that Ts’o was a dynamic visionary who made it no secret that he was out to get the Nobel Prize. I heard him say a number of times that his lab was working on everything from “atoms to man” and we were, and this was the problem with his program. I quickly discovered that Paul Ts’o had been interviewed by Kolata while I was away on vacation and that he had exaggerated our work which was ultimately published and relatively inconsequential (Leavitt et al., Biochimica et Biophysica Acta , 435:167-183, 1976).

About 6 years later Paul Berg’s lab at Stanford placed the SV40 promoter in front of the bacterial gene that I was trying to insert into to mammalian cells (the E.coli gpt gene) and succeeded in repairing human Lesch-Nyhan cells for the defect in hypoxanthine-guanine phosphoribosyltransferase. Between my work and Berg’s, recombinant DNA cloning had emerged and provided Berg with a solution that allowed him to express the bacterial gene in mammalian cells. Using a recombinant bacterial antibiotic resistance gene for neomycin resistance, Berg showed that a small amount of bacterial DNA could integrate into the human genome of a vast minority of ‘infected’ cells to replicate to remain in the transfected mammalian cells for ever.

Kolata’s article was chilling for me. Later Kolata left Science Magazine for the New York Times and made great waves in her article about cold fusion. In a later article in the Times her quote of James Watson that Harvard scientist, Judah Folkman (at Harvard), was “going to cure cancer” because of his discovery of angiostatin and endostatin was sensational. She hadn’t realized that Watson was pulling her leg.

My interest in inserting bacterial genes into mammalian cells stemmed from a paper published in Nature in 1971 by NIH scientists, Carl Merril, Mark Geier, and John Petricciani, entitled “Bacterial Virus Gene Expression in Human Cells.” (Nature 233:398-400). Merril and colleagues presented experiments that claimed to show that a bacterial gene encoding galactosyltransferase, transduced into a bacteriophage DNA molecule, could be ‘transfected’ into human fibroblasts taken from patients with the genetic disease, galactosemia. In other words, the authors claimed to have repaired a human genetic defect with a bacterial gene which was the only kind of gene that could be isolated at that time.

Merril’s initial manuscript was rejected by Nature because the reviewers needed ‘direct’ evidence that the bacteriophage gene was actually transcribed within mammalian cells. Mark Geier, a late comer in Merril’s lab saw the prospect of getting his name on an important paper by performing this experiment – not an easy experiment to perform in those days. Nevertheless, Geier quickly produced RNA hybridization experiments that demonstrated that the bacterial transcripts of the galactosyltransferase gene were being synthesized. This experiment was added to the Nature manuscript naively by Merril, the manuscript was resubmitted, and then published by Nature. The publication of this paper made national news. Since this came from National Institute of Mental Health on the NIH campus just up the street from the White House, President Nixon took notice and awarded Carl Merril with a small presidential grant to help fund his laboratory.

There was wide skepticism about this work among scientists in the field and the experiments in this paper were never reproduced although numerous labs tried. In 1976, in my final days in the Ts’o lab, I struck up a short collaboration with Carl Merril, who later became a longtime friend, productive investigator at NIH. Together we devised a rigorous experiment to determine if bacterial genes could be expressed in mammalian cells using the same type of approach taken later by Paul Berg – but without an essential recombinant mammalian gene promoter driving the expression of the bacterial gpt gene.  We found no evidence of expression of the bacterial gene although we did find genetic revertants of the mutant mouse hprt gene already present in the mouse cell we were using. This more positive story was published much to Merril’s chagrin by me with colleague Greg Milman in Experimental Cell Research (123:402-406, 1979).

There is one more oddity to this story. In 1972, I went into Paul Ts’o office with Merril’s Nature paper and said to Ts’o “Why don’t we do this?” Ts’o read the paper and quickly responded to the effect that he was not interested. A few weeks later Ts’o called me into his office to suggest that we get into the field of gene transfer (as though the earlier conversation had never taken place). Ts’o had overnight realized that he could achieve great success if we could learn how to repair human genes with bacterial genes. So I wrote a small grant proposal that was submitted to the March of Dimes. This grant application was reviewed by Howard Temin (later a Nobel Prize winner) and funded to support my initial research in gene transfer.

My Escape from Johns Hopkins

During one of my numerous train rides from Paul Ts’o’s lab at Johns Hopkins to Aaron Bendich’s lab at Sloan-Kettering in 1974 (mentioned in Part 1) another major case of fraud hit the national news. Since this happened at Sloan-Kettering, I learned the inside scoop a few floors away from where the incident happened.

William Summerlin, an MD, was working in the lab of Robert Goode then Director of the prestigious Sloan-Kettering Memorial Institute in Manhatten. Both had their eyes on the Prize but Goode was honest with himself and the world. Summerlin, on the other hand, saw the glory but could not wait for the results. Summerlin had talked openly about having success at transplanting skin from a black (haired) mouse onto an albino mouse without transplant rejection. His break-through was to culture the black pigmented skin in-vitro before transplantation. If this simple step solved the problem of transplant rejection, then the advance could aid in improving skin transplantation for burn victims and possibly aid in organ transplantation. 

At some point Dr. Goode asked to see the white mice bearing black patches of transplanted skin. Summerlin wheeled a cart of mice in small cages into an elevator to take them upstairs to Dr. Goode’s office. Before the doors could close, an associate who was in the hall outside the elevator noticed Summerlin ‘touching up’ some of the mice with a black magic marker. I imagine that Goode gave the mice a causal glance and noticed the black patches of hair, then asked Summerlin to take the smelly critters back to the animal room. Shortly afterwards, the associate who saw Summerlin’s artwork, reported what she had seen to Goode. Goode came forward publicly, took blame for his in attentiveness and misuse of federal funding, and needless to say Summerlin was fired.

Several years later I left Paul Ts’o’s  lab in January of 1977 and moved upstairs to the Biochemistry Department and Greg Milman’s lab for nine months. One Monday morning, I was walking down the main hall of the Biochemistry Department with Larry Grossman who was chairman of the department. Larry asked “what’s going on down in Ts’o’s lab?” – I asked what he was talking about. Larry had met a Hopkins psychiatrist at a cocktail party over the weekend, who revealed to Larry that “half the people in Ts’o’s lab” were seeing him for something akin to traumatic stress syndrome. I was astonished, but I added that quite a few in the lab had ulcers and I knew of one postdoc whose wife was having mental problems. After talking to Larry, I scurried down a few floors to Ts’o’s lab and snagged best friend in the lab, Ron, into a small room off the main hall. It was a “guess what I heard” kind of conversation – immediately Ron became agitated. He uttered plaintively, “John, I’m one of them”. I then realized that I was not the only one in that lab that felt stress from Ts’o’s limitless ambitions. Nearly everyone there was suffering from the realization of their career-ending move with projects that were virtually impossible. The frustration was compounded by the realization that Ts’o could block anyone’s move out of the lab by failing to give them a decent letter of reference for their years of toil. He had tried to do this to me but I was able to move on by selling myself and my ideas.

We already knew that he had done this with others essentially sabotaging their efforts to move forward in their careers. I should have figured out the situation in my first year as his postdoc. After only two months in the lab, I was working with another postdoc who had been there for 5 years. On one Friday, the two of us planned in detail an experiment that would be started on Monday. However, my colleague did not show up for work on Monday and never returned to the lab even though he remained on the payroll for the better part of a year. One of my other colleagues in the lab found him driving a taxi cab in downtown Baltimore.

 While at Johns Hopkins I met a number of graduate students and postdocs who languished for years trying to earn their Ph.D. or as postdocs trying to establish independent careers without success – they eventually burned out and left like my colleague, the taxi driver. Although this is usually not because of fraud or over-zealous ambition, it’s a dark side of graduate-level academic science that is rarely talked about. Perhaps this environment pressures some to commit scientific fraud. It seems to me that the institution bares some responsibility for prolonging these situations when failure seems certain especially when no limiting benchmarks exist from start to finish. Universities will argue the opposite, but there are many professors that take advantage of ‘free’ help without considering the impact of the exploitation. The victim becomes so time-invested in the endeavor that he or she feels that there is no option to turn back or go in a different direction.

One of the worst examples of this problem is in the case of Theodore Streleski, a 19-year graduate student in the Math Department at Stanford. In his ninetieth year as a graduate student, in 1978, he bludgeoned to death his advisor, head of the math department, with a ball-peen hammer. He later called the killing “…a protest over treatment of graduate students…” Streleski was convicted of second degree murder based upon diminished capacity and was paroled after seven years in prison in 1985. During three parole hearings he indicated no remorse and refused to agree to never return to Palo Alto or the Stanford campus. For this reason he was denied parole at his first two hearings. At his third parole hearing by law the authorities had no choice but to set him free. Explaining his lack of remorse he stated “I say Stanford treats students criminally. If I express remorse, I cut the ground out from under that argument. I would not only be a murderer but a dirty lying dog. I am a murderer. I am not a dirty lying dog.”

In the early 1980s I found myself at the Pauling Institute in Palo Alto a few blocks from the Stanford campus. My colleagues and I usually took a coffee break at the “Printer’s Inc” where many Stanford people hung out. I can remember audibly complaining about the unfathomable mindset of professors who would keep a graduate student for 19-years – period of time where many, if not all, would lose their minds. I was quickly met with words of reproach by two nearby Stanford people who overheard my remark who then opined that they were on his “hit list”.

From that time to when I left Palo Alto in 1996, I never heard a word about Streleski returning to town.  Although the years spent in Ts’o lab were frustrating and less productive than they should have been given the federal investment in his program, some good scientists emerged from the lab and prospered independently in their careers. Brian Crawford became an Oppenheimer Fellow (with my reference) at Los Alamos after receiving his Ph.D. in 1981, Bob Moyzis followed Brian to Los Alamos after a decade as a graduate student and receiving his Ph.D. in 1983, and Paul Miller remained at Johns Hopkins and rose to Professor. Eventually Johns Hopkins shut down Ts’o’s lab. I was fortunate to have made contact with researchers on the outside – Bendich, Milman, and Merril who became my mentors during these dark years – and I had published some papers that formed the basis of my future work. I took a position with the FDA’s Bureau of Biologics at NIH in the fall of 1977, Aaron Bendich died unexpectedly in 1979, Greg Milman left research and Hopkins and eventually became Director of the NIH Office of Innovations and Special Programs, and Carl Merril built a highly successful and productive career at NIMH after weathering the storm of the early 1970’s.

