Interview Problem Solved: An Interview with Sir Edwin Southern

Jane Gitschier* Departments of Medicine and Pediatrics and Institute for Human Genetics, University of California San Francisco, San Francisco, California, United States of America

Yes. In answer to your question, yes, Gitschier: When you look back over to divide the lab into two. One group this is an interview with the Southern, as the course of your research career, what would go to Bristol and become a meat in the eponymous blot. Devised in the would be—for you—the highlights? research institute, which had no appeal to mid-1970s, Southern’s technique for Southern: I think that for me the me whatsoever. transferring DNA from gels onto nitro- thrills are really what happens in the lab. Gitschier: Which, Bristol or the meat? cellulose paper allowed single-copy, eu- It’s not necessarily when you look back and Southern: Either of those, actually. karyotic genes to be discerned for the first say, ‘‘Well that was a fantastic thing!’’ It’s And the other would go to Norwich, time. It quickly became a mainstay for a the day-to-day things that happen when which is better, and would become a food generation of molecular biologists and you are really doing the experiments and research institute. spawned a template for mapping the you see the results and you say, ‘‘Wow, But as it happened, a friend of mine was human genome. that’s a great result!’’ going to Edinburgh and he met Peter But the blot was no one-off for South- So from that way of looking at things, Walker, who was looking for somebody to ern, who is now entering his sixth decade it’s probably the earliest stuff that gave me join his group. So I had a chat with Peter at the bench. Working in Edinburgh in the the biggest kicks. When you are younger, and that was it, really. We hit it off. Medical Research Council (MRC) Mam- you are probably more pleased by small It was an MRC group. Peter was a malian Genome unit, he was one of the results than when you get older. Those are university professor, but he had his first to sequence eukaryotic DNA and to really hard to recall, exactly, because they research group in the Department of appreciate the genetic architecture of happen all the time! There are things on a Zoology. Pretty unusual for zoology in satellite DNAs. He was also a strong and weekly or monthly basis that give you a big those days to be doing molecular work, but productive proponent of physical mapping kick. he had that sort of vision. of the human genome as a complement to Gitschier: You are lucky if that’s true, He was interested in what was called the genetic map. In the late 1980s, while actually. A lot of people have lots of ‘‘satellite DNA’’, and he gave me the job Professor of Biochemistry at Oxford, failures weekly and monthly! of sequencing mouse satellite DNA. One Southern conceived of oligonucleotide Southern: Well, I may be easy to problem with that was that there weren’t microarrays for DNA sequencing and please. any sequencing methods at the time! I was was issued a patent for the invention. This In my earlier career, I was a radiation very lucky that Ken Murray had just award led to his founding of a small chemist, and I had some very nice arrived in Edinburgh from Fred Sanger’s company, Oxford Gene Technology experiments in looking at the effects of lab, and he was working on the develop- (OGT), and through successful lawsuits radiation on proteins, actually polyami- ment of DNA sequencing methods. He to defend his patent from infringement, to noacids, which I used as a model for basically held my hand and led me into licensing the patent as a source of funding proteins. In that period, people wanted to DNA sequencing as he developed it. for two highly successful philanthropic find peaceful uses of atomic energy, and It was guinea pig that gave me the first trusts: the Edina Trust, which supports sterilization of food was one. Some of the results, and speaking of which, going back science education in the United Kingdom, discoveries I made then were, I think, to your question, seeing those fingerprints and the Kirkhouse Trust, which develops really quite original. But it’s a field that of the satellite DNA was such a moment, disease- and pest-resistant legume crops in never went anywhere. because you could see just instantaneously Africa and India. I was then very lucky to go into that it was a very simple sequence, much I visited Southern in November during molecular biology in Edinburgh when I simpler than was thought by reassociation one of the wettest years in Oxford history, joined Peter Walker’s group in 1967. It kinetics. That was a big moment for me. as this grey day sprinkled its contribution was quite late in my career; I was 29 at At that time, the Murrays—Ken and into the record books. We met at the that time. Noreen—were working on restriction en- Trinity Gate, where I was given shelter by Gitschier: That doesn’t sound too late donucleases and they introduced me to the porter as Southern arrived under a to me, and I’m sure it doesn’t sound too those fantastic enzymes. What a real tool large umbrella (Image 1). We headed for