John Leavitt
[Ed Note: This is part one of a two part series of guest posts written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]
There was an article about Linus Pauling in Time magazine in early 1981 about the fact that at the age of 80 he was still seeking a grant from the National Institutes of Health (NIH) to fund his research on ascorbic acid for treating diseases. This news caught my attention and I looked into the possibility of joining Dr. Pauling’s institute. Toward the end of the summer I was invited to visit the Pauling Institute in Palo Alto, CA to give a seminar on my research at NIH.
In late August Koloman Laki, an aging scientist at NIH, called me up and invited me over to his lab in NIH Building 10, a short walk across the campus from my lab in NIH Building 37. He was interested in talking to me about my recent discovery of mutations in human non-muscle cytoskeletal actin that was published in Cell in late 1980. This protein is the major architectural protein of all eukaryotic cells and we had shown that it was the most highly conserved protein in evolution of the species from yeast to humans. This fact made these mutations even more interesting.
Koloman was a protege of the Hungarian Nobel Prize winner Albert Szent-Györgyi who, I later learned, was much admired by Dr. Pauling because he had discovered both vitamin C and actin. Koloman described how Szent-Györgyi discovered muscle actin. When I mentioned that I was to visit the Linus Pauling Institute in late September, he told me about Emile Zuckerkandl’s and Dr. Pauling’s work on the ‘biological clock,’ which provided evidence in support of Charles Darwin’s theory on divergence of the species.
In the last week of September I flew to Oakland, CA and was picked up at the airport by Emile who was President of the Linus Pauling Institute of Science and Medicine. The next morning I stood up in front of Dr. Pauling and the institute staff to tell them about my discovery of a mutant human beta-actin and my speculation on its involvement in neoplastic transformation. The evidence suggested that I had actually discovered at least two mutations in the same gene, each of which caused a progression to a higher malignant state.
Linus Pauling was in the front row and was all smiles. He asked me if I knew who discovered actin. I was prepared to answer that question thanks to Koloman Laki. In the afternoon I met with Emile who offered me a Senior Scientist position at the Institute, which I accepted. At the time it would be me and Dr. Pauling with separate research interests. Nevertheless, Dr. Pauling could appreciate my discovery as, 23 years earlier, he had described the molecular basis for sickle cell anemia, which predicted that mutations in hemogloblin governed the sickled shape of red blood cells which caused the disease, sickle cell anemia. Likewise ,human cancer cells exhibit altered shapes.
So I resigned my secure job-for-life at NIH and moved to Palo Alto to join the struggling Linus Pauling Institute. My technician, Patti Porecca, hired from Bob Gallo’s lab at NIH, would follow me to the Pauling Institute.
Cloning of the Human Beta-Actin Gene
After I arrived at the Pauling Institute, two of my colleagues at NIH and I published a comprehensive study of the changes in protein expression between normal and neoplastic cells in Carcinogenesis using high-resolution computerized microdensitometry to analyze the complex protein patterns (my first paper from the Pauling Institute). This was the first time that such a study had been published, e.g. the comparative profiling of expression of a large number of proteins in neoplastic cells. It was a study of the 1,000 most abundant proteins in normal and neoplastic human cells which revealed potential biomarkers and causative genetic events for human cancer. At the time it was staggering to view these patterns but perfect for my dyslexic brain and mind’s eye. In addition, we published another paper in Cell that described, for the first time, the progression of a neoplastic human cell to a higher malignant cell following a second mutation in the same beta-actin gene. Early in 1982, Steve Burbeck and Jerry Latter at the Institute set up the same computerized microdensitometry platform I had exploited at NIH.
Jerry Latter gave a stirring talk at Argonne Labs in Chicago demonstrating that computerized microdensitometry of protein profiles could be used to determine the identities of unknown proteins based upon determining their amino acid compositions in situ in protein profiles. This paper was published in Clinical Chemistry in 1984. At the same meeting, Steve Burbeck described a truly innovative invention that could measure beta-particles emitted from radioactive protein profiles to produce a direct image of the protein profile pattern. As a group we had entered an exciting period of discovery and innovation at the Linus Pauling Institute.
When I got to Palo Alto in December 1981, I called Professor Larry Kedes at Stanford and we embarked on a collaboration to clone the human beta-actin gene. His impressive postdoctoral fellow, Peter Gunning, taught me some basic recombinant DNA techniques, and I was off to the races. The difficulty was to identify the functional gene in a sea of actin pseudogenes (sometimes referred to as junk DNA). I used an elegant method of homologous recombination developed in Tom Maniatis’ lab at Harvard that had never been used before to clone a novel gene (In fact, cloning of human genes was just getting started at the time). This was smart because Professor Maniatis would be the chairman of the NIH study section that reviewed my first grant proposal submitted from the Pauling Institute. I did not know it at the time but within a month or two I had cloned the functional beta-actin gene a week before Christmas in 1982.