Later at the National Institutes of Health

In 1979 I was ensconced in Building 29A in the center of the NIH campus with a career appointment at the FDA’s Bureau of Biologics. I had arrived there in November 1977 at a Senior Fellow and was the first staff member to successfully transition to a career civil service position. My friends and colleagues, Carl Merril and David Goldman at the National Institute of Mental Health across the street, and I were on a roll. I had made a discovery that was going to fuel my research for the next 15 years by exploiting protein profiling using high-resolution 2-dimensional electrophoresis. Protein profiling produced an abstract image of greater than 1000 proteins from individual cell-types – a perfect approach for surveying gene expression with my dyslexic brain. The three of us were heavily invested in this technique and applying it to many different areas of investigation. The technique had been developed by Patrick O’Farrell at the University of Colorado in 1975. In 1976 Greg Milman demonstrated elegantly that this technique could detect point mutations in genes. At the same time Dr. Norman Anderson from the federal Argonne Labs in Chicago was starting to swing his weight around in this field. He had started publishing numerous papers on the many different applications of 2-D gels, in effect ‘wallpapering’ over the historical facts in development of this technique.

The Director of NIH invited Anderson to present a seminar in Building 1, as a part of the Director’s special series of invited speakers who were doing something innovative in the biosciences. It was unusual for such attention to be given on this prestigious occasion to a laboratory technique that had not produced any great discoveries as yet.  Nevertheless, those at NIH who were interested in exploiting the technique attended the seminar with great enthusiasm, and, indeed, the auditorium was almost full.

I found myself near the front of the auditorium at the edge of the center aisle and Carl Merril found a seat on the opposite side of the aisle in the same row. Because this was a special series, Anderson’s talk was scheduled to last an hour and a half – an unusually lengthy talk – as a rule of thumb, the more eloquent the presentation the less time it should take. Anderson took every last second to present every thought that had come to mind about the importance of protein profiling as a tool for elucidation of the genetic basis of human traits and disease population-wide. After about an hour, I started to imagine hearing a crescendo of goose-stepping soldiers in jackboots marching in the background. The tone of Anderson’s talk started to sour in my mind and I was thinking that he would have been much better off had he ended his talk after the usual 50 to 55 minutes. I lost count of the number of slides shown during his presentation, but it seemed like at least 50. At this point Anderson ended his talk, but the projectionist did not cooperate and produced one more slide. The speaker looked back at the screen and saw something that immediately rattled him – in an agitated fashion he asked the projectionist to shut down the slide presentation because he didn’t want to show anymore slides.

The slide, mistakenly shown, displayed the technique of using a silver nitrate-based stain to enhance an image of a protein separation pattern in a polyacrylamide gel – a soon to be published technique that had just been discovered by Carl Merril. Carl, an avid photographer, recognized that the process of film development could be applied to polyacrylamide films and had shown that one could enhance, by 100-fold, the resolution of a complex mixture of proteins in an electrophoresis separation pattern thus allowing the investigator to make observations that couldn’t be made otherwise. The technique was especially useful for analyzing body fluids such as plasma and cerebral spinal fluids. The problem here was that Carl had submitted his first manuscript to Science on the discovery of this method. With manuscript submissions to Science, the author can recommend informed scientists as potential reviewers of the manuscript and often Science will choose from such recommendations. Carl had recommended Anderson as a reviewer. Peer reviewers of grants and journal submissions traditionally respect and honor the proprietary nature of unpublished manuscripts. Anderson rejected the paper for Science on the basis that it was not important enough for this journal. However, the showing of the slide revealed that Anderson had added the technique to his long repertoire of research accomplishments and incorporated the unpublished technique into his well-toured seminar presentation.  There were perhaps only two people in the audience that knew what had happened – Carl and I glanced at each other with wry smiles. At the end of all of this, as I walked out, Anderson leaped off the stage to explain himself to Carl.  

 After being rejected by Science, Carl had quickly resubmitted his manuscript entitled “A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels” to Analytical Biochemistry which published the paper without hesitation on September 15^th, 1979 (Volume 98, pages 231-237).

My Move to Palo Alto CA

When I resigned my position at the FDA many of my colleagues asked why. After all I was a tenured scientist at NIH, supposedly the best place to be. The answer was that I left for “purely scientific motives.” And, it turned out to be the best career and life move I could have made so I am happy to relive it here. The move from Bethesda in late 1981 was driven by my interest in cloning a gene that I had discovered, the human beta-actin gene and the mutant genes that I had discovered. At the time few human genes had been cloned. After I settled into Palo Alto, I discovered that the town was a wonderful place to be.

Four years earlier I had landed a job with the FDA in a division that regulated vaccines on the campus of the National Institutes of Health where I had the freedom, more or less, to conduct my own research but not to build my own program. Being at NIH seemed like the best place to be. However, I stumbled onto a discovery that opened up my entire career. Hopefully not being too technical, the gene was the human beta-actin gene – I had discovered mutations in this gene using the profiling technique described above. These mutations published in the most presitious journal Cell stirred a lot of interest in the cancer research community.

In September 1981, I was invited to give a talk at the Pauling Institute in Palo Alto at the bottom of Page Mill Road near the intersection of El Camino Real. I was picked up at the airport the night before and deposited at that motel on the opposite corner on El Camino Real.  The next morning I got up at dawn and strolled down to Stickney’s restaurant past the entrance to Varian for breakfast. I was struck by how green the grass was on Varian’s lawn and that image lasted in my mind. From the outside, the Pauling Institute building looked like a warehouse but inside, labs were being built during my visit.  After Pauling died in 1993 the Insitute left Palo Alto and now thrives at Oregon State University on the leadership of Steve Lawson who was a technician during my days there.

Before I made the trip, I was contacted by an old Hungarian molecular biologist, Koloman Laky, who knew Linus Pauling and who had worked with Szent-Gyorgyi. Szent-Gyorgyi (admired by Pauling) had discovered both Vitamin C and Actin back in the 1930’s and 40’s respectively. Actin is the protein that along with myosin governs muscle movement (contraction). However, it is also the most abundant protein in all replicating cells. The mutations that I discovered were implicated in malignancy and metastasis.

Koloman explained to me the history of both of these discoveries (C and actin) and some history about Linus Pauling and his associate Emile Zuckerkandl the inept President of the Pauling Institute (appointed by Pauling). So I went to the Pauling Institute armed with some background. During my talk Pauling who was in his early 80s sat in the front row with a big smile on his face. In the middle of my talk he interrupted me to ask if I knew who discovered actin. I responded that it was his friend Szent-Gyorgyi and told him about my colleague at NIH, Koloman Laky. As silly as this was, we hit it off and I was offered a position as a senior scientist by the end of the day. By December ‘81 although still employed by the FDA for two more months, I was working at the Pauling Institute.
 Below Ewan Cameron a Scottish physician and good friend of Becki and me stands together with Linus Pauling at the Pauling Institute. I have a hand written letter from Pauling that states that he “took my advice”. I won’t say what the advice was, but I can be sure that such a letter from a two-time Nobel Prize winner has to be special.

A few months before leaving NIH I had met Larry Kedes who was a Professor of Medicine at Stanford. Larry’s lab was the only lab in the country that was interested in human actins at the time, a protein/gene boring to most. Few at that time knew that actin was the major architectural protein of all non-muscle cells. When I arrived at the Pauling Institute, Larry and I began a very productive collaboration that led to my cloning of the normal and mutant actin genes and then our patenting of this gene promoter for genetic engineering (the part of the gene that drives this abundantly expressed gene), and Stanford licensed our promoter to the biotech industry. It eventually got used in gene therapy in cancer patients at MD Anderson. We also distributed the promoter to hundreds of academic investigators around the world. This whole experience was very gratifying, as esoteric as it may sound.

My friends and colleagues at NIH said that I was crazy to leave a ‘safe’ government position but I was willing to take some risks. Pauling’s notoriety helped fund my research until I could raise my own grants which I managed to keep funded until we left Palo Alto in December of 1995.

On the personal side, I rented a small apartment on Byron, a block west of Middlefield and the Community Center near Embarcadero. It was a simple but productive period for me. I would walk or bike to work, work for 12 hours, and then go back to the apartment seven days a week. By December 1982, I had cloned the beta-actin gene, one of the first human genes to be isolated. Over the next five years we proceeded to publish many papers on our research. Then I moved on to the next gene and so on and et cetera.

In the middle of all of this Becki and I got together. We moved into a house on Maureen, a block off Cowper and two blocks from East Meadow and the elementary school on the edge of Mitchell Park in central Palo Alto. We had Mariah in Oct. 1988. A few years later we moved next door to Rambow (better palms and Eucalyptus). Becki was the caterer for the docs at Stanford Children’s Hospital and I had left the Pauling Institute to run the California Institute for Medical Research in San Jose, and then later to Adeza Biomedical in Sunnyvale. Becki walked Mariah to her first day of kindergarten. Mariah is not camera shy and was interviewed on her first day of school by an SF news station. This is when she announced to the Bay Area that she didn’t miss her parents, “uh uh!” Mariah is now 22 and living in Virginia Beach … and not missing us too much.

Life in Palo Alto was great for us but for the last 14 years we have adapted to the quieter life-style of northeastern CT. It’s less social here except for the local politics about which we opine at the Café. Becki goes back to Burlingame every year and visits all of our friends and relatives around the Bay Area, but I’ve only managed to get back once. We usually treat everyone to dinner at Casa Isabelle’s on Park. Whenever possible I watch a Stanford football game on TV like last night’s Notre Dame game just to get a glimpse of Palo Alto.

 My Role in the Emergence on Proteomics

Over the past 10 years I have been somewhat amused by the seemingly abrupt emergence of the fields called “genomics” and “proteomics” and all of the other “…omics”. The hype surrounding their emergence seems similar in effect to drinking too much espresso in the morning.

The earliest review on the subject of “proteomics” (with an “s”) that I could find was published in 1997 by James entitled “Protein identification in the post-genome era: the rapid rise of proteomics” in the Quarterly Review of Biophysics (30: 279-331), a year when only a few papers mentioned this word. The word “proteome” was actually first trademarked in 1995 by Jim Garrels at Proteome Sciences, Inc. in 1995. Simply put, “proteomics” now defines an industry that is focused on linking newly identified proteins to disease states for the purpose of improved diagnosis and treatment.