late to you anymore either! those proved to be! We heard about Ham the tower, then up a flight of stairs to a Southern: No, you are right. The Smith’s work on the type II restriction cozy room for coffee and discussion. It was place where I was working made a endonucleases. Ken had set up this club, just as the reader might imagine: oak- decision to move out of Cambridge and and the rules were if you made a paneled walls chock-a-block with portraits of former fellows, dons, and benefactors, Citation: Gitschier J (2013) Problem Solved: An Interview with Sir Edwin Southern. PLoS Genet 9(3): e1003344. upholstered chairs, rugs, and an attractive doi:10.1371/journal.pgen.1003344 grandfather clock that allegedly chimes Published March 14, 2013 twice at 10 and, as we witnessed, 10 times Copyright: ß 2013 Jane Gitschier. This is an open-access article distributed under the terms of the Creative at 11. Southern is soft-spoken, his voice Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, low and gravelly, and I hung on every provided the original author and source are credited. word. * E-mail: [email protected]

PLOS Genetics | www.plosgenetics.org 1 March 2013 | Volume 9 | Issue 3 | e1003344 was coined by Waddington in 1942 to describe the transition from embryonic state to differentiated state as a way of thinking about how the environment might influence phenotype during devel- opment.] Southern: And in Edinburgh there was also Eric Reeves working on plasmids and Bill Hayes in the Department of Molecular Biology. We didn’t appreciate it at the time, but looking back, it was a stunning collection of people. Some of the first recombinant DNA stuff was done there. Edinburgh was just an amazing place and still is. So, where were we? Gitschier: We’re talking about the highlights along the way. Southern: Yeah, the satellite DNA. The guinea pig sequence work established this idea that there is a lot of sequence in higher eukaryotes which couldn’t possibly have a function that depended on its sequence; it couldn’t be coding because it didn’t make any sense. I think that triggered in some people’s minds that there was probably a lot of junk DNA. Francis Crick, Susumu Ohno, Peter Walker, all had that idea. And there is still a debate about that, of course, and I think the debate now is how much of the genome is junk, not the question of is there junk or not. This business recently published, this ENCODE [Encyclopedia of DNA Ele- ments] project, I don’t know if you read any of it? Gitschier: Well, I printed a few of them out. Image 1. Sir Edwin Southern. Photograph by Jane Gitschier. Southern: Well done! Well, I was doi:10.1371/journal.pgen.1003344.g001 slightly depressed by the reports about it, because they spent so much on that. There restriction enzyme, you could then have out of the darkroom and said, ‘‘You have a are 400 or so authors on the papers— access to all the restriction enzymes that ladder!’’ thirty-some papers. And the main conclu- had joined the club, as it were. Edinburgh Gitschier: Actually, I’m working on a sion seems to be, according to the was a very lively place in those days, so book project about the ’70s, and I’m newspapers, that there’s not quite as much there were plenty of people in the club. provisionally calling it ‘‘The Thrill in a junk DNA as we thought there was! That’s It turned out that EcoRII gave this Dark Room.’’ an awful lot of money to spend on that beautiful pattern with mouse satellite Southern: Well, there you go. You conclusion. No doubt, it will come. DNA—sort of a ladder pattern, which know, that was a really interesting period. So this notion of junk DNA came out of gave a huge amount of information about There were so few labs working on that work, largely from the guinea pig the structure of mouse satellite DNA at a genomic problems. There was Roy Britten satellite DNA. But there is a nice irony glance. You could tell the repeat length, at the Carnegie Institute of Washington, here because a few years later, Bob Moyzis you could tell there was divergence of the Brian McCarthy, an Englishman that published the sequence of the human sequence, you could measure the diver- worked in San Francisco, Don Brown at telomere, which turned out to have the gence of the ladder components, you could Carnegie. And then in Edinburgh there same sequence as the guinea pig alpha- tell there were internal repeats within the were Max Birnstiel and John Bishop, and satellite. So, although I said firmly that major repeat, and so on. Just at a glance! there was also a famous epigenetics group there is no way this could be a functional Gitschier: Just by ethidium bromide that was set up by Conrad Waddington in sequence, actually there was a bit of mud staining. the genetics department. He was a re- on my boots there. Southern: Yes! It was very simple. Just markable man, a real visionary. Gitschier: OK, I have a feeling we’re digest the DNA, run it on the gel, take a Gitschier: I’m thinking even the word now coming to blotting. photograph, and that’s it. And it was my ‘‘epigenetics’’ seems ahead of its time. Southern: Right. That came out of a colleague Gerard Roizes, who brought it [According to Wikipedia, ‘‘epigenetics’’ collaboration with Peter Ford, who was in