I developed a scheme to identify the correct gene among 300-400 clones of pseudogenes that Patti and I had cloned and the strategy worked. We gave Dr. Sun-Yu Ng the task of sequencing the DNA clone that we were betting on. Rather quickly we determined that we had cloned the functional human beta-actin gene because the DNA sequence that Sun-Yu determined from our candidate clone accurately encoded the amino acid sequence of human beta-actin protein that I had published in Cell in 1980 (with Klaus Weber). Quite coincidentally another lab discovered a rat oncogene that was a fusion of part of an actin gene with a tyrosine kinase gene. I sent this information off to the study section that was reviewing my grant in January 1984 as added evidence that the actin gene was in some way relevant to neoplasia.
My colleagues and I at the Pauling Institute and Stanford published our successful isolation of both the mutant and wildtype human beta-actin genes in Molecular and Cellular Biology in October 1984. As shown below, we had given Armand Hammer’s name to our cancer research program because of his generosity in helping to fund the Linus Pauling Institute.
In January 1984 I was awarded a grant of about $110,000 a year for two years from the American Cancer Society…what a relief. Later in the spring I received word from Professor Maniatis’ NIH study section that our program would also be funded in June by a grant of about $150,000 a year for 3.5 years from the National Cancer Institute for the same work. I was able to hire Dr. Ching Lin from Iowa State University and Dr. Ng (Sun-Yu) from Kedes’ lab. By 1985 Sun-Yu finished the complete DNA sequencing of the human beta-acid gene and Ching sequenced the copy of the beta-actin gene that had two mutations to formally prove the mutations at the level of the gene. Everything that we had learned about the genetic code and amino acid sequences of proteins made our findings predictable. I had learned from my own research how Darwin’s theory of evolution and natural selection worked.
This was the year I finally successfully transferred in recombinant gene inside a cell in culture. I transferred the mutant human actin gene into a rat fibroblast cell line to show that I had cloned the functional gene which could abundantly express its protein the way the natural endogenous beta-actin gene worked (shown in a protein profile below).
At this point I had a brief meeting arranged by Emile with Alex Zafferoni, founder and CEO of Alza Corporation, a block away on Page Mill Road. Zafferoni recommended Bert Roland as a patent attorney. I arranged a meeting with Roland, also a block away, for that afternoon to discuss patenting the human beta-actin gene promoter because of its strong constitutive nature (the engine of the gene that drives its expression). I told Bert that this was a collaboration with Peter Gunning and Larry Kedes at Stanford. Roland was famous for filing Boyer’s and Cohen’s genetic engineering patent which created Genentech and eventually funded Stanford with hundreds of millions of dollars.
We published Sun-Yu’s work on the sequence, structure, and chromosomal location (chromosome 7) of the human beta-actin gene in Molecular and Cellular Biology and we published Ching’s work locating three mutations in this gene in the Proceedings of the National Academy of Sciences, sponsored by Linus Pauling. A patent was filed on the beta-actin promoter and over the years it was licensed to about 15 biotech companies by Stanford University. This patent was prosecuted for the full 17 years (the life of a patent) but never issued. The Institute’s first royalty check was about $10,000 in 1986, but most of the royalties were earned by Stanford’s patent attorneys.
Peter, Larry and I published a paper in PNAS on the use of the human beta-actin gene promoter for expression of other genes. This vector was distributed to anyone who asked for it – and many did – and to those companies that licensed the invention. At last count this paper had more than 1,000 reference citations.
Our paper popularized the actin promoter as a strong constitutive promoter of foreign gene expression. Soon the rice actin promoter would be used to make Round-up Ready crops by DeKalb Genetics and Monsanto, and giant tilapia fish would be engineered with growth hormone under the control of the fish beta-actin promoter. There were even fluorescent mice running around in Japan created with firefly luciferase expressed by the beta-actin promoter (which I called “the cat’s meow”). Since cytoplasmic actins are the most abundant proteins in most cells you could use the promoter to abundantly express foreign genes in most cells of any animal.
In 1987 we also published the culmination of my research on the mutant beta-actin gene in Molecular and Cellular Biology. When I introduced this gene into non-tumor forming immortalized human fibroblasts they became tumorigenic. The results showed that the more abundant the expression of the mutant beta-actin, the more tumorigenic the non-tumorigenic cells became and the cells that came out of the tumors were enhanced further in the level of mutant beta-actin expression. This was a sensational finding that was the goal of research which began with the discovery of the mutant beta-actin in 1978 at NIH.
