Faculty Profile: Boris Rotman, Chem. Eng. - Ph.D.

Boris Rotman
Boris Rotman, Chem. Eng. - Ph.D.
Professor Emeritus of Medical Science
Molecular Biology, Cell Biology, & Biochemistry

Research Description

Biographical Sketch
A Scientific Career Guided by Empirical Results
"By the time most scientists have reached age thirty they are trapped in their own expertise." (Francis Crick, What Mad Pursuit, 1988)
Certainly, this is not my case. My scientific career is far from typical since it has followed a multidisciplinary trajectory including (in chronological order) Organic Chemistry, Microbiology, Biochemistry, Immunology, Immunogenetics, Oncology, and Biotechnology.
Changing disciplines, however, was not due to any whimsical decision, but resulted from applying major discoveries of my laboratory to solve problems outside of my own research specialty. For example, the methodology for measuring single molecules of β-galactosidase led to discovering a new class of antibodies capable of repairing (activating) inactive mutant enzymes, which, in turn, "force" me into Immunology.
Entering new research fields is most exhilarating if and only if one enjoys steep learning curves and doesn't mind (or is fond of) humbling experiences as a neophyte.
On the negative side, changing disciplines may be harmful to scientific careers because one has to deal with new sets of peers (including grant reviewers) who are unaware of one's past endeavors. Another unattractive aspect is losing contact with scientific friends.
Here, I briefly describe how specific discoveries led me into new research areas. I have listed below pertinent publications and also indicated how they triggered my decisions. For a complete list of publications, please refer to PubMed or Google Scholar at
http://scholar.google.com/citations?hl=en&user=BMr4daMAAAAJ&cstart=40&view_op=list_works&is_public_preview=1
Research field: Mechanisms of protein synthesis in Escherichia coli (1952-53)
Publication Impact
On the origin of the carbon in the induced synthesis β-galactosidase in Escherichia coli.
Rotman, B., and Spiegelman, S. 1954. J. Bacteriol. 68: 419-429. This groundbreaking work answered a central question concerning protein synthesis: Are induced enzymes made from inactive precursors?
This work was central to my obtaining a post-doctoral position in Joshua Lederberg's laboratory at Madison, WI (http://www.nobelprize.org/nobel_prizes/medicine/laureates/1958/lederberg-bio.html). My project was to investigate a puzzling phenomenon: why intact cells of E. coli exhibit less than one percent of their intracellular β-galactosidase activity.
The dilemma was solved two years later by the discovery of the first bacterial transport system termed lactose permease (LacY) in Monod's laboratory. (http://www.nobelprize.org/nobel_prizes/medicine/laureates/1965/monod-bio.html).
In 1955, I moved to the Medical School of the Universidad de Chile where I continued studying the LacY permease. Our work led to the discovery of a second permease, the methyl-β-D-galactoside (MeGal) permease that turned out to be mechanistically different from the LacY (see article below). Subsequently, our laboratory studied the genetics and mechanism of the MeGal for about 25 years.
New research field: Transport systems (Permeases) in E.coli (1955-1980)
Publication Impact
Separate permeases for the accumulation of methyl-β-D-galactoside and methyl-β-D-thiogalactoside in Escherichia coli. Rotman, B. 1959. Biochim. Biophy. Acta 32:599-601. The MeGal permease was the first transport system of a broad superfamily (across phyla) presently known as ABC transporters.
New research field: Fluorogenic substrates for enzymes (1960-present)
While still a post-doctoral fellow at Lederberg's lab, I thought of improving the sensitivity of the β-D-galactosidase assay (which was done with ONPG (o-nitrophenyl- β-D-galactoside), a chromogenic substrate) by using a newly synthesized substrate, fluorescein di-β-galactoside (FDG). I coined the term "fluorogenic" (in analogy to chromogenic) for enzyme substrates that are not fluorescent per se but produce fluorescent products upon enzyme action. At present, there are hundreds of fluorogenic substrates for many diverse biological applications.
FDG remained a curiosity on a shelf of my laboratory for about five years,. However, I enjoyed showing to visitors how an FDG solution becomes instantaneously fluorescent in the presence of the enzyme.
A breakthrough occurred in 1959 while working as Research Associate at Harvard Microbiology Department. In a seminar presented to a group of Boston biochemists, I discussed a speculative feat of measuring the activity of individual molecules of β-galactosidase by using FDG in a microfluidic device. Surprisingly, the seminar was well remembered, and (to my embarrassment) I was often asked "how does the single enzyme molecule coming along?"
For this reason, upon my return to the States in 1960, I began to work in earnest on developing the single molecule assay. I didn't make much progress until a second breakthrough occurred during a visit to the Medical Research Council (MRC) at Mill Hill (North London). I was fortunate to meet John F. Collins who had developed a novel, simple microfluidic system for measuring penicillinase in single cells of Bacillus licheniformis. When we applied the Collins microfluidic system to measure β-galactosidase in the presence of FDG, we were highly surprised to discover that the system was not only capable of measuring single E. coli cells but also individual molecules of the enzyme!