Some would argue that there is more to proteomics than this, but the emphasis remains in defining protein targets for diagnostic use and targeted therapies. The essential component of proteomics is determination of the identity of a protein in the midst of the entire proteome (consisting of roughly 20,000 proteins) in relation to its presence or absence or modulation accompanying a disease state. Initially this hinges on defining the protein by its amino acid sequence. Once the identity of the protein target is determined by its sequence, all other issues dealing with the importance of this target can be settled, albeit with a lot financial investment and hard work. Did proteomics begin in 1997? Indeed, the title of the early review suggests that this field of investigation was just beginning.

I believe proteomics or at least proteomic thoughts began with Patrick O’Farrell’s doctoral project in Edmund McConkey’s lab at the University of Colorado. His paper presenting high-resolution 2-D polyacrylamide gels was published in the JBC in 1975 (250: 4007-21). This was the first presentation of a “protein profile” of a cell. The importance of this technique was elegantly displayed by Greg Milman (PNAS 73: 4589-93, 1976) who demonstrated that one could predict occurrence of mutations in the HRPT polypeptide by positional changes in the 2-D gel in the midst of the proteome. Then in 1980, Joel Vandekerckhove in Klaus Weber’s lab, incollaboration with me at the US National Institutes of Health, was the first to sequence a polypeptide ‘variant’ isolated discovered in a pattern of  >1000 polypeptide spots in a 2-D gel (Cell 22: 893-899). My notion that the polypeptide that captivated my interest was a”variant” stemmed directly from Milman’s work. Vandekerckhove, Weber, and I not only identified the protein as human beta-actin by complete sequencing; we pin-pointed the mutation that caused a positional shift in both dimensions of the 2-D gel to an exchange at amino acid 244 – a Gly to Asp. This was the first mutation event discovered using 2-D protein profiling, and the first protein to be sequenced after discovery in a 2-D gel. A few years later I cloned the mutant and wildtype genes as a consequence of this work and proved the mutation formally. In addition, gene transfer of the mutant beta-actin converted non-tumorigenic (immortalized) human fibroblasts to tumorigenic cells (Leavitt et al MCB 7: 2467-76, 1987). A few years after Vandekerckhove’s work, Jerry Latter, Steve Burbeck and I demonstrated that it was possible to predict the identity of an unknown polypeptide by producing multiple gels of the same protein profile each with a different amino acid label (Clinical Chemistry 30: 1925-32, 1984).

During the mid-1980s, Lee Hood’s group at Cal Tech was developing more sensitive protein microsequencing techniques to advance the field that became “proteomics”. In 1987 Reudi Aebersold in Hood’s lab and I were the first to determine internal peptide sequences from an unknown polypeptide “spot” directly from a 2-D gel (PNAS, 84: 6970-4, 1987); then we identified the protein “spot” as a chaperonen based upon the sequence of the fragments. That same year Ruedi and I produced 8 internal peptide sequences from another unknown protein (L-plastin) that appeared in many tumor-derived cells and also in-vitro SV40-transformed human fibroblasts. Within a month the plastin mRNA sequences were cloned using a nucleic acid probe designed from one of these short peptide sequences (Lin, Leavitt et al. MCB 8: 4659-68, 1988). Five years later the full structure and chromosomal location of the 3 one-hundred kilobase genes of the plastin multigene family were published (Lin, Leavitt et al JBC 268: 2781-92, 1993). The sequences and domains of this family of proteins revealed their role in regulating actin filament assembly in the cellular cytoskeleton. Since 1993 numerous papers have been published on these plastin isoforms in relation to tumor cell expression, lymphocyte activation, cytoarchetecture, and even intracellular transport of disease causing bacteria. In addition to diagnostic applications (examples: Otsuka et al. BBRC 289: 876-881, 2001; Zhou et al. Cancer Res. 63: 7122-7127, 2003; and world patent WO2005015227) several therapeutic applications have been described for plastins – the plastin protein as a drug target (Rosales et al, PNAS 91: 3534-8, 1994) and the plastin promoter for targeting gene therapy to tumors (Deisseroth et al. Cancer Gene Therapy 10: 388-395, 2003).

Today these steps in the evolution of a proteomics project would move much faster because of advances in techniques but the hard part will always be producing useful knowledge. So, really, nothing has changed except that the world is moving faster.    

How Dekalb Genetics Made Roundup Ready Corn and other Roundup Ready Veggies

I’ve been doing some reading on Monsanto’s glyphosate-resistant Roundup Ready crops – soybeans, canola, corn, alfalfa, etc (because of Scott’s stimulating ruling against Monsanto). There are a number of controversial aspects to this class of crops. There is a baseless fear of recombinant (genetically modified crops; GMO) crops combined with their more complicated unknown consequential impact on the ecology of farm lands. Then there is the never settled fear of environmental toxicity of glyphosate itself which is the most widely used herbicide in agriculture in the presence or absence of a GMO crop. Decades of research tend to support this herbicide’s safety if it is used under the guidelines of the product. Nevertheless it is always possible to raise concern by showing that at some level glyphosate can be shown to be toxic; for example, in the far east including India, ingestion of this chemical has been used commonly to commit suicide. The only chemical that I know that is not toxic when stirred in water in ascorbic acid (Vitamin C) as Linus Pauling used to ingest 18 grams a day (without supervision).

I understand the basic principal of how the combination of glyphosate and Roundup Ready crops work effectively because I used the same principle in the lab repeatedly to detect transfer of recombinant genes into cells in tissue culture throughout the 1980s and early 90s. The common practice was to engineer a plasmid (small supercoiled circular DNA that can be introduced inside cells) that carries a transgene conferring a useful trait and an antibiotic-resistance gene that renders a particular antibiotic ineffective (in my case usually neomycin). Both the useful transgene and the antibiotic resistance gene are incorporated in a low percentage of transfected cells but these transfected cells are easily enriched into a pure culture by adding neomycin to the culture medium – the cells that did not acquire the neomycin resistance gene (or the useful transgene) simply died in the presence of neomycin. This process is now widely used even in laboratory classrooms to teach recombinant microbial genetics (personal communication from Ron Sills J). 

At the end of the 1970s, Paul Berg at Stanford made such a plasmid carrying the neomycin-resistance gene and a useful transgene isolated from the E. coli bacterium called the gpt gene. This was one of the earliest applications of recombinant DNA engineering which was made possible because of the Nobel Prize winning work of Hamilton Smith and Dan Nathans at Johns Hopkins who demonstrated that you could splice foreign genes into plasmids using restriction enzyme cutting and a DNA ligase. It was common knowledge that the gpt gene was defective in people with the rare genetic disease, Lesch-Nyhan Syndrome. So Berg obtained the simian viral SV40 gene promoter that Nathans had isolated and fused it to the coli gpt gene and then put it into his neomycin-resistance plasmid. The role of the promoter is to drive the expression of the gpt gene by providing a site for a mammalian RNA polymerase to initiate transcription of a messenger RNA that acts as a template for synthesis of the gpt protein/enzyme.

In human cells (or any mammalian cell in culture) you can block the pathway for de novo purine synthesis with the anti-cancer drug methotrexate – purines are the precursors of DNA and RNA – but the cells don’t die if you add exogenous purines.  This is because these cells, if they are normal and have a gpt enzyme, can salvage purines from the medium (hypoxanthine and guanine). [In mammalian cells the gpt enzyme is called hprt for hypoxanthine-guanine phosphoribosyltransferase.] So Berg obtained a human fibroblast culture from a patient with Lesch-Nyhan Syndrome and transfect the cells with his plasmid containing the neomycin-resistance gene and the coli gpt gene. He selected only the cells that would grow in the presence of neomycin and then tested them for gpt activity which they had. He then was able to show that these cells could also grow in the presence of methyltrexate if the two purines were added to the culture. In doing this Berg was the first to demonstrate the correction of a human genetic defect with a recombinant gene fused to a functional mammalian gene promoter.

We all knew of this approach to demonstrating correction of a genetic defeat so as dramatic as Berg’s demonstration was, it was a very doable experiment by that time. In fact a year or two earlier when I was at Johns Hopkins, I obtained the transduced bacteriophage from which Berg harvested the gpt gene and tried to transform mouse cells which had the gpt defect but I was unable to detect bacterial gpt expression. Instead I found that the mouse endogenous gene had incurred reversing mutations that led to the selection of cells in which the hprt gene had reactivated. In retrospect I should have walked across the street to ask for Nathan’s help. So Berg got the Nobel Prize because he was smart enough with the timely development of recombinant DNA to fuse the gpt gene to a mammalian gene promoter that would work in mammalian cells. At least Berg later gave me his neomycin vector which we used extensively.

In 1978 at NIH, I discovered an unusual and unprecedented mutation in human beta-actin, and that beta-actin along with another lesser isoform, gamma-actin, were the most abundant proteins of all normal mammalian cells that could replicate. This discovery was made in human fibroblasts routinely grown in culture. Before this time many researchers were not even aware that actin was more than just a muscle protein (when you eat meat, you are eating a lot of actin and myosin). Around the same time, my brother Andy and I published a paper that showed the same two isoforms of actin were the most abundant proteins of lymphocytes (white blood cells) and that the ratios of these two isoforms could change from the normal lymphocyte to leukemic cells. In collaboration with Klaus Weber at the Max-Planck Institute at Goettingen Germany we obtained the sequence of the mutant and wildtype beta-actins, along with the sequence of the normal gamma-actin (there were only 23 aa differences out of 375 aa between humans and slime mold). Klaus came to the states, and we met for the first time. We were collaborating on something else which he wanted to pursue in great depth (and led to the demonstration of the Tuschl II siRNA invention in 2001). He asked me ‘what I am going to do next?’  I said ‘I’m going to clone the human beta-actin gene and the two mutant genes that I had found’ and try to establish their relationship to cancer … and he was free to carry on with the other work.

So I moved on to Palo Alto CA and Linus Pauling’s Institute after meeting Peter Gunning and Larry Kedes (Stanford) at a symposium at Cold Spring Harbor Labs in Long Island. I told them what I wanted to do because they were already working on the muscle actin genes and could help me with their know-how. So we decided to join forces. By December of 1981, I had cloned the human beta-actin gene (and the promoter of the gene) and the cloning of the mutant genes quickly followed (this work was published in 1984).

Here is where our work became relevant to Roundup Ready J.

