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• W2074549262 abstract "In many ways, I went from being on the outside with a smattering of techniques to the inside of the world of molecular biology with the study of RNA processing. Much of my current work grew out of what I learned as a graduate student and as a postdoctoral fellow. In that I was very lucky. I studied with masters of DNA starting in the decade that began with the Watson-Crick model of DNA. My graduate work was done with Leonard Lerman as my thesis advisor and my first postdoctoral experience was in Matthew Meselson's laboratory. How I wandered into molecular biology and my second postdoctoral experience with Sydney Brenner and Francis Crick are described elsewhere (1Altman S. Genetics. 2003; 165: 1633-1639Crossref PubMed Google Scholar). It is appropriate to begin with my experiences in Lerman's laboratory in the small Department of Biophysics, chaired by T. T. Puck, at the University of Colorado Medical Center in Denver. Leonard Lerman discovered the intercalation of acridine derivatives in DNA (2Lerman L.S. J. Mol. Biol. 1961; 3: 18-30Crossref PubMed Scopus (1757) Google Scholar, 3Lerman L.S. Proc. Natl. Acad. Sci. U. S. A. 1963; 49: 94-102Crossref PubMed Scopus (507) Google Scholar, 4Lerman L.S. J. Mol. Biol. 1964; 10: 367-380Crossref PubMed Scopus (57) Google Scholar). In my view, that was an astounding finding, partly because one knew so little about how small molecules bound to DNA in a quite specific manner and also because of the biological relevance of the discovery. Critical experiments that proved that the acridines intercalated occurred when Lerman was on sabbatical at the MRC Laboratory of Molecular Biology in Cambridge, England in 1960. At that time, Crick and Brenner and their colleagues were carrying out the monumental experiment that showed that the genetic code was triplet in nature (5Crick F.H.C. Barnett L. Brenner S. Watts-Tobin R.J. Nature. 1961; 192: 1227-1232Crossref PubMed Scopus (622) Google Scholar). Actually, Lerman's experiments on intercalation gave them some notion of the kind of mutations that the acridines might produce. Crick and Brenner masterly used the idea of addition and deletion mutations to explain their data. Over the next few years, Lerman proved aspects of the intercalation model in many ways. Originally, he used viscosity and some x-ray diffraction data to confirm the model, and later he also used the lack of reactivity of amino groups on acridine derivatives to further the proof. I met Lerman, in a serendipitous manner, in the spring of 1962 when I was inquiring about the prospects for a potential graduate student in the University of Colorado department in Denver and a description of the work done there. Shortly after our meeting, I was accepted as a student in Denver. The department had six or seven faculty members and one very active student, Barry Egan. In a way, it was a classic research department: no courses; everybody was in the laboratory working. That was an ideal change for me because I had left a very large physics department in which there was little opportunity for new students to do any laboratory work... and so began my education as a student of molecular biology. Before I continue with my own view, faulty as that can be at this time, it is worthwhile documenting some of the other accomplishments of Lerman's. As a postdoctoral fellow with Leo Szilard, he invented affinity chromatography on columns to purify mushroom tyrosinase (6Lerman L.S. Nature. 1953; 172: 635-636Crossref PubMed Scopus (18) Google Scholar). Subsequently, he plunged into studies of DNA in the late 1950s. Lerman and Tolmach succeeded in labeling transforming DNA with 32P and demonstrated that the radioactivity was incorporated into a genetically transformed strain of Pneumococcus (7Lerman L.S. Tolmach L.J. Biochim. Biophys. Acta. 1957; 26: 68-82Crossref PubMed Scopus (34) Google Scholar, 8Lerman L.S. Tolmach L.J. Biochim. Biophys. Acta. 1959; 33: 371-387Crossref PubMed Scopus (25) Google Scholar). These were bold experiments in a time when DNA was not yet routinely treated as both a biological and physical material at the same time, notwithstanding the earlier Avery et al. (9Avery O.T. MacLeod C.M. McCarty M. J. Exp. Med. 1944; 98: 451-460Google Scholar) and Hershey and Chase (10Hershey A.D. Chase M. J. Gen. Physiol. 1962; 36: 39-56Crossref Scopus (605) Google Scholar) experiments. Shortly after I started in work in Lerman's laboratory, some pieces of small equipment had to be purchased. This was a puzzle for me; I was sent out to buy a particular kind of screwdriver. As I recall, there were two prices in the hardware store I visited, $7 and $17. The agony of choice consumed me and forced me to call Lerman. His answer to the question I posed represented his attitude as a teacher perfectly: “You decide.” Fortunately, we agreed on a thesis topic rather soon—the effect of acridine derivatives on T4 DNA replication. The idea behind the topic was to understand how the acridines acted as mutagens. As a consequence, I became extremely familiar both with the acridines and phage T4 over the next few years (11Altman S. Lerman L.S. J. Mol. Biol. 1970; 50: 235-261Crossref PubMed Scopus (37) Google Scholar, 12Altman S. Lerman L.S. J. Mol. Biol. 1970; 50: 263-277Crossref PubMed Scopus (10) Google Scholar). This was extremely important for both my postdoctoral experiences, and certainly I also became adept at various ultracentrifugation techniques, including buoyant density centrifugation. An additional and important teacher during my graduate years was Rose Litman, a research associate affiliated with the Lerman laboratory. Rose had characterized the mutagenic effect of bromodeoxyuridine during her thesis work (13Litman R.M. Pardee A.B. Virology. 