MY SCIENTIFIC CAREER FOLLOWED AN ATYPICAL MULTIDISCIPLINARY TRAJECTORY INCLUDING (IN CHRONOLOGICAL ORDER) ORGANIC CHEMISTRY, MICROBIOLOGY, BIOCHEMISTRY, IMMUNOLOGY, IMMUNOGENETICS, ONCOLOGY, AND BIOTECHNOLOGY.
TO LEARN HOW BREAKTHROUGHS OF MY LABORATORY "FORCED" ME TO WORK IN A NEW DISCIPLINE, SEE MY AUTOBIOGRAPHY UNDER CURRICULUM VITAE
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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."
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.