Since beta-actin was the most abundantly expressed gene/protein in all mammalian cells, it didn’t take a genius to predict that the promoter in this gene was highly active and might have applications for genetic engineering. But Genentech was already developing recombinant proteins using the powerful cytomegalovirus early gene promoter so we were too late to promote universal application of our promoter. Nevertheless we filed a patent application on its use and succeeded in licensing its use to multiple biotech companies. We also published multiple papers touting the strength of this promoter and the fact that it was constitutive in nearly all cells (continuously turned on). One of these papers has been cited over 1000 times.

This prompted others to take an interest in actin promoters from other species. Don Cleveland at Johns Hopkins jealously reproduced the chicken beta-actin promoter. I told others that if they used Cleveland’s promoter in humans, they might cluck like a chicken J. Around this time Genetic Therapy, Inc., in Gaithersburg tested the human beta-actin promoter driving a tumor suicide gene in patients with brain cancer at MD Anderson Hospital in Houston. And since the mid-1980s our promoter was freely distributed and used widely in academic research labs around the world.

So, how does this relate to Roundup Ready?

Today I spent some time reading about how Dekalb engineered Roundup ready corn for Monsanto. The herbicide, glyphosate, inhibits the growth of plants by blocking the activity of the enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (epsps) which participates in the synthesis of aromatic amino acids that are essential for growth of plants. Dekalb identified a mutant version of this gene/protein (mepsps) that still functioned properly but was resistant to the inhibiting effects of glyphosate (it had 2 amino acid differences from the wildtype enzyme). They engineered a recombinant gene that would be expressed highly and constitutively in plants, they placed the gene into a plasmid, and they introduced the plasmid into corn germ cells which thereby became resistant to glyphosate. This strain of corn as well as soybeans, alfalfa and other oil and food producing plants became Roundup Ready. These strains of corn have been tested in the US since 1994 and in the European Union since 1996. They were commercialized in the US in 1998 and in Canada in 1999 and have been cultivated ever since (over 6 million acres) “with no reported instability”.

To allow for adequate expression of mepsps, Dekalb placed this coding gene under the control of the rice actin promoter and a neighboring intron (flanking non-coding DNA sequence) from the native rice actin gene. By the mid 1980s we had determined that the presence of this intron was best mode for high level constitutive expression of a recombinant transgene gene. A Dekalb publication stated “The expression of mepsps gene is expected to occur throughout the corn plant because the rice actin promoter drives constitutive gene expression in corn.”

Satisfaction with My Career

Besides the Eureka moment of making an important or, at least, useful discovery the most satisfying experience I had in research was receiving a fundable rating on my federal grant applications from other scientists. In this process, notable scientists would sit in committees in Washington DC, called study sections, and discuss the merits of hundreds of grant applications and then vote on them resulting in scores that were above or below the fundable level. On a few occasions I was invited to become an ad hoc member of such committees because of the nature of my expertise. The next most satisfying experience was having a research paper accepted for publication; this also happened as a result of peer review; and I published about 60 research papers during my career – the more prestigious the journal, the greater the feeling of accomplishment. The next most satisfying event was seeing others pick up on my findings and extend them to something more valuable. This form of satisfaction usually comes several years later. I checked the citations of my papers recently and found that some of my papers are still being cited as references some 15 to 30 years after the papers were published.

One protein that I discovered and named plastin continues to be investigated as a biomarker and possible drug target for diagnosing and treating various forms of cancer. In 1978 I performed an experiment that led to the discovery of plastin and another interesting protein. This led to NIH funding of my research program for 13 continuous years after I left NIH several years later. In 1985, I submitted a paper to Cancer Research and the reviews came back positive, but one reviewer suggested that I give the protein that was the subject of the paper a name. I was tickled by the idea of actually naming a human protein (and its gene).

I had a concept for the function of the protein because of its normal expression in all white blood cells and its mysterious appearance in tumor cells from solid tissues (carcinomas and sarcomas). I thought that it may make adherent cells that are sort of flat behave more like white blood cells that are sort of round and able to migrate throughout the body through the circulatory system and interstitial space.

In cancer biology the migration of a carcinoma cell to other tissues – like breast cancer spreading to the lymph nodes – is called “metastasis”. It was my suspicion that this protein gave adherent cells more plasticity in their shape and motility so I named the protein “plastin”. I got the idea for the name from the movie The Graduate which was a big hit in 1967.

Remember that scene…
When we cloned the gene for plastin, we discovered in the process that there were two slightly different plastins, one found only in white blood cells (L-plastin) and one found only in solid tissues (T-plastin). Today both isoforms of plastin are being investigated for their roles in tissue adhesion, cell motility, cell architecture, their respective roles in different forms of cancer. I was really surprised when one researcher on the West Coast discovered that T-plastin was inappropriately expressed in Sezary lymphomas which derive from T-lymphocytes – a white blood cell involved in immune and inflammatory responses due to infections and tissue damage. Sezary cell lymphoma is often lethal. This lymphoma is distinct from other forms of lymphoma and leukemia because it invades the skin and behaves sort of like a solid tumor on the surface of the skin. As the result of this finding there is now an approach to making drugs that could conceivably lead to a treatment for Sezary lymphoma by targeting T-plastin. This new technology which was first published in 2001 is called “interfering RNA” or siRNA for “short interfering RNA”. It would be hard to fit an explanation of how siRNA works as a drug into this story so I won’t try.

In April I began covering a major lawsuit at a website called RNAiLitigation . The lawsuit was filed in June 2009 by the prestigious German university system known as the Max-Planck Institute against MIT’s prestigious Whitehead Institute and the University of Massachusetts. This litigation is a fight over who has the right to use and sell the siRNA invention previously assumed to be owned by Max-Planck. I had to read carefully the seminal papers and patents on this invention to prepare myself for telling the story behind the lawsuit and covering the testimony and developments in the upcoming trial. When I read the seminal paper by Thomas Tuschl on his discovery of siRNA and how it works, I found that the paper was co-authored by Klaus Weber a highly revered long-time professor at Max-Planck in Goettingen. I was somewhat pleased to see that Klaus was still around and doing great things in science, especially because we did something great together back in 1980.

I knew of Klaus Weber and his wife Mary Osborne long before I ever conceived of contacting him in 1979. Weber and Osborne were famous for their work at Harvard which I won’t go into here. I used a technique they developed in the late 1960s when I was a grad student working on my Ph.D. In the mid 1970s, Klaus received a professorship at Max-Planck and developed his well-known research program investigating the nature of mammalian cellular architecture and motility. His lab had published the amino acid sequences of muscle actins from various species down to single cell eukaryotes (cells with nuclei). In 1978 on that day when I discovered plastin, I also discovered the first mutation event in a human actin called beta-actin that along with gamma-actin was the most abundant architectural protein of all non-muscle cell types. I wanted to determine the sequence of this mutant actin to prove that a mutation had occurred. This required expertise that was out of the scope of my own area of research and I was advised by a specialist in this field who work downstairs from me at NIH. He suggested that the only way to get this done was to find a laboratory that specialized in sequencing actins. There was only one in the world, Klaus Weber’s lab at the Max-Planck.

Back in 1979 the only way to communicate what I had found effectively was to write Klaus a letter. Two weeks later I received a letter back saying that he and his post-doc Joel Vandekerckhove would be willing to sequence my mutant actin and he told me how I should prepare the cell extract containing the mutant actin so that they could purify it and sequence it. The speed of Klaus’ response told me that he was seriously interested. Years later after more experiences like this, I had learned many times over that when you collaborate with sharp people, extraordinary things happen. In December of 1980 we published the complete amino acid sequences of the normal and mutant human beta-actins and the other gamma-actin in the top biomedical journal Cell. The mutant beta actin had incurred a single amino acid exchange that allowed us to predict the actual mutation in the gene sequence.

In the summer of 1981 before I moved to Palo Alto CA, Klaus called me up and proposed that we meet for the first time at the Shoreham Hotel just north of Rock Creek Park in DC just down the road from NIH in Bethesda. So we got together one morning in the lobby of the hotel. I had made a lot of monoclonal antibodies against human architectural proteins (this architectural structure is called the “cytoskeleton”) as a by-product of something else I was doing. I had little use for these antibodies and shipped them off to Goettingen since Klaus was well known for using immune antisera to visualize the cellular cytoskeleton. The method for making epitope-specific monoclonal antibodies was new and this had not been tried in Klaus’ lab. It turned out that he wanted to show me some pictures of cells stained with antibodies that I had sent him. I think that he was also trying to find out what I intended doing with them which was a proper thing to do. The photos were very impressive and suggested that these antibodies could be used to map the cytoarchitecture of all cell types. He asked me vaguely “What are you going to do next?” I told him that my only interest was in cloning the human actin genes to reassure him that he could move forward with the monoclonal antibodies. That was our last communication until April 2010.

As I mentioned above, Klaus was a co-author on the seminal paper describing the invention of siRNA in 2001 – still today the most exciting biomedical discovery since the mid-1980s. I had notified the Technology Transfer Office for the Max-Planck in Munich about our coverage of the litigation they were involved in. I had mentioned Klaus Weber’s involvement in the discovery of siRNA at the website.

In April I received an email from “Mary Osborne”. It turned out to be Klaus using his wife’s email much in the same way I use Becki’s email. I had said at the website that “It’s is a small world” and he said it that “indeed it was a small world” and explained to me his simple contribution to Tuschl’s discovery of siRNA. He also said that he remembered our exchanges. Needless to say, I was thrilled to hear from him this one last time.

I went back to that seminal paper to look more closely at his ‘small’ contribution from his own description. When I looked at the particular experiment that was used to demonstrate that Tuschl’s invention worked, I realized a lasting connection. When we met at the Shoreham Hotel nineteen years earlier, one of the antibodies that I had provided to him stained the nuclear envelope, a protein called “lamin”. I think before showing me the pictures, Klaus had already realized the power of mono-specific monoclonal antibodies. His lab proceeded to make them and use them extensively in his research. Although not the very same antibody that I had prepared, his anti-lamin antibody was the one that was used elegantly to demonstrate shutdown of lamin expression with siRNA targeting the lamin messenger RNA, e.g. gene silencing. This entire story is emblematic of how discovery science works.

In the ensuing nine years that seminal siRNA paper has received close to 6000 citations from other research papers, and siRNA-based drugs are now progressing through the early stages of clinical development.

My Experience with the Early Invention of Monoclonal Antibodies and How I Passed this on to a Great Scientist
NYTimes Obit

When I saw an obit for Hilary Koprowski recently, I was surprised that it did not mention his benchmark work with “monoclonal antibodies” or “hybridomas.” Then I searched Google and found other pages that didn’t mention “monoclonal antibodies” or “hybridomas.” I found some websites that summarized his accomplishments emphasizing his role in development of the oral polio vaccine which stated almost as an after-thought “In the late 1970s he and his Wistar colleagues were granted the first monoclonal antibody patents which he used to co-found Centocor, one of the first biotechnology companies to commercialize monoclonal antibody diagnostics and therapeutics.”

