COMMENTARY LASKER CLINICAL MEDICAL RESEARCH AWARD Tools for genomics Edwin Southern Genomics, in its early days, used a range of trifugation, the technique used famously by atmosphere of excitement and kept us abreast techniques that were developed to explore the Meselson and Stahl2 to prove the semiconserva- of developments beyond our own fields of composition and sequence organization of tive nature of DNA replication. The major sat- specialization. the nuclear DNA. High-throughput methods ellites are absent from most genome sequences Michael Swann’s Zoology Department also changed that, and most research in genomics because their structure, which comprises very housed a remarkable number of scientists is now done in factory-like laboratories, with long stretches of tandem repeats, makes clones interested in molecules; there was a strong robots doing much of the work. I have adapted unstable, and sequence assembly from short immunology group under James Howard; http://www.nature.com/naturemedicine to working in this way, but miss the immediacy stretches is nearly impossible. But in 1967, it Murdoch Mitchison studied the cell life cycle of direct involvement with experiments. I want was the simple nature of the sequences that of the fission yeast Schizosaccharomyces pombe; to see the data before the computer does. But made it seem worthwhile to give sequencing Peter Walker headed the Medical Research solving problems at the bench has always given a try. The difficulty that stood in the way was Council Mammalian Genome Unit. Peter was me as much enjoyment as the more cerebral the lack of a method for DNA sequencing. Here a professor of zoology, but also a qualified engi- aspects of science. This practical approach is, I was an opening for someone with an enthu- neer, and for his PhD studies in the 1950s he think, deeply rooted in my childhood: I was born siasm for method development, so I packed had built an ultraviolet microspectrophotom- in the year before the outbreak of World War II, a my old car with my possessions and returned eter with a spatial resolution of 1 µm, which he period of austerity and ‘make do and mend.’ My north to join Peter’s group in Edinburgh. used to measure the DNA and RNA contents of father was a Royal Air Force aircraft mechanic, The University of Edinburgh is very strong intact cells6: one of the most interesting puzzles who took great pride in improvising solutions in science. In 1967, when the word ‘genome’ presented by higher eukaryotes was the c-value to the problems he faced daily, fixing damaged was understood by very few, Edinburgh was paradox; the amount of DNA in the genome ‘kites’ as they limped back from their missions notable for the high concentration of scientists can vary by orders of magnitude in organisms 2005 Nature Publishing Group Group 2005 Nature Publishing to his base in Reykjavik. I learned from him that working in genomics. The sizes of eukaryotic that are closely related. © if the tool for a job is not at hand, you make genomes presented a puzzle and major chal- So when I joined Peter in Edinburgh to one. My theoretical contributions to genomics lenge to the molecular biologist. Edinburgh sequence satellite DNA, largely ignorant of may endure longer, but I have to accept that I was exceptional in the number of scientists nucleic acids, I turned for help to Ken Murray, am better known for developing tools and it is developing molecular methods that could who was adapting to DNA the methods Sanger for one of these that I am delighted to have been be applied to eukaryotic genomes. Conrad had developed for RNA sequencing7. These recognized with the Lasker Award. Waddington had built one of Europe’s premier methods, with some of my own variations, genetics laboratories. With great foresight, he lacked the power of modern methods, but were The beginnings of genomics founded a Department of Epigenetics, to which good enough to give a picture of the structural As a postdoctoral research assistant in he recruited such pioneers in the molecular organization of satellite DNA sequences8. We Cambridge, I studied the effects of ioniz- biology of eukaryotes as Max Birnstiel, John showed that the sequences did indeed comprise ing radiation on polypeptides at the Low Bishop and Ken Jones. Max was the first to iso- short repeats joined together in tandem, with Temperature Research Station. The Medical late a eukaryotic gene—the ribosomal genes of variants introduced by point mutation. Research Council Laboratory of Molecular Xenopus laevis3; John devised ingenious meth- Ken and Noreen had a keen interest in restric- Biology was not far away and its luminar- ods for studying gene expression4; and Ken tion and modification and when the type II ies often