LASKER~KOSHLAND SPECIAL ACHIEVEMENT IN MEDICAL SCIENCE AWARD ESSAY On the road from classical to modern molecular biology Tom Maniatis I graduated from the University of Colorado in a Boulder in 1965, and, while browsing through the university bookstore, I noticed a new book called Molecular Biology of the Gene by James Watson. When I read it, I was captivated by the elegance and power of an exciting new field that combined structure, genetics and biochemistry. Delving more deeply, I was fas- cinated by the classic paper of Francois Jacob and Jacque Monod on the operon theory and the subsequent identification of the Lac and l phage repressors by Walter Gilbert and Mark Ptashne, respectively. These breakthroughs in our understanding of gene regulation in b prokaryotes, which were based on a combi- nation of ingenuity, genetics and biochemis- try, defined classical molecular biology and inspired me to seek a PhD in the new field and, ultimately, to study the mechanisms of gene regulation in eukaryotes. On the basis of my interest in molecular biology, Joseph Daniel, my undergraduate research advisor at Colorado, suggested that I contact Leonard Lerman, who had recently moved from the University of Colorado Medical School to the new Department of Figure 1 Photos of me and my colleagues taken circa 1976–1978. (a) Tom Maniatis, Richard Axel and Molecular Biology at Vanderbilt University. Hal Weintraub. (b) Argiris Efstratiadis and Fotis Kafatos. Courtesy of the Cold Spring Harbor Archives. I took Joe’s advice and ultimately joined Leonard’s lab, where I studied the structure of My postdoctoral project was the characteriza- and mentor. Fred’s quiet brilliance and modesty highly compacted DNA by small-angle X-ray tion of the l repressor/operator protein–DNA affected everyone fortunate to work with him. scattering. Leonard was an excellent men- complex. I found that the repressor binds coop- It was a particularly exciting time in Fred’s lab, tor who encouraged critical thinking, and I eratively to multiple adjacent DNA sequences in as he and Allen Coulson were developing the enjoyed learning a new field. Toward the end the l operators and that mutations in these sites Sanger sequencing method (and back home, at of my thesis research, Mark Ptashne visited weakened DNA binding in vitro and increased Harvard, Allan Maxam and Walter were devel- Vanderbilt, and during his dinner with the expression in vivo. This early use of restriction oping an alternative approach to sequencing). graduate students I had the opportunity to enzymes and DNA binding assays presaged As these methods were still in development, discuss my interests in his work. That conver- my later studies of eukaryotic promoters and Mark and I worked with Bart Barrell and John sation ultimately led to an offer to join Mark’s enhancers. To determine the DNA sequence Donelson using an older, more cumbersome lab at Harvard for postdoctoral studies. of the l operators, Mark trapped Fred Sanger procedure to determine the DNA sequence of a on a ski lift at Keystone and coerced him into 27–base-pair λ operator, which required nearly Tom Maniatis is in the Department of Biochemistry hosting us in his lab at the UK Medical Research a full year to complete. Over the next several & Biophysics, Columbia University, New York, Council laboratory in Cambridge. We were ini- years, Mark and his lab went on to show how New York, USA. tially the unwelcome American duo, but Fred the organization of the operators has a funda- e-mail: [email protected] quickly warmed to us and was a gracious host mental role in the l ‘genetic switch’. I benefited NATURE MEDICINE VOLUME 18 | NUMBER 10 | OCTOBER 2012 xxix ESSAY Globin mRNA Plasmid PMB9 agated in bacteria, and Dale Kaiser and Paul 5ʹ 3ʹ AAAA Berg’s labs had shown that double-stranded dT 15 Reverse DNA molecules could be joined by dA:dT transcriptase joining. David Hogness exploited this method to clone and characterize random fragments of AAAA dT Drosophila genomic DNA, and his laboratory cDNA 15 EcoRI went on to transform Drosophila developmen- Alkali 5ʹ 3ʹ tal biology through the elegant application of genetics and genomic cloning methods. dT Our strategy for cDNA cloning was to 15 λ exonuclease perfect a method for generating full-length DNA polymerase I double-stranded cDNA, which could then be inserted into a plasmid by dA:dT tailing. cDNA Terminal However, our goal of cloning cDNAs was sDNA transferase derailed by the recombinant DNA controversy in Cambridge, Massachussetts, which led to a SI nuclease dA 100 moratorium on recombinant DNA research in dA 100 July 1976. The moratorium was the result of a complex mix of genuine concerns for safety, Anneal Double-stranded post-Vietnam social activism, historic town- globin DNA and-gown antagonisms, city and scientific