 

The invention of monoclonal antibodies using the hybridoma technique was reduced to practice by César Milstein at the Medical Research Council (MRC in England) and colleagues Georges Köhler and Dane Niels Kaj Jerne in 1975 who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery.

 

The Table to the left (not shown) shows that publications mentioning monoclonal antibodies (mabs) started to appear in the mid-1970s and rose sharply after 1978. Koprowski used the phrase “monoclonal antibodies” and/or “hybridoma” in an April 26, 1978, paper in the high profile journal Proceedings of the National Academy of Sciences of the United States of America (PNAS) entitled “Study of antibodies against human melanoma produced by somatic cell hybrids” and in a second paper he published in August 1978 in PNAS entitled “Monoclonal antibodies against rabies virus produced by somatic cell hybridization” three years after Milstein’s invention. The first patent on preparation of mabs issued to Koprowski and the Wistar Institute in Philadelphia (US4172124) entitled “Method of Producing Tumor Antibodies.” This patent was filed on April 28, 1978, and issued on October 23, 1979. The National Research Development Corporation in Britain (NRDC), the body responsible for patenting MRC inventions, had simply flubbed the opportunity to patent the invention of mabs.  Koprowski’s applications of monoclonal antibodies were enticing and made cell biologists realize the importance of this invention, not to mention stimulating entrepreneurs to see dollar signs. Early in the AIDS epidemic, this invention led to development of the first test for HIV infection first developed by Luc Montagnier (Pasteur Institute) and shared with Robert Gallo (NIH).

 

The abstract of Koprowski’s patent states “Antibodies demonstrating a specificity for malignant tumors are produced by somatic cell hybrids between hypoxanthine phosphoribosyltransferase (HPRT) deficient myeloma cells and (HPRT-positive) spleen or lymph cells derived from an animal previously primed with tumor cells.” I wondered if anyone had noticed that Koprowski’s patent priority date was two days after he supposedly disclosed the invention in PNAS on April 26^th.

 

In 1977 I joined the FDA’s Bureau of Biologics (now Center for Biologics Evaluation and Research or CBER) located on the center of the campus of the National Institutes of Health in Bethesda, Maryland. I had published research surrounding applications of HPRT-deficient tumor cells so the procedure for producing monoclonal antibody (mab)-secreting hybridomas was not foreign to me – that is, fusing normal HPRT-positive antibody-producing B-lymphocytes with HPRT-deficient immortalized B-myeloma cells to produce clones of immortalized hybrid (fused) myeloma cells secreting a single antibody species.

 

In late 1978 I obtained the x63-Ag8 mouse myeloma strain developed by Milstein and used by Koprowski and immediately began trying my hand at making hybridomas against vaccine viruses (Influenza A, poliovirus, and cytomegalovirus). Within a few weeks we had hundreds of hybridoma clones secreting mabs specific for these viruses, some of which neutralized the flu virus and even poliovirus. I thought, ‘no wonder the polio vaccines that had been developed in the 50s worked so well.’ As a result of the polio work I confirmed that I had had a polio infection as a young kid in the late 40s because in order to grow the virus everybody at the Bureau had to be tested for immunity. Those of us that had the natural infection had titres of poliovirus antibodies a thousand-fold higher than those who achieved immunity by vaccination alone.

 

Screening hybridoma clones for mabs that bound to viral epitopes was very easy and fast. I would grow infected cells on glass slides which had eight round patches where the cells could adhere and uninfected cells were used as negative controls. Then I would fix the cells in acetone and dry the slides for later use. To screen the hybridoma clones, I would simply take a drop of culture medium out of each of hundreds of culture wells containing individual hybridoma clones and place the drop on the slide wells containing the infected or uninfected cells. This incubation only required 10 to 15 minutes. After that I flicked away the medium and washed the slide in three or four washes of saline. Then I repeated this procedure with a saline solution containing a fluorescently tagged antibody that bound to the mouse antibody produced by the hybridoma clone. At this stage I was eager to look at the cells to see if there was a mab reaction with the cells. If there was a reactive mab, the cells would light up like Christmas trees under a fluorescent microscope.

 

I set up a specialized lab at the Bureau to teach and practice the hybridoma technique for physicians who reviewed biologic drug applications and I sat on the FDA committee which wrote the first guidelines for clinical use of mabs. My boss, Frank Ennis, was so thrilled with these developments that he had me stand up in front of the Commissioner of the FDA and describe the technology and its potential applications during his annual one-hour visit. Soon after our meeting with the Commissioner began, the fire alarm rang and we all had to leave the building. When we were allowed to return there was only 15 minutes left so Frank snagged me  to give the only presentation. I remember using a wooden pointer to describe the mab-specificity to a specific epitope on a linear molecule. We all had a chuckle and both the Commissioner and my boss were pleased.

 

My early experience with mabs was probably reproduced in hundreds of labs by the early 1980s. The unique patterns of fluorescent mab staining of cells were fascinating because influenza has eight proteins. It became obvious that we had isolated eight different anti-influenza mab types, one for each of the influenza proteins because there were eight different patterns of mab staining. For example, a mab that stained primarily the cell nucleus was specific for the viral nucleocapsid protein and a mab that stained the entire cytoplasmic shape turned out to be a hemagglutinin-specific mab, the antigen that gives us immunity.

 

While screening hundreds of hybridoma clones, I quickly discovered that some of the mabs were not specific for virus infected cells. These mabs turned out to be targeting cyto-architectural antigens (proteins) in intermediate filaments (such as vimentin) and microfilaments (actins, tropomyosins, and actin-binding proteins) which are the major structural proteins of all eukaryotic cells. I was fascinated by the myriad of structures that were stained by these antibodies. Since I had just discovered the first mutation of an actin, in this case a mutant human beta-actin (the most abundant protein of fibroblasts, epithelial cells, endothelial cells, white blood cells, etc.), I realized quickly that these mabs could be used to dissect the structural components of the human cellular cytoskeleton, an endeavor that I would never undertake because of my interest in cloning of the genes I discovered. So I packaged the mabs frozen in solution and shipped them off to Klaus Weber at the Max-Planck Institute in Goettingen Germany since Klaus was well known as a cell biologist who was interested in the cyto-architecture of cells. Klaus and I had just published the amino acid sequences of both beta- and gamma- cytoskeletal actins, and the mutant beta-actin as well, in Cell. This was work done by Klaus’ post-doc Joel Vandekerckhove in Goettingen. We discovered that these cytoplasmic actins were the most highly conserved proteins from yeasts to humans with very few amino acid changes throughout evolution.

A short time later Klaus asked me to meet him at the Shoreham Hotel near Rock Creek Park in Washington DC in 1981. This was the first and only time we met face-to-face. He had brought with him photographs of the cytoskeleton staining patterns produced with my mabs. He was fascinated by them as much as I was, including the mab that stained the nuclear envelope protein, nuclear lamin. He looked at me ernestly and said ‘What are you going to do next?’ I told him that I was moving to Palo Alto, California, to Linus Pauling’s Institute and I was going to clone the mutant beta-actin gene. I encouraged Klaus to carry on with the mab work. A year and a half later I cloned the mutant and wildtype human beta-actin genes with some valuable help from my colleagues at Stanford.

<!–[if gte vml 1]> <![endif]–><!–[if !vml]–><!–[endif]–>Klaus and wife Mary Osborne are well-known for their work at Harvard that started in the late 1960s which continued at Cold Spring Harbor Labs in the 1970s, and during their Professorships at the Max-Planck Institute in the late 1970s through to the present. So I should not have been surprised when he and Thomas Tuschl used a nuclear lamin mab to show for the first time that siRNA-mediated gene silencing worked in mammalian cells in 2001, a paper that has since been cited close to 9000 times. Klaus and I exchanged emails in April 2010 over his role in Tuschl’s discovery of siRNA-mediated gene silencing.

By the mid 1980s Genentech had started development of the first therapeutic antibody Trastuzumab (Herceptin) that bound to the extracellular HER2 domain of the tumor surface antigen ERBB2, a receptor for epidermal growth factor. In June of 1992, Genentech began phase I clinical trials of humanized Trastuzumab for treating breast cancer by intravenous infusion. This drug was launched in August of 1999. In May of 2000 Genentech and ImmunoGen began collaborating on the development of Trastuzumab conjugated with a small molecule oncology drug linked to the antibody using Immunogen’s proprietary linker. Trastuzumab as a monotherapy, adjuvant therapy, and combination therapy is now approved for use in gastric cancer in addition to breast cancer. The drug conjugate Trastuzumab-emtansine has been approved as a second line therapy for breast cancer in the USA, and is currently in preregistration in the European Union and Japan. Since the first approval of Herceptin, 43 other therapeutic antibody formulations which target novel drug targets have been approved for various disease indications.

A quick examination of the therapeutic antibody pipeline reveals that at least 466 therapeutic antibodies are in development for treating disease indications, including at least 80 drug-conjugated antibodies (ADCs). Herceptin/ Trastuzumab from Genentech/Hoffmann-La Roche had revenues of about $6.3 billion in 2012. Other leading therapeutic antibodies include Adalimumab (Humira by Abbott Labs, ~ $9.1 billion in 2012), Rituximab (Rituxan by Hoffmann-La Roche, ~ 7.2 billion in 2012), Becacizumab (Avastin by Hoffmann-La Roche, ~ $6.1 billion in 2012), and Infliximab (Remicade by Johnson & Johnson, ~ $6.1 billion in 2012). These five mabs by themselves represent a $35 billion dollar industry.

My Views on Stem Cell Research

This interview was first published in May of 2005 by Thought Mechanics, a nationally recognized blog. It has been updated to include a comment on the recent success of scientists at Stemagen (La Jolla CA) who announced their success at cloning human embryonic stem cells.

Thought Mechanics: What is an embryonic stem cell?
John Leavitt: An embryonic stem cell is a cell that is toti-potent. In other words, these cells are the earliest stage embryonic cells that develop after the egg is fertilized. The unique property of an embryonic stem cell is that it has the capacity to differentiate into any tissue in the body.