gave seminars at the university that developed a method of in situ hybridization restriction endonucleases were discovered by were open to all comers. I recall attending talks and used it to show that satellite DNAs clus- Ham Smith and his colleagues at Johns Hopkins by Sydney Brenner, Frances Crick and, most tered in the heterochromatic regions close to University9, they introduced these enzymes influentially, Fred Sanger. The Laboratory of centromeres5. The Department of Molecular to the Edinburgh community. Ken founded Molecular Biology also attracted star speakers Biology, the first in any UK university, also had a restriction endonuclease club. To join, you and it was here that I met my future mentor, its share of scientists working on genes and had to purify an enzyme and make it available Peter Walker, who had come to talk about his genomes in the more genetically amenable to other members. I was given EcoRII as my work on mammalian satellite DNAs. These prokaryotic systems. Ken and Noreen Murray entry ticket. This really was a piece of good luck: major fractions, which can make up as much were particularly important to my own devel- EcoRII is one of the few enzymes that finds a site as half of the genome1, can be separated from opment as a molecular biologist, but there in the sequence of mouse major satellite DNA. the bulk of the DNA by density-gradient cen- were many others who contributed to the My colleague Gerard Roizes and I had shown NATURE MEDICINE VOLUME 11 | NUMBER 10 | OCTOBER 2005 1029 COMMENTARY abcd 5S RNA genes and the hunt for their into thin slices and eluting the DNA, which I promoters then bound to nitrocellulose discs. Each disc It may have been the possibility that the satel- was hybridized with radioactive RNA in a vial, lites had no functional importance that turned washed and counted in a scintillation counter my interest to a DNA that certainly did have an using the hybridization method of Gillespie important function. The sequencing methods and Spiegelman15. This whole process was available to us were developed for analyzing extremely tedious, but worse, the background small RNA molecules and I used them in a col- was so high that it was difficult to discern the laboration with Peter Ford, which set me on the signal. So I set out to devise a method of ‘in path to developing the gel-transfer or blotting situ’ hybridization that could detect the bands Tetramer method. Peter had found that the o ocytes of X. in the gel and attempted, with Julia Thomson, laevis store large amounts of 5S ribosomal RNA to hybridize to DNA fragments trapped in Trimer in 40S particles13, ready for the rapid burst of cell dried gels. Though Oliver Smithies and his cleavage and protein synthesis that takes place colleagues did get this method to work16, in Dimer after fertilization. We showed that the 5S RNA our attempts, the fragments diffused out of the in this particle has a different sequence from the gel. The story of how I stumbled on the realiza- 5S RNA of somatic cells14. This indicated that tion that agarose gels are permeable enough to there were two types of 5S RNA genes and that allow the DNA to be soaked out and trapped Monomer they were differentially regulated. It seemed a onto membranes has been written elsewhere17. good system in which to study eukaryotic gene Once I realized the gel was permeable, it was a regulation. Our plan was to isolate the two simple matter to set up the transfer (Fig. 2). Figure 1 Restriction endonucleases and ‘selfish types of 5S gene so that we could sequence the My first sight of bands on the autoradiograph DNA.’ Digestion of mouse major satellite with http://www.nature.com/naturemedicine EcoRII reveals the repetition of a short sequence upstream regions, in the expectation that this was another great thrill. The full range of possi- motif, with variation introduced by mutation, would lead us to the promoter regions. At the bilities that was opened up by visualizing genes which characterizes this type of DNA. Selfish time there was a great deal of excitement about in this way was beyond my imagination, but I DNAs require mechanisms for expanding the the potential of cloning DNA sequences. But could think of enough to know that I was onto sequence. In this case, the mechanism is as there were fears that this powerful technol- a good thing. And it was immediately clear that 11 probably unequal crossover . ogy could somehow get out of hand and wreak it was a simple and robust method; it worked genetic havoc, it was shelved while regulatory well at the first attempt. mechanisms were set in place. I did some experiments with bacterial that gel electrophoresis of restriction fragments The signs were that the 5S genes, like the 5S genes (Fig.