Figure 1 Daniel Linzer at Princeton in 1979. Figure 1 Daniel Linzer at Princeton in 1979. As a first-year graduate student in the fall of 1976, I completed a research rotation on DNA tumor viruses in Arnie Levine's laboratory at Princeton and then decided on that research area and research group for my doctoral work. Arnie welcomed me into his laboratory with his typically infectious enthusiasm and high energy. At the time, graduate students and postdoctoral fellows in the group were working on a number of DNA viruses. Among those was simian virus 40 (SV40), and Arnie pointed me toward a set of questions on this virus's infectious cycle in monkey cells and its ability to induce tumor formation in mice. It turned out that these separate questions converged. One question concerned the identification of proteins encoded by the very small SV40 genome. Animals with SV40-induced tumors produced antibodies that could be used to bind to and precipitate these viral proteins. SV40-infected cells could be labeled with a radioactive amino acid, the proteins extracted and mixed with the antiserum, and finally the immunoprecipitated, radioactively labeled proteins identified as bands on gels exposed to X-ray film. Angie Teresky, Arnie's long-time research associate and laboratory manager, was an expert at producing these antisera, and she provided me with sera from several tumor-bearing mice to start my analysis. The large tumor (T) antigen had been reasonably well characterized already by the approach of tumor serum immunoprecipitation of virus-infected cell extracts. More recently, a smaller protein recognized by tumor antibodies had been discovered and was referred to as the small tumor (t) antigen. For the closely related DNA tumor virus, polyoma, a third protein had been described and called middle T antigen. The expectation in the Levine laboratory (and probably in most DNA tumor virus laboratories) was that SV40 would similarly encode a middle T antigen of ~50?kDa. So, I began a search for that presumed SV40 middle T antigen. A second question was to identify the biochemical steps in the infection pathway by comparing cells in which SV40 infection proceeded to different extents. Previous research had shown that SV40-infected mouse cells failed to produce mature virus particles and furthermore that the number of steps in the infectious cycle completed after introduction of SV40 into mouse fibroblasts differed from the number of steps completed after introduction of the virus into a set of tumor cells known as embryonal carcinomas. Why would the infection fail in mouse cells but go to completion in monkey cells, and why would the infection proceed to two different degrees in non-tumor mouse fibroblasts compared to these mouse tumor cells? The third question was how the SV40 tumor antigens, especially large T antigen, interfered with normal cellular regulation to convert a non-tumor cell into a tumor cell. At the time, much of the excitement around DNA tumor viruses was that they might serve as a molecular window into the transformation of a well-behaved cell into a cancer cell. But how? Surely, the SV40 tumor antigens must interact with the cellular growth regulatory machinery, yet it remained to be determined if T antigen, and perhaps other tumor antigens, altered the expression of certain cellular genes, stimulated a higher level of activity of certain enzymes, or interfered with the function of certain proteins. Some 40?years later, these questions might in hindsight seem straightforward to attack. At the time, of course, our (or maybe it was just my) understanding of the complexities of the SV40-infected cell and the toolbox of analytical techniques that could be deployed were limited. In any event, I struggled to make headway. I do not recall any sense of frustration, though, primarily because Arnie was such an effective cheerleader. It did not matter if the results of an experiment were negative; his response was to praise what did work (`Hey, that is a great looking gel!’). And, if I (or someone else in the laboratory) happened on a potentially interesting result, the response might be to get a group together to go celebrate with a beer at the student center (where, more often than not, Arnie would intend to treat, only to find his wallet empty). That constant support and enthusiasm, coupled with the excitement of kicking around ideas in an informal manner in the laboratory or in Arnie's office or at the student center at any hour, made it fun to work in the laboratory. It therefore came as a shock to me that one day when I brought to Arnie a dripping wet film fresh out of the darkroom that his reaction was `I don't believe it.’ Not, `I don't believe it! Wow, this is great!’ Rather, `I don't believe it’ with the tone saying `this can't be right.’ Arnie, the master motivator, recognized the potential importance of the result, but by reacting as he did, he lit a fire under me to demonstrate that this result wa
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