Thought Mechanics: We heard in the news 2 years ago about the Korean fraud regarding their claim to have successfully cloned human embryonic stem cells. How has this impacted on the future of this field of research?
JL: Fraud in academic research is rare. It’s unfortunate but I have heard of no negative fallout when it comes to the academic and public desire to move forward with embryonic stem cell research. On the positive side, the false claims of the Korean ‘success’ led to lots of productive discussion about the importance of embryonic stem cell research. It gave us an opportunity to envision the benefits and the downside and weigh them against each other.

Thought Mechanics: Very recently we heard in the news about Stemagen, a small company in La Jolla California. In January 2008 Stemagen scientists announced their success in cloning of human embryonic stem cells. How has this impacted on the future of stem cell research?
JL: This is a stunning break-though if their work can be reproduced. Assuming the credibility of this success, they have shown that, indeed, human embryonic stem cells can be cloned just like stem cells from many animal species. This success will stimulate human stem cell cloning worldwide. Besides this country the United Kingdom has especially strong expertise in cloning as well as strong government support for stem cell research. 

Before this success there were fears that it couldn’t be done for some mysterious reason. Not only did the Stemagen erase that concern, but they showed that stem cell cloning could be done efficiently. The only limitation that exists now is the willingness of a woman to donate oocytes so that another individual, male or female, can have their own stems cells cloned and mass produced.

Thought Mechanics: In the news we’ve heard that there are other stem cells called “adult stem cells”. How do these differ from embryonic stem cells?
JL: Adult stem cells can be harvested from various fetal and adult tissues. For example, umbilical cords contain ‘pluripotent’ stem cells that have been shown to be capable if differentiating in several types of tissue-specific cells. Adult stem cells act as a reservoir inside tissues to replace cells that age. The liver, for example, has progenitor cells that replace the liver epithelium. In the fetal brain there are neuronal stem cells that can become neurons or glial cells which form the myelin sheath that protect the neuronal connections.

Thought Mechanics: Then why do we need to have research on embryonic stem cells if we can use the so-called adult stem cells?
JL: Adult stem cells are limited in their ability to form the myriad of cell types in the body. Also, if you need liver or neuronal stem cells, who is going to step up and donate their liver or brain? Besides cells from another person would not be autologous and would be rejected. Sources of embryonic stem cells are also limited in that one needs human oocytes to prepare them. A woman would have to donate her eggs for this to happen. But the tremendous advantage of embryonic stem cells is that one can prepare large populations of them to match any human being histologically. These autologous stem cells are prepared by removing the nucleus from an unfertilized egg and replacing it with the nucleus of an adult cell (like a skin cell) from any human being. This process is called “nuclear transfer”. The stem cells that are grown from this artificially fertilized egg will match the person from which the adult cell was taken and can be used to replace damaged or diseased tissues of that person.

Thought Mechanics: Is there a way to overcome the limitation of availability of female eggs?
JL: This is a considerable limitation but there has been discussion of the possibility through research of discovering a way to differentiate embryonic stem cells into oocytes to generate an endless supply of unfertilized eggs.

Thought Mechanics:Why is stem cell research important to the public?
JL: One could treat diseases that are caused by the loss of a particular cell function or tissue function and if you have stem cells that can differentiate into that missing or defective tissue, then in theory you can do something called cell replacement therapy where you replace the defective cells with the healthy cells and the healthy cells would then be able to regenerate the defective tissue.

Thought Mechanics: What kinds of diseases could potentially be cured with stem cell technology?
JL: Any disease where tissue is destroyed or defective and there is no ability to regenerate those tissues by the body. We all know that livers regenerate to some degree but adult brain cells don’t, adult neurons don’t. So, when you think of the diseases that might be addressed first, it’s the neurodegenerative diseases that would be addressed like Parkinson’s and Alzheimer’s. Spinal cord injury is another key area. In the news we heard about Christopher Reeve and his wife, and also Nancy Reagan essentially pleading for more stem cell research. It’s because the general belief is that spinal cord injury and/or Alzheimer’s disease could be addressed.

Thought Mechanics: At the center of the debate about embryonic stem cells is the issue of “when does human life begin?” What do you have to say about that?
JL: One position is that human life begins with the existence of an egg; a second position is that human life begins with the fertilization of the egg; and a third position is that human life begins with the recognition of self. Within the last year I listened to a Harvard stem cell scientist describe the following scenario to illustrate validity of the third position – ‘Imagine an IVF clinic that has caught fire and will burn to the ground. There is one person who is left trapped within the burning building along with thousands of unfertilized and fertilized eggs stored in liquid nitrogen tanks. Who will the firemen attempt to save?’

Thought Mechanics: President Bush says that embryonic stem cell research destroys life. Is there any validity to that claim? If so, why? If not, why not?
JL: When I was culturing human cells in the lab there’d be an occasion where I was finished with the culture and we destroyed it. We’d take the cells, we’d do the experiment and then we’d throw them away in an efficacious manner. We’d sterilize them so they were dead, but is that destroying life? Those cells are living cells, so that’s life in a sense. Bacteria are living, and viruses are living by the definition, they can self-replicate. So, what kind of life are we talking about here? Most of the public has really no concept of what is done in the laboratory today. Not that there’s anything wrong with that, but there are people that are very familiar with cells and the embryology behind the development of the fetus and those are the people that really, I think, understand what we’re doing in the laboratory when we’re manipulating cells. So President Bush says that we’re destroying life… well I ask what kind of life are we talking about here?

Thought Mechanics: Well, I think the perception that people have is that the cells used for stem cell research are cells that would otherwise be used to create life, to create babies, and if those cells are being used for experimentation, they aren’t being used to create life.
JL: Every egg has the potential to be a human being, so that’s probably what we’re talking about, but should the woman decide not to go in that direction, then it has no potential. An egg will be produced each month and the woman may not choose to try and get pregnant. Once somebody has freely donated an egg, once you take that egg and plant a nucleus into the egg with no intention of implanting it into the uterus of a woman, then it becomes a cell culture process. To me, it’s no different than culturing amniotic cells from an unborn child to see whether the child has any genetic defects. It’s done routinely. I don’t see a distinction between those processes and the process of developing a cell culture from an egg that is not from sexual reproduction. Since the eggs were donated for this purpose, no one is being harmed. The only source for these types of eggs would be from a bank of cells where women had donated eggs for the purpose of storing them and producing children later on in life, or helping someone else to produce a child. Today there are many IVF clinics, hundreds of them, that store eggs in liquid nitrogen tanks just for this purpose. Then at some point the mother of the eggs says she doesn’t need them anymore and they’re thrown away, is that destroying life?

Thought Mechanics: How is stem cell research currently being funded?
JL: Embryonic stem cell research is not being funded by NIH, the predominant source of funding for academic research. But it’s interesting that states, like California and Connecticut have taken steps to override that. States are rebelling, congress passed a new bill recently to override Bush’s personal decision to block embryonic stem cell research, but Bush vetoed it. This is the policy of this administration; it’s not a policy that the majority of the public or congressmen accept. It’s been foisted on us and there hasn’t been serious political debate about it. It’s more of a religious position but I wouldn’t label them as idealogs for having this belief. Even Republican congressmen are trying to override Bush’s opinion on this. They’ve looked into it and have been persuaded that the research is valuable. And there are others articulating what the value of this research is. Christopher Reeve’s wife was talking about it; Mrs. Reagan spoke openly about the need to move forward, so it’s not simply a Republican/Democrat issue, it’s the policy of the Bush administration because of Bush’s religious or even niave point of view. Does this make the rest of us pagans? There are brilliant scientists just sitting on their hands and impeded from doing productive research in this area. Until that bill is passed, NIH is not going to fund the type of research that supports this important therapeutic area. Government funding of academic research is what drives progress in all fields.

Thought Mechanics: So basically the Bush administration has literally shut this down, and we are out of luck until Bush changes his mind, we get enough votes to override his veto, or we get someone else in office?
JL: That’s true. We’ve already lost seven years in this area; it has retarded progress in human embryonic stem cell research dramatically.

This has been especially agonizing for the afflicted who might benefit. I’ve had occasion to talk to some of these people and it’s a strenuous conversation to have when you are forced to explain why nothing is being done in this area. It’s a shame because many people are misinformed and it is complex, that’s for sure, and not everyone can be a cell biologist or an embryologist, but you see remarkable naivety and bias on this issue. Yeah I have my own bias too, but I come to the table with some first-hand experience in the area. But it’s my impression that the people who are voting on this issue are very naïve and not well educated on the subject and the value of this research. This will be argued for decades, it will never be settled. But what might happen is that if people start to see the benefits of the technology, they might start to understand it better. We saw this happen with test-tube babies and recombinant DNA. And, we are now growing transgenic corn.

Thought Mechanics: How soon could we expect to see cures for diseases if the government put its support behind the technology?
JL: I was a cancer researcher for 25 years and I was funded by the National Cancer Institute and the American Cancer Society and people were always coming up to me and saying, “When are we going to cure cancer?” Early on in my naïve beginning, I would say, “well, by 1980? or something like that.
(Laughter)
The truth of the matter is that after decades of research, we still can’t cure certain types of cancer, but people are living longer and surviving cancer at a higher frequency because of it, so there have been great benefits to the amount of research that’s gone into cancer research. To me harnessing embryonic stem cells is an equally difficult problem and this will require a tremendous amount of research. The research will focus on improving, first, the efficiency of generating embryonic stem cells from the egg by asexual fertilization. Then the major area of development in this area will be to figure out how to control the differentiation of these cells into the type of tissue that you’re interested in growing. These are complicated matters, but there has been a lot of progress in understanding differentiation even without studying stem cells directly. So, to put a number on how many years a successful treatment will take, I can’t tell you.

Thought Mechanics: Will we see someone with injuries similar to Chris Reeve walk in our lifetime?
JL: Well, walking is going all the way. It could be that we’ll see small improvements, like people regaining the use of an arm or fingers. There should be hundreds of defined goals, because there are many levels of success. Once we allow this whole thing to go forward, we’ll be able to better measure the timing of success, but the success should be measured in increments, not just getting up and walking. I don’t want to be the one to say that it’s going to be five years or 10 years, but it’s a sure thing that eventually there will be huge scientific and medical benefits derived from embryonic stem cell research.