1959; 8: 125-127Crossref PubMed Scopus (0) Google Scholar) and then went on to study transformation with Harriet Ephrussi-Taylor. Later, she characterized a UV-induced DNA polymerase by an affinity method (14Litman R.M. J. Biol. Chem. 1968; 243: 6222-6233Abstract Full Text PDF PubMed Google Scholar) very similar, and simultaneous with, the method that Alberts devised for studying T4 DNA polymerase (15Alberts B.M. Amodio F.J. Jenkins M. Gutmann E.D. Ferris F.L. Cold Spring Harbor Symp. Quant. Biol. 1968; 33: 289-305Crossref PubMed Scopus (254) Google Scholar). Alberts had used DNA cellulose that had been prepared by heating whereas Rose used a DNA cellulose that had been prepared by UV radiation. Rose was a warm and very literate colleague and a good teacher. Several students had come to the department after I joined it, and Rose gave a course essentially on molecular genetics; it was excellent. Indeed, the course was so stimulating that it encouraged me to study genetics, including Drosophila genetics, on my own. My studies of acridine-treated T4 DNA showed very quickly that the DNA was made in foreshortened pieces, possibly because of acridine inhibition of DNA molecules with many single-stranded breaks in preparation for recombination. It became a task to understand how the size of the pieces fit in with what we knew of acridine mutagenesis. There was a correlation between molecular weight of the T4 DNA and the concentration of acridines (12Altman S. Lerman L.S. J. Mol. Biol. 1970; 50: 263-277Crossref PubMed Scopus (10) Google Scholar). I did succeed in an interpretation of the experiments, but I think the real explanation of the mutagenic event fell beyond my capabilities at the time. Ripley went further in this regard with respect to the enzymology and function of the acridines during the mutagenic event (16Ripley L.S. Annu. Rev. Genet. 1990; 211: 63-74Google Scholar, 17Kaiser V.L. Ripley L.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2234-2238Crossref PubMed Scopus (18) Google Scholar). Lerman left Colorado in 1965 to join the faculty at Vanderbilt University. A few months later, I joined him (while remaining a student at Colorado) in order to finish up my experiments. I became very friendly with Leonard and his family and our friendship has continued. We became much more than a student-teacher pair. I can only hope that has happened to many graduate students and their advisors. As a final note here, let me also say that when I was applying for a postdoctoral fellowship, after my failure with two private foundations, Leonard suggested that he better take a more liberal attitude toward the truth and, I think, omitted the various faults and warts he could detect in my character. After I left the laboratory, as a consequence of my outrageous attempt first to join the Brenner-Crick group (no room at that time) and then Meselson's group, Tom Maniatis became a student of Leonard's, and they went on to study a collapsed form of DNA, akin to the structure one finds in phage heads (18Maniatis T. Venable Jr., J.H. Lerman L.S. J. Mol. Biol. 1974; 84: 37-64Crossref PubMed Scopus (162) Google Scholar). The methods and analysis used were subtle, and the experiments produced a good version of a collapsed DNA. At this time, the intercalation system was used more and more widely, and the name Lerman began to be forgotten by the new experts on DNA and its manipulations. Lerman's ideas were always novel and creative. With great good fortune, I did succeed in getting a fellowship that enabled me to become a postdoctoral fellow with Matthew Meselson. Matt was, of course, a famous name in molecular biology, having done his great experiment with Frank Stahl (19Meselson M. Stahl F.W. Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 671-682Crossref PubMed Google Scholar) when he was a graduate student. He seemed a rather formal person when I first met him at a meeting in Gatlinburg, Tennessee; very few people wore suits and a tie but Matt did. There was a swimming pool where we were staying, and one evening, Matt was at the side of the pool, looking in, fully dressed. Someone shouted, “Go on Matt, you can do it—just step across the water!” It did seem possible. Matt's description of how he came across buoyant density centrifugation of DNA has been written about extensively (20Holmes F.L. Meselson and Stahl and the Replication of DNA. Yale University Press, New Haven, CT2001Crossref Google Scholar). It was no accident, it seemed, when he understood the problem of separating pieces of DNA made in different generations in bacteria by density labeling with 15N. A lecture by Monod on adaptation of enzymes and his own personal education on rare earth salts as a high school student provided some of the clues for the experimental method of buoyant density centrifugation. Both in this case and Lerman's case with acridines, the combined nature of deep intelligence with a true knowledge of the nature of the physical-chemical properties of DNA in solution allowed for the discoveries to be made. Most importantly, in both Lerman's (2Lerman L.S. J. Mol. Biol. 1961; 3: 18-30Crossref PubMed Scopus (1757) Google Scholar) and Meselson and Stahl's experiments (19Meselson M. Stahl F.W. Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 671-682Crossref PubMed Google Scholar), unique theories were presented regarding an explanation of the data; in both cases, only one possible model fit the data really well. It is important to point out that Lerman was a graduate student of one of Pauling's collaborators at Caltech and Meselson was a graduate student of Pauling himself. Matt Meselson's ideas revolutionized the steps one could take to study DNA (and later RNA and complexes with proteins (21Stark B.C. Kole R. Bowman E.J. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3717-3721Crossref PubMed Scopus (205) Google Scholar)). This breakthrough certainly dwarfed many new methods we see today. Meselson has said that he was only following what the Watson-Crick model suggested in terms of what he wanted to show: first, how does DNA replicate, and second, does breakage and reunion occur in recombinant molecules? The latter was proved, again with the use of buoyant density centrifugation (22Meselson M. Weigle J.J. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 857-868Crossref PubMed Scopus (100) Google Scholar), a few years after the first discovery by Meselson and co-workers. I came to Matt's laboratory as the outsider from the boondocks. Shortly after I arrived, there was some problem in the laboratory with equipment. I was approached immediately as the person responsible even though I had nothing to do with the equipment. I recognized the situation but it did not repeat itself very often. The laboratory was populated by several postdoctoral fellows and a few graduate students. Among the postdoctoral fellows were Ray Kaempfer, Marc Rhoades, Toshio Nagata (a senior visitor), and Bob Yuan. I recall that we were a chummy group but very hard working. Ray had just shown that ribosomal subunits went through a cycle of unattached subunits that assembled into a ribosome and recycled through the subunit state after use in the complete ribosome (23Kaempfer R.O.R. Meselson M. Raskas H.J. J. Mol. Biol. 1968; 31: 277-289Crossref PubMed Scopus (64) Google Scholar). He used a modification of the traditional velocity and buoyant density centrifugation to prove his point. Bob Yuan was working with Matt on the first purification of a restriction enzyme (24Yuan R. Meselson M. Nature. 1968; 217: 1110-1114Crossref PubMed Scopus (483) Google Scholar). Their method, using velocity sedimentation, proved to be very powerful and became a widely used step in general purification procedures. Both Marc Rhoades and I were working on endonucleases coded for by phages λ and T4 (25Rhoades M. Meselson M. J. Biol. Chem. 1973; 248: 521-527Abstract Full Text PDF PubMed Google Scholar, 26Altman S. Meselson M. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 716-721Crossref PubMed Scopus (13) Google Scholar). We found such nucleases, although there certainly was more than one produced by T4. The one I characterized, a nuclease that induced single-stranded breaks in native T4 DNA, was made during the mid-phase of T4 infection, but it was not sufficiently characterized to link to any particular gene sequence at that time. We used column chromatography to purify the enzymes with velocity gradients waiting to be employed too. From my point of view, I learned how to assay for and purify an endonuclease, something with which I became very familiar with on my next stop at the MRC laboratory in the England, and I certainly had a good education in biochemical purification. We had various activities in the laboratory that were amusing. I recall, at the beginning of my stay, trying to calibrate one of Matt's centrifuges with a stroboscope. It required turning all the lights in the laboratory off from time to time. Certainly, not a popular activity. We also had a few sessions of birling on 5-gallon toluene drums. Two reagents added to toluene in the counting of electrons that made the solution fluorescent were poured into the drum and the 5 gallons were mixed, supposedly by hand. With a small amount of balance involved, these drums could be rolled around the floor by someone standing on them and persuading the drums to roll. Ray and I became very good friends, and when his physician wife, Miep, gave birth (after a heavy Italian dinner loaded down with garlic) to their first child I became his godparent. Matt, whose mind worked at an extremely fast pace and seem to outstrip all of us in thinking about scientific problems, was very involved with activities in chemical and biological warfare at the time. This did not hurt the postdoctoral fellows too much because we were accustomed to fewer interactions