poli- tics and intellectual dishonesty. The latter led to λ Exonuclease AA an infamous recital of a ‘slow-death scenario’ A T T A TT A T T A T at a city council meeting by an opponent of the A A T T T A T A research in the guise of providing ‘scientific’ advice to the mayor. The end result was that we Terminal transferase were unable to insert our in vitro–synthesized DNA into a bacterial plasmid. dT 100 Fortunately, however, we were rescued dT 100 by Jim Watson, who offered me a place to work at the CSHL, where recombinant DNA Figure 2 Schematic illustrating the synthesis and cloning of double-stranded cDNA from purified rabbit b-globin mRNA using the sequential activities of reverse transcriptase and DNA polymerase I. Reverse research was allowed. CSHL was a beehive transcriptase generates a short hairpin that functions as a primer for second-strand synthesis. S1 nuclease of activity when we arrived. Jim Watson had cleaves the hairpin, the ends are prepared for the terminal transferase reaction by l exonuclease treatment, recruited an exceptional group of young and oligo-dT is added to the ends. The plasmid PM9 vector is cleaved by EcoRI and subjected to oligo-dA investigators, including Joe Sambrook, Phil addition by terminal transferase. The cDNA is annealed to the plasmid DNA through dA:dT base pairing, Sharp, Rich Roberts, Mike Botchan and and the hybrid DNA molecule is introduced into E. coli. Redrawn from ref. 2. many more. Restriction enzymes were being used to create genetic and physical maps of enormously from my time in Mark’s lab. Mark ball shoes, and only rarely could we accom- viral and eukaryotic genomic DNA in Joe was an excellent and intellectually demanding modate Richard’s expensive tastes like dining Sambrook’s lab, and these tools contrib- mentor, and he deeply influenced my think- at a Michelin-rated restaurant. On one occa- uted to the discovery of RNA splicing by ing and writing. He is the best I have known sion, Hal appeared very much out of place in a Rich Roberts and his colleagues, who were at designing clever, incisive experiments that spectacular restaurant when an elegant French also producing dozens of new restriction address deep, mechanistic questions. gentlemen sitting at the next table turned to enzymes (RNA splicing was independently While still a postdoc at Harvard, I attended Hal and said “nice shoes.” The gentleman was discovered in Phil Sharp’s lab at MIT). CSHL the 1976 Cold Spring Harbor Symposium on François Mitterrand, the President of France. also provided an endless source of new infor- chromatin. This meeting would profoundly Hal was the most imaginative and creative mation and interesting people and science influence the future directions of my science biologist I have known. through its meetings program. It was in this and lead to life-long friendships. During the Upon returning to Harvard from the UK, atmosphere that we succeeded in generating annual lobster bake on the Cold Spring Harbor I met Argiris (Arg) Efstratiadis, who was a and thoroughly characterizing the first full- Laboratory (CSHL) beach, I met Richard Axel graduate student (and MD) in Fotis Kafatos’s length cDNA clones of b-globin mRNA1,2. and Hal Weintraub (Fig. 1a). I felt an imme- lab at Harvard (Fig. 1b). Arg and I connected This required the purification of a number diate intellectual and personal connection immediately, and I came to enjoy his humor, of enzymes that were not commercially avail- with them, and we remained close scientific intellectual intensity and madness. An initial able, the observation that reverse transcrip- colleagues and friends until Hal’s untimely brief conversation led to a highly productive tase leaves a short hairpin DNA at the 3ʹ end death at the age of 50 in 1995. We spoke collaboration with Arg and Fotis, the ultimate of the transcript and the establishment of every week on the phone, often late at night goal of which was to clone full-length comple- conditions for cleaving the hairpin by the discussing the latest data, paper or idea. We mentary DNAs (cDNAs). At that time, Herb single-strand–specific S1 nuclease. (Fig. 2). often attended meetings together, including Boyer and Stanley Cohen had shown that a The production and faithful propagation of some in fancy locales overseas. Hal was always eukaryotic ribosomal gene provided by Don full double-stranded DNA clones provided dressed in jeans, t-shirt and high-top basket- Brown could be joined to a plasmid and prop- powerful tools for isolating genes from xxx VOLUME 18 | NUMBER 10 | OCTOBER 2012 NATURE MEDICINE ESSAY genomic DNA and for the production of transient transfection method in collabora- DNA technology to a generation of emerging mammalian proteins (see ref. 1 for a more tion with Yasha Gluzman at the CSHL6 and molecular biologists.