Some Minor Details

EDUCATION
1961-1966 B.S. Chemistry & Math, Bethany College, Bethany WV
1966-1971 Ph.D. in Biochemistry, University of Pittsburgh School of Medicine
1971-1977 NIH-sponsored Post-Doctoral Fellow, Dept. of Biochemistry, Johns Hopkins University, Baltimore MD

EMPLOYMENT
1977-1982 Senior Fellow & Research Biochemist – Bureau of Biologics (CBER), FDA, NIH campus, Bethesda MD
1982-1988 Senior Scientist – Linus Pauling Institute, Palo Alto CA
1988-1991 Scientific Director – California Institute for Medical Research, San Jose CA.
1991-1993 Director of Research – Adeza Biomedical, Sunnyvale, CA.
1991-1996 Senior Scientist – Palo Alto Medical Foundation Research Institute, Palo Alto CA.
1994-1995 Adjunct Faculty – Dept. of Biology, US Air Force Academy, Colorado Springs CO
1996-pres  Biotech Analyst – NERAC, Inc., Tolland CT.

FELLOWSHIPS – NIH Predoctoral Fellowship, Univ. Pittsburgh School of Medicine, 1966-1971; NIH Postdoctoral Fellowship, Johns Hopkins University, Dept. of  Biochemistry, 1971-1973; Natl. Cancer Inst. Postdoctoral Fellowship, 1973-1976; Senior Staff Fellow, FDA, 1977-1979

FEDERAL GRANT & CONTRACT AWARDS ($2.4 million in funding)
March of Dimes Grant to John Leavitt to investigate gene transfer 1974-1976,
ACS-NP-433 to John Leavitt from the American Cancer Society “MUTANT Beta-ACTIN GENE STRUCTURE AND FUNCTION IN NEOPLASIA” Jan. 1st, 1984 to Dec 31st, 1986.

From CRISP (NIH grants):
R01CA034763-01-04 to John Leavitt from the Natl. Cancer Inst. “MUTANT Beta-ACTIN GENE STRUCTURE AND FUNCTION IN NEOPLASIA” June 15th, 1984 to Feb 29th, 1988.
RO1CA034763-05-07 to John Leavitt from the Natl. Cancer Inst. “ROLES OF MICROFILAMENT PROTEINS AND PLASTIN IN NEOPLASIA” March 1st 1988 to Nov. 30th 1990.
R01CA034763-08-10 to John LEAVITT from the Natl. Cancer Inst. “ROLES OF MICROFILAMENT PROTEINS IN NEOPLASIA/METASTASIS” May 1st 1991 to April 30th 1994.
R01CA049423-01 to John LEAVITT from the Natl. Cancer Inst. “ROLE OF METASCHEMATIN PROTEINS IN NEOPLASIA” May 10th 1989 to April 30th 1992. S07RR005768-10 John LEAVITT from the Natl. Ctr. Research Resources BIOMEDICAL RESEARCH SUPPORT GRANT April 1st 1988.
S07RR005768-11 John LEAVITT from the Natl. Ctr. Research Resources BIOMEDICAL RESEARCH SUPPORT GRANT April 1st 1989.
S07RR005768-12 John LEAVITT from the Natl. Ctr. Research Resources BIOMEDICAL RESEARCH SUPPORT GRANT April 1st 1990 .
S15AG008028-01 John LEAVITT from the Natl. Inst. on Aging Small Instrumentation Grant July 1st 1988.
S15CA053652-01 John LEAVITT from the Natl. Cancer Inst. Small Instrumentation Grant Aug 1st 1990.

Contract to John Leavitt from the Laser and Optics Research Center, US Airforce Academy, Sep 22nd 1994 to Sep 21, 1995.

FEDERAL PEER REVIEW STUDY SECTIONS (grant reviewing and approvals)
Army Breast Cancer Research Program (USAMRMC) study section 1993, NIH.
AdHoc SBIR study section 1987.
NIH Cytology Study Section 1989.

CONSULTANTSHIPS – Channing-Weinberg Venture Fund 1986.

Other lay articles:

“Many of Us May Already Be Protected From the Lethal Effects of the H1N1 Swine Flu and Possibly the Dreaded H5N1 ‘Bird Flu’ “
“Gene Doping in Athletics – Prospects for the 21st Century”
“Interfering RNA”
“Flu Hysteria”
“Flu Mania”
“Human Embryonic Stem Cell Cloning – a Stunning Breakthough“
“Bird Flu in Perspective“
“Prion Diseases“
“Better Monkeys“

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Selected Peer-Reviewed Papers published by John Leavitt  (>3500 citations of all 70 published papers as of Jan. 2001)

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1. The use of the L-plastin promoter for adenoviral-mediated tumor-specific gene expression in ovarian and bladder cancer cell lines.  
Peng, X. Y.; Crystal, R.; Leavitt, J. ; Deisseroth, A et al.  
JOURNAL NAME- Cancer Research VOL. 61    2001    PP. 4405-13.      
Corporate Author- Genetic Therapy Program,Yale Univ. Sch. Med., New Haven, CT 06520, USA
Citation references – 26
  
2. Use of L-plastin promoter to develop an adenoviral system that confers transgene expression in ovarian cancer cells but not in normal mesothelial cells.  
Chung, I.; Schwartz, P. E.; Crystal, R. G.; Pizzorno, G.; Leavitt, J. ; Deisseroth, A. B.  
JOURNAL NAME- Cancer Gene Therapy  
VOL. 6    NO. 2    1999 Mar-Apr    PP. 99-106     CORPORATE AUTHOR- Yale University School of Medicine, New Haven, Connecticut 06520 Citation references – 39
 
3. Mutagenic activity of high-energy 532 nm ultra-short laser pulses.   
Leavitt, J. ; Fatone, M.; Hestdalen, C.; Obringer, J. W.; Tillinghast, H. S. Jr  
JOURNAL NAME- Radiation Research  
VOL. 147    NO. 4    1997 Apr    PP. 490-4    
CORPORATE AUTHOR- Department of Biology, United States Air Force Academy, Colorado Springs, Colorado 80840, USA. Citation references – 2  
 
4. Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin.   
Adams, A. E.; Shen, W.; Lin, C. S.; Leavitt, J. ; Matsudaira, P.  
JOURNAL NAME- Mol Cell Biology  
VOL. 15    NO. 1    1995 Jan    PP. 69-75  
CORPORATE AUTHOR- Department of Molecular and Cellular Biology, University of Arizona Tucson
Citation references – 22

5. Discovery and characterization of two novel human cancer-related proteins using two-dimensional gel electrophoresis.  
Leavitt, J.   
JOURNAL NAME- Electrophoresis  
VOL. 15    NO. 3-4    1994 Mar-Apr    PP. 345-57  
CORPORATE AUTHOR- Laboratory of Cancer Cell Biology, Palo Alto Medical Foundation Research Institute, California 94301.
Citation references – 16
   
6. Regulation of synthesis of the transformation-induced protein, leukocyte plastin, by ovarian steroid hormones.   
Leavitt, J. ; Chen, Z. P.; Lockwood, C. J.; Schatz, F.  
JOURNAL NAME- Cancer Research  
VOL. 54    NO. 13    1994 Jul 1    PP. 3447-54  
CORPORATE AUTHOR- CORPORATE AUTHOR- Laboratory of Cancer Cell Biology, Palo Alto Medical Foundation Research Institute, California 94301
Citation references – 5
   
7. Activation of the leukocyte plastin gene occurs in most human cancer cells.   
Park, T.; Chen, Z. P.; Leavitt, J.   
JOURNAL NAME- Cancer Research  
VOL. 54    NO. 7    1994 Apr 1    PP. 1775-81  
CORPORATE AUTHOR- Laboratory of Cancer Cell Biology, Palo Alto Medical Foundation Research Institute, California 94301.
Citation references – 20
 
8. Characterization of the human L-plastin gene promoter in normal and neoplastic cells.   
Lin, C. S.; Chen, Z. P.; Park, T.; Ghosh, K.; Leavitt, J.   
JOURNAL NAME- J Biol Chemistry  
VOL. 268    NO. 4    1993 Feb 5    PP. 2793-801  
CORPORATE AUTHOR- Laboratory of Cancer Cell Biology, Palo Alto Medical Foundation Research Institute, California 94301.
Citation references – 38
 
9. Human plastin genes. Comparative gene structure, chromosome location, and differential expression in normal and neoplastic cells.   
Lin, C. S.; Park, T.; Chen, Z. P.; Leavitt, J.   
JOURNAL NAME- J Biol Chemistry  
VOL. 268    NO. 4    1993 Feb 5    PP. 2781-92  
CORPORATE AUTHOR- Laboratory of Cancer Cell Biology, Palo Alto Medical Foundation Research Institute, California 94301. 
Citation references – 65
 
10. Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: towards an integrated protein database.   
Aebersold, R.; Leavitt, J.   
JOURNAL NAME- Electrophoresis  
VOL. 11    NO. 7    1990 Jul    PP. 517-27      
 CORPORATE AUTHOR- Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Citation references – 54  

11. Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins.
de Arruda, M. V.; Watson, S.; Lin, C. S.; Leavitt, J. ; Matsudaira, P.  
JOURNAL NAME- J Cell Biology  
VOL. 111    NO. 3    1990 Sep    PP. 1069-79  
CORPORATE AUTHOR- Whitehead Institute for Biomedical Research, MIT, Cambridge, Massachusetts.
Citation references – 102 

12. Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.  
Lin, C. S.; Aebersold, R. H.; Leavitt, J.   
JOURNAL NAME- Mol Cell Biology  
VOL. 10    NO. 4    1990 Apr    PP. 1818-21  
CORPORATE AUTHOR- California Institute for Medical Research, San Jose 95128.
Citation references – 35   

13. Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.   
Lin, C. S.; Leavitt, J.   
JOURNAL NAME- Mol Cell Biology  
VOL. 8    NO. 1    1988 Jan    PP. 160-8  
CORPORATE AUTHOR- Linus Pauling Institute of Science and Medicine, Palo Alto, California 94306 Citation references – 31

14. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts.   
Gunning, P.; Leavitt, J. ; Muscat, G.; Ng, S. Y.; Kedes, L.  
JOURNAL NAME- Proceedings Natl Acad Sci U S A  
VOL. 84    NO. 14    1987 Jul    PP. 4831-5  
CORPORATE AUTHOR- Dept. of Medicine, Stanford University, CA
Citation references – 801 

15. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.   
Lin, C. S.; Aebersold, R. H.; Kent, S. B.; Varma, M.; Leavitt, J.   
JOURNAL NAME- Mol Cell Biology  
VOL. 8    NO.11    1988 Nov    PP. 4659-68  
DOCUMENT TYPE- JOURNAL ARTICLE    
CORPORATE AUTHOR- California Institute for Medical Research, San Jose, California 95128.
Citation references – 75 

16. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.   
Aebersold, R. H.; Leavitt, J. ; Saavedra, R. A.; Hood, L. E.; Kent, S. B.  
JOURNAL NAME- Proceedings Natl Acad Sci U S A  
VOL. 84    NO. 20    1987 Oct    PP. 6970-4  
DOCUMENT TYPE- JOURNAL ARTICLE  
CORPORATE AUTHOR- Division of Biology, California Institute of Technology, Pasadena 91125.
Citation references – 745

17. Expression of transfected mutant beta-actin genes: transitions toward the stable tumorigenic state.   
Leavitt, J. ; Ng, S. Y.; Varma, M.; Latter, G.; Burbeck, S.; Gunning, P.; Kedes, L.  
JOURNAL NAME- Mol Cell Biology  
VOL. 7    NO. 7    1987 Jul    PP. 2467-76  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA
Citation references – 58 

18. Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.   
Leavitt, J. ; Ng, S. Y.; Aebi, U.; Varma, M.; Latter, G.; Burbeck, S.; Kedes, L.; Gunning, P.  
JOURNAL NAME- Mol Cell Biology  
VOL. 7    NO. 7    1987 Jul    PP. 2457-66  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA
Citation references – 68 

19. Tropomyosin isoform switching in tumorigenic human fibroblasts.   
Leavitt, J. ; Latter, G.; Lutomski, L.; Goldstein, D.; Burbeck, S.  
JOURNAL NAME- Mol Cell Biology  
VOL. 6 NO. 7   1986 Jul    PP. 2721-6  CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA
Citation references – 79  
 
20. Identification and order of sequential mutations in beta-actin genes isolated from increasingly tumorigenic human fibroblast strains.   
Lin, C. S.; Ng, S. Y.; Gunning, P.; Kedes, L.; Leavitt, J.   
JOURNAL NAME- Proceedings Natl Acad Sci U S A  
VOL. 82    NO. 20    1985 Oct    PP. 6995-9 DOCUMENT TYPE- JOURNAL ARTICLE  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 28

21. Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes.  
Ng, S. Y.; Gunning, P.; Eddy, R.; Ponte, P.; Leavitt, J. ; Shows, T.; Kedes, L.  
JOURNAL NAME- Mol Cell Biol  
VOL. 5    NO. 10    1985 Oct    PP. 2720-32  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 411

22. Abundant synthesis of the transformation-induced protein of neoplastic human fibroblasts, plastin, in normal lymphocytes.   
Goldstein, D.; Djeu, J.; Latter, G.; Burbeck, S.; Leavitt, J.   
JOURNAL NAME- Cancer Research  
VOL. 45    NO. 11 Pt 2    1985 Nov    PP. 5643-7  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 43 

23. Smooth muscle alpha-actin is a transformation-sensitive marker for mouse NIH 3T3 and Rat-2 cells.   
Leavitt, J. ; Gunning, P.; Kedes, L.; Jariwalla, R.  
JOURNAL NAME- Nature  
VOL. 316    NO. 6031    1985 Aug 29-Sep 4    PP. 840-2  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 114   

24. Tumorigenic potential of human fibroblasts as a function of ability to express a novel form of influenza A nucleocapsid protein.   
Leavitt, J.   
JOURNAL NAME- Carcinogenesis  
VOL. 4    NO. 10    1983 Oct    PP. 1229-37  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 8   

25. Molecular cloning and characterization of mutant and wild-type human beta-actin genes.
Leavitt, J. ; Gunning, P.; Porreca, P.; Ng, S. Y.; Lin, C. S.; Kedes, L.
JOURNAL NAME- Mol Cell Biology
VOL. 4 NO. 10 1984 Oct PP. 1961-9
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 113

26. Identification of polypeptides on two-dimensional electrophoresis gels by amino acid composition.
Latter, G. I.; Burbeck, S.; Fleming, J.; Leavitt, J.
JOURNAL NAME- Clinical Chemistry
VOL. 30 NO. 12 Pt 1 1984 Dec PP. 1925-32
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 34  
 
27. Neoplastic human fibroblast proteins are related to epidermal growth factor precursor.   
Burbeck, S.; Latter, G.; Metz, E.; Leavitt, J.   
JOURNAL NAME- Proceedings Natl Acad Sci U S A  
VOL. 81    NO. 17    1984 Sep    PP. 5360-3  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 20   

28. Variations in expression of mutant beta actin accompanying incremental increases in human fibroblast tumorigenicity.  
Leavitt, J. ; Bushar, G.; Kakunaga, T.; Hamada, H.; Hirakawa, T.; Goldman, D.; Merril, C.  
JOURNAL NAME- Cell  
VOL. 28    NO. 2    1982 Feb    PP. 259-68  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 92

29. Changes in gene expression accompanying chemically-induced malignant transformation of human fibroblasts.  
Leavitt, J. ; Goldman, D.; Merril, C.; Kakunaga, T.  
JOURNAL NAME- Carcinogenesis  
VOL. 3    NO. 1    1982    PP. 61-70  
CORPORATE AUTHOR- Linus Pauling Institute, Palo Alto, CA 94306
Citation references – 37 

30. Coexpression of a mutant beta-actin and the two normal beta- and gamma-cytoplasmic actins in a stably transformed human cell line.  
Vandekerckhove, J.; Leavitt, J. ; Kakunaga, T.; Weber, K.  
JOURNAL NAME- Cell  
VOL. 22    NO. 3    1980 Dec    PP. 893-9  
DOCUMENT TYPE- Max-Planck Institute, Goettingen, Germany Citation references – 112  

31. Dissimilar modes of expression of beta- and gamma-actin in normal and leukemic human T lymphocytes.  
Leavitt, J. ; Leavitt, A.; Attallah, A. M.  
JOURNAL NAME- J Biol Chemistry  
VOL. 255    NO. 11    1980 Jun 10    PP. 4984-7  
DOCUMENT TYPE- Bureau of Biologics, FDA, National Institutes of Health, Bethesda, MD
Citation references – 57  

32. Expression of a variant form of actin and additional polypeptide changes following chemical-induced in vitro neoplastic transformation of human fibroblasts.  
Leavitt, J. ; Kakunaga, T.  
JOURNAL NAME- J Biol Chemistry  
VOL. 255    NO. 4    1980 Feb 25    PP. 1650-61  
DOCUMENT TYPE- Bureau of Biologics, FDA, National Institutes of Health, Bethesda, MD
Citation references – 130  
THESIS – Doctor of Philosophy, Biochemistry “Stimulation of In-vitro RNA Synthesis by Ribosomes.” University of Pittsburgh, School of Medicine 1971. Sponsor – Kivie Moldave, Chairman of the Department of Biochemistry.

PRODUCTIVE COLLABORATIONS (leading to major papers) – Aaron Bendich, Memorial Sloan Kettering Inst. NY, Klaus Weber, Max-Planck Inst., Goettingen GR, Takeo Kakunaga, National Cancer Inst., Bethesda MD, Carl Merril, Natl. Inst. Mental Health, Bethesda MD, David Goldman, Natl. Inst. Mental Health, Bethesda MD, Larry Kedes & Peter Gunning, Stanford Univ., Brian Crawford, Los Alamos Natl. Laboratory, Ueli Aebi, Biozentrum, Univ. Basel, Reudi Aebersold & Lee Hood, California Inst. Technology, Paul Matsudaira, Whitehead Inst. MIT, Alison Adams, Univ. Arizona, Charles Lockwood & Fred Schatz, Mount Sinai Medical Center NY

PUBLISHED PATENTS

L-PLASTIN PROMOTER REGION AND ITS USES FOR REGULATING GENE EXPRESSION
INVENTOR(S) LEAVITT, JOHN C .   
PCT INT. APPL., AUG 04 1994, 66 PP.   
PATENT NUMBER- 94 17182   
PATENT APPLICATION NUMBER- 94-US436   
DATE FILED- JAN 25 1994   
DOCUMENT TYPE- PATENT   
PATENT ASSIGNEE(S)- RESEARCH INSTITUTE OF THE PALO ALTO MEDICAL FOUNDATION   
PATENT APPLICATION PRIORITY- 930126US9167   
DESCRIPTOR(S)- PLASTIN PROMOTER PROGESTERONE ESTROGEN GENE EXPRESSION

Plastin isoforms and their uses
INVENTOR(S)- Leavitt, John C .; Lin, Ching-Shwun; Aebersold, Ruedi H.   
PATENT NUMBER- 05360715   
PATENT APPLICATION NUMBER- 642983   
DATE FILED- 1991-01-10   
PATENT DATE- 1994-11-01   
NUMBER OF CLAIMS- 16    
PATENT CLASS- Invention (utility) patent   
INVENTOR COUNTRY/ZIPCODE- .; .; CAX   
PATENT ASSIGNEE(S)- California Institute of Technology   
ASSIGNEE CITY- Pasadena CA   
ATTORNEY, AGENT, OR FIRM- Skjerven, Morrill, MacPherson, Franklin & Friel   
 
Plastin isoforms and their use
INVENTOR(S)- Leavitt, John C .; Lin, Ching-Shwun; Aebersold, Ruedi H.   
PATENT NUMBER- 05002870   
PATENT APPLICATION NUMBER- 495256   
DATE FILED- 1990-03-16   
PATENT DATE- 1991-03-26   
NUMBER OF CLAIMS- 4   
SUPPLEMENTARY NOTE(S)- 25611   
PATENT CLASS- Invention (utility) patent   
INVENTOR COUNTRY/ZIPCODE- .; .; CAX   
PATENT ASSIGNEE(S)- California Institute for Medical Research   
ASSIGNEE CITY- San Jose CA   
ATTORNEY, AGENT, OR FIRM- Terlizzi, Laura    

beta-Actin gene and regulatory elements, preparation and use.   
INVENTOR(S)- Leavitt, J. C. ; Kedes, L. H.; Gunning, P. W.   
PATENT NUMBER- EP 0174608   
PATENT DATE- 1986 19 Mar   
DOCUMENT TYPE- Patent   
CORPORATE AUTHOR- Leland Stanford Junior University, USA   
PATENT PRIORITY INFO- Appl. US 650958, filed 13 Sep 1984   
PATENT TYPE- European Patent Appl.   
ATTORNEY, AGENT, OR FIRM- Bert Rowland
(licensed to the Biotech Industry 1985-2002)