with our mentor than were the graduate students. We got along and Matt was a pleasure to be with. Although I was trained mainly as a biochemist, I was deeply impressed by the power of genetics both as a consequence of Matt's own work listening to his lucid lectures in a genetics course and speaking to other friends, Mark Ptashne and Vincent Perrotta. This certainly helped in my talks with Matt. After I left the laboratory, Matt became involved with heat shock genes in Drosophila and, more recently, with the evolution of Bdelloid rotifers. At one point during my stay in Matt's laboratory, Sydney Brenner came by for a visit. He now walked with a cane, the result of a serious motorcycle accident in England. He and I and Matt went to lunch at “Au Bon Pain” in Harvard Square. Sydney commented “That's what I have— bon pain.” It was on this visit that Sydney asked if I wanted to come to his laboratory when I finished my time with Matt because there was now room for another postdoctoral fellow. I agreed in a flash. Of course, the topic we had discussed for my work was obsolete (1Altman S. Genetics. 2003; 165: 1633-1639Crossref PubMed Google Scholar) when I arrived in Cambridge, England, but I, myself, soon settled on another: the effects of acridine mutagenesis on tRNA structure! I presumed I knew a lot about acridines and how they worked, and it seemed to be one of the few choices I had, given the general topics of research in the Brenner-Crick group and my limited upbringing in molecular biology. In fact, my view on the world certainly had been colored both by Lerman and Meselson, two individuals who had a wide perspective on the DNA world and who chose only to work on topics of undeniable general importance. Their hardheaded view of how to look critically at experiments was uncompromising. In England, I started making acridine-induced mutations in a suppressor tRNA with the hope that I would generate mutant tRNAs with small additions or deletions in their structure. Although I was not far off from this goal (I made large additions, it seemed), it pushed me on the right road to further, rewarding experiments (27Altman S. Kirsebom L. Gesteland R. Cech T. Atkins J. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999Google Scholar). Initially, I used an acridine half-mustard derivative to make non-suppressing mutants of the tRNA. The mutated tRNA gene products were ultimately examined during infection by phage ϕ80 carrying these genes (28Altman S. Nat. New Biol. 1971; 229: 19-21Crossref PubMed Scopus (81) Google Scholar). In this manner, I discovered the first radioactive tRNA precursor molecule. In the MRC Laboratory, only wide sample well slots in a slab gel were then used, but after a while when I was investigating cell extracts for enzymes that cleaved the tRNA precursor I isolated, a local machinist quickly made template sample well slots that contained at least 12 wells. In fact, I was so excited that now any number seemed possible. Of course, I soon learned that Bill Studier had already developed such “analytical” slab gels concurrently (29Studier F.W. Science. 1972; 176: 367-376Crossref PubMed Scopus (355) Google Scholar). Nevertheless, my previous experience with small T4 DNA molecules induced by acridines and the biochemistry of a T4 endonuclease led perfectly to the required state of mind for analysis of RNase P and the cleavage of tRNA precursor molecules. In fact, one lesson stayed with me until today: if you want to learn what happens to a large molecule (a tRNA precursor, for example) in biochemical analysis of nucleolytic enzymes, you must always have an assay that displays the effect of enzymes on any part of the large molecule. Subsequently at Yale, another lesson from my earlier days led to a convincing experiment that RNase P had an RNA component. One of my graduate students, Ben Stark, had purified that enzyme and had shown that micrococcal nuclease could inactivate the RNase P function by attacking, presumably, an RNA component (21Stark B.C. Kole R. Bowman E.J. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3717-3721Crossref PubMed Scopus (205) Google Scholar). At least one other method of proof was required. To me that meant a physical test in a buoyant density gradient. We were exceedingly lucky that RNase P from Escherichia coli did not dissociate in CsCl, and we measured the buoyant density of the complex at 1.71 g/ml, a reasonable compromise between an RNA and a protein component. Although we were now dealing with an enzyme that had an RNA component, many of the rest of the experiments we did for the next 10 years or so were perfectly straightforward from a biochemical point of view. The enzyme kinetic constants, ion requirements, subunit properties and their function, and the outline of the catalytic center (and nucleotide sequence) became clear as the years passed. What I learned as a graduate student and during my first postdoctoral years was essential in preparing me for what came afterward in the world of science. What I could not learn from anyone was the complete variety, good to bad, of human reactions to a novel idea: an enzyme had a catalytic RNA subunit." @default.
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