Publications Impact
Measurement of activity of single molecules of β-D-galactosidase. Rotman, B. 1961. Proc. Natl. Acad. Sci. USA 47, 1981-1991.
Fluorogenic substrates for β-D-galactosidase and phosphates derived from fluorescein (3, 6-dihydroxyfluoran) and its monomethyl ether. Rotman, B., Zderic, J.A., and Edelstein, M. 1963. Proc. Natl. Acad. Sci. USA 50, 1-6. The 1961 article demonstrated for the first time the feasibility of measuring single molecules of β-galactosidase using microfluidics and fluorogenic substrates. The second publication extended fluorogenic substrates to other enzymes.
About 30 years later there was a surge of interest in studying enzymes as single molecules (see V. I. Claessen et al. Single-Biomolecule Kinetics: The Art of Studying a Single Enzyme (2010) Ann. Rev. Anal. Chem. 3: 319-340).
New research field: Fluorochromasia in mammalian cells
In 1965, Ben W. Papermaster, a post-doctoral fellow at Stanford Medical School, made a short visit to our laboratory to explore the possibility of measuring β-galactosidase activity in single mouse lymphoma cells.
I vividly recall the occasion. It was a Saturday afternoon and Ben was busy doing experiments. Out of curiosity since I had never seen under the microscope a mammalian cell, I mixed lymphoma cells with a series of newly synthesized fluorogenic substrates. To our great surprise, the cells that had been contact with fluorescein diacetate (FDA) for a few minutes had become highly fluorescent!
At first, both Ben and I thought that our observation was not unique because FDA was known since 1871. However, after thoroughly searching the literature, we concluded that we were dealing with an unprecedented cell membrane phenomenon and called it "fluorochromasia" for "becoming fluorescent."
Publication Impact
Rotman, B., and Papermaster, B.W. (1966). Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci USA 55, 134-141. The outstanding feature of fluorochromasia is that only occurs in living cells that have a healthy membrane. Consequently, fluorochromasia has been widely used (more than 25,000 papers) to easily distinguish between living from dead cells. Over the years, fluorochromasia has been shown to occur in all types of cells including mammalian, microbial, and vegetal.

New research field:Immunology, Immunogenetics
The discovery of fluorochromasia was pivotal to my becoming interested in tumor cells. In 1966, I took a sabbatical leave at the Tumor Biology Institute of the Karolinska Institute. There, I learned about tumor cells first hand since I shared a lab with Gorge Klein, the head of the Institute.
At the Karolinska, I met Franco Celada, an immunologist working on tissue transplantation in mice. Franco and I used fluorochromasia to develop a new immune cytotoxicity assay (Celada, F., and Rotman, B. (1967). A fluorochromatic test for immunocytotoxicity against tumor cells and leucocytes in agarose plates. Proc Natl Acad Sci USA 57, 630-636) that was used as a model for human histocompatibility testing.

Grants and Awards

Rhode Island Governor's Award for Scientific Achievement, 1990.

Emeritus Professor, School of Medicine-Brown University, July, 1990.