PLOS Genetics | www.plosgenetics.org 2 March 2013 | Volume 9 | Issue 3 | e1003344 the Department of Molecular Biology in liquid come through the gel on the top, blotting to map the human genome. He Edinburgh at the time. Peter had studied through osmosis really, and that told me had this idea of using restriction enzymes the 5S RNA from frog oocytes. When the the gel was permeable. So if I just turned it and these restriction enzyme polymor- embryo starts its development, it synthe- upside down and put salt underneath the phisms, and so on. I didn’t understand sizes a huge amount of ribosomal RNA, gel and put the membrane on top, it would what he was talking about. It made no but the 5S RNA has already been soak the stuff through, which is what I did. sense. But he was really enthusiastic about synthesized and stored. He had shown And that worked the first time, it was it, so I thought, ‘‘Well there must be that the sequence of the 5S RNA in the just so easy. That was another of those something in this!’’ oocytes is different from that in the moments, a thrill in the dark room, as it So, when I went back, I thought well, somatic cells. That shows that there are were. This was quite early on, in 1973, the obvious thing to do, alongside the two kinds of 5S genes, differentially actually. genetic map, is to make a physical map. regulated in frogs, with two different Gitschier: But you didn’t publish till And I had ideas about how to do that: promoters. That was what everybody was ’75. cosmid libraries, fingerprinting, that sort of interested in then: what are the eukaryotic Southern: I’m pretty slow to publish at thing. promoters? the best of times, but I was rejected the And then there was a lot of talk about We thought it would be interesting to first time I submitted it! I wanted it to go sequencing the genome. Sydney Brenner isolate the DNA from both kinds of genes somewhere where it would get noticed. got an interest in the whole area. Sydney for sequencing, and my thinking was we Methods papers tend to get buried, so I and I were pushing the idea of just could use restriction enzymes to enrich for sent it to the Journal of Molecular Biology. sequencing the coding regions first of all. 5S genes. It was known that the genes Sydney Brenner was the editor and he But actually it was Fred Sanger who were repeated, it was quite likely they rejected it because they didn’t accept should get the credit for this genomic would be tandemly repeated, like satellite methods papers. But he said if you get sequencing idea, because Fred not only DNA. The idea was that we would start some biologically relevant result, then we developed a sequencing method, he also with high molecular weight DNA and look can publish it. developed this idea that you just start with for restriction enzymes that didn’t cut the That delayed the publication, but in the a genome and you sequence it completely. 5S genes, so we’d end up with a block of mean time, we had a visitor in Edinburgh, He started with phiX174 genome and just repeats that would stay close to the origin Mike Mathews from Cold Spring Harbor. sequenced the whole of that. Out of that when you ran the stuff through a gel. Mike saw all the things we were doing and came things like alternative codons and so You’d greatly enrich for the 5S genes, and asked me if he could use the method in his on. Then, mitochondrial DNA, he se- once you’d done that, you could cut with work, and I said ‘‘sure’’ and sketched out quenced that. And then, he went on to enzymes that did cut the 5S, and you’d get how to do it. He went back to Cold Spring phage lambda and sequenced that. He just a nice band of 5S genes. Harbor, and they got it to work straight went to more and more complex genomes But the problem that we were faced away and then showed it to visitors, and and without any original hypothesis. He with is how did you know where the 5S they all said, ‘‘Well, that’s great, can we do said, ‘‘Well, let’s just sequence the whole genes are? We were cutting the gel up, it?’’ thing and see what comes out of it.’’ eluting the DNA, and hybridizing with the I said it’s fine to tell people as long as John Sulston, of course, was a colleague 5S labeled RNA on [nitrocellulose] fil- you tell them where it came from. So they of Fred’s, and he went on to sequence the ters—the Gillespie and Spiegelman tech- were very honorable and did that. They nematode genome. It was an extraordi- nique—that’s how you measured genes in acknowledged it as ‘‘Southern’s tech- narily courageous thing to do, I think. But those days. And of course it was terribly nique’’. So that is how my name got hung he was determined and pushed it through. noisy, and it’s just a mess. So that was onto it. He was then pushing for sequencing the when I thought, ‘‘Well, if I could just Gitschier: Ah! I see. Now, at some whole human genome. That became the hybridize the gel, then we might see a point you start to think about using UK effort. band.’’ restriction enzymes to make a physical There was a lot of talk in the mid-’80s We tried drying the gels down and map of DNA. that Sanger’s method was too expensive. I hybridizing to the gels, and that was very Southern: I had the idea that we went to a meeting in Japan, organized by messy. Nothing came of it. would establish a group that would have Akiyoshi Wada specifically to answer the Then I thought about transferring it out the remit of mapping the human genome. question of whether there is a quicker, of the gel onto cellulose nitrate paper, That idea really came from—this is going cheaper way than Sanger sequencing. because Charles Thomas had developed back to 1979—when I went to Woods Wada himself gave a paper. A colleague this way of dissolving agarose gels in a high Hole for the summer. of his had shown that if you put an concentration of sodium perchlorate, a I wound up collecting all these bizarre oligonucleotide on a column and pass a little known fact. My idea was to float the creatures you can get in Woods Hole and tRNA down it, then you can fractionate, membrane on this 6M sodium perchlorate extracting DNA from them, but the essentially, a nucleic acid that differs by a and then put the gel on top of it, and the unfortunate thing was that when I came single mismatch. I’m not sure that he said sodium perchlorate would diffuse through to look at the DNA, it was all degraded! this, but basically, you are reading a bit of the membrane, dissolve the gel, and the Because I hadn’t realized that the purified sequence by doing that. If a nucleic acid contents would sink and adsorb to the water was just filtered seawater! So all that sticks to the column under the conditions paper. experimental work was wasted, but I had a they were using, then you’d essentially Gitschier: Was that part of that wonderful time. read that bit of sequence of the stuff that publication? While I was there, I had a visit from sticks to the column. So it occurred to me Southern: No. Because what hap- Dave Botstein, and I vividly remember that if you had a complete set of all pened was that as I was watching the this. He perched himself on the stool in the oligonucleotides—every sequence of a thing sit and float in a boat, I saw a drop of lab and told me that he wanted to use given length, each one in a column—then

PLOS Genetics | www.plosgenetics.org 3 March 2013 | Volume 9 | Issue 3 | e1003344 you could sequence. I can remember company had wanted to take through We find the crop breeders who are talking to George Church about it while exploitation. And they went into a license working on those crops in the region and hanging onto a strap on a bus. That was agreement with Beckman, but really, it bring them together into a consortium, one of those moments, having the first idea wasn’t part of Beckman’s mainstream and we say, ‘‘We want to help you that if you had a lot of oligonucleotides, interests. So eventually we brought the establish molecular techniques in your this was essentially a sequence reading licensing back to the university. breeding programs.’’ So we set up basic tool. That was very pleasing. And then the university weren’t being labs in Africa. We give them equipment I sat down and thought about that and very energetic about licensing it, and there for doing DNA extraction, PCRs, running realized that that would be an awful lot of were people out there who were infringing gels, taking gel photographs, interpreting columns! Because you know, if you chose it, I thought. If you have a patent and the data, and we train the people in those octanucleotides, then there are 65,536 somebody’s infringing it, the only recourse methods. The effect of that is remarkable; different octanucleotides. you have is to sue! And suing is an extremely all kinds of things fall out. You can look at Gitschier: That’s a lot of columns! expensive business. It’s not what a university the diversity of the crops, which is an Southern: But then I thought, if you should be spending their time and energies important first step in any breeding had these on a spot on a membrane, you on. I had to make a decision. Do I want to program, but you can also track the gene could do it that way. You’d label up your just let this thing go? Or carry on with it? through the generations of the breeding genomic DNA and you’d hybridize it to I decided to carry on with it. I program in a very robust way. this piece of paper or whatever kind of negotiated with the university to take it We’re interested in resistance to pests matrix you had. and set up my own company and to assign and diseases. And remarkably, there are a So I started looking at the chemistry for a license to my own company, Oxford lot of these genes already present in the making oligonucleotides. Beautiful chem- Gene Technology. And then we got into species. Take the bean, for example, we’re istry: the reactions are quick, they go to this whole business of licensing and working on five different ‘‘constraints’’ 99%, and it’s done on glass! This wonder- litigation and so on. We sublicensed to a [pests or diseases], as they are called. ful material called controlled pore glass, a lot of people; Agilent and Affymetrix have And there are resistances known to all of mixture of ordinary glass and borosilicate a lifetime license. those, mainly coming from South Amer- glass, in which all the silicate glass is That licensing gave me quite a substan- ica, where beans originated. And with the etched away, leaving this mesh of borosil- tial income, and that gave me opportuni- molecular techniques you can combine icate glass with a massive surface area. ties, then, to develop other things. It resistances. That is really difficult unless Finding out about the chemistry was a helped set up OGT in a more substantial you use the molecular methods, tracking real thrill, as well, and then realizing we way, and I set up two trusts, the Kirkhouse the genes. It speeds things up enormously could very simply adapt the chemistry for Trust and the Edina Trust. and it’s working! making oligonucleotides to print them on When I first set up Kirkhouse Trust, my We already have cowpeas that we glass. With a German PhD student, Uwe idea was to go into medical areas that I developed in West Africa that are out in Maskos, we developed a way to leave them was familiar with. But Paul Nurse, who is the farmers’ fields! There, the main pest is attached to the glass. And that worked now President of the Royal Society, was a a parasitic weed called Striga, or witch- beautifully. It showed that you could trustee and he said, ‘‘That is such a weed. This is a parasite that latches itself synthesize oligonucleotides on the glass at crowded area, why don’t you do some- onto the outside of the roots of the high yield, that they would hybridize, and thing different?’’ cowpeas and sucks out all of the goodness, off we went. We worked on different ways And so I said, ‘‘Well, what have you got and the cowpea dies. And there is very of making arrays of different sequences, in mind?’’ little you can do about it once it’s and then moved from there into applica- And he said, ‘‘Well, what about crops?’’ established. On average, it destroys about tions. We thought that sequencing would And I said, ‘‘Well, I don’t know 30% of the crop. be an application, but we didn’t have the anything about crops!’’ Two hundred million people in West means to make the size of arrays that you And he said, ‘‘Good reason to get Africa are dependent on this crop for their would need for sequencing. going!’’ source of high-quality protein. In some In 1987 I wrote a grant application to Gitschier: Well, did he know anything years, 100% of the crop is destroyed by the MRC to develop this methodology. about crops? this weed. Very quickly, within 3 years, we And that was successful, but at the same Southern: Only in a general way. I were able to establish resistant varieties. time the university was pressing us to mean, his first degree is in botany. There are seven different races of Striga, patent any new ideas. I was on the What we [Kirkhouse Trust] do is decide and each one has a different resistance committee, actually, that set up this group on a crop species, and we gather together gene associated with it, apart from one. to exploit intellectual property. So they a group of breeders across a region. We’re Gitschier: I think one of things that is came to me and said, ‘‘Do you have any in West Africa for a crop called cowpea, so interesting about life is that you can’t ideas about a patent?’’ And the grant which you would call a black-eyed pea, always anticipate where it is going to lead application became a patent application. and then in East Africa for common bean, you. This is clearly an example for you. And that’s been a long story. which is kidney bean or navy bean. We go Southern: You couldn’t say that I had Gitschier: In that it’s been so success- to those regions with somebody knowl- a planned career. It’s just evolved. And ful? edgeable in the area, from the States I’ve followed things as they have arisen, Southern: Yes. But we had a few usually. We have Paul Gepts from UC and I think that is the best way to go. I’ve battles along the way. [University of California] Davis and Mike certainly ended up in places where I’ve Gitschier: Battles in terms of patent Timko from UVA [University of Virgin- never imagined. infringements. ia]. They give the leadership to the whole Gitschier: I’m just wondering. If you Southern: Yeah. This was the first thing because they are molecular geneti- could just start over today, do you have patent that the university’s tech transfer cists, as well as plant breeders. any idea what you might want to do?

PLOS Genetics | www.plosgenetics.org 4 March 2013 | Volume 9 | Issue 3 | e1003344 Southern: Yeah. I’d be an engineer. energy systems, there are tremendously It’s mathematical skills and imagination. Definitely! interesting engineering problems. That’s That’s what it needs and it must be Gitschier: What kind of engineer? what I would want to do. tremendous fun, I think, to see your ideas Southern: Well, I think a lot of the I like DIY [do it yourself], building made into something that works. problems that we are faced with have things in the lab, that sort of thing. engineering solutions, for example, in new Engineering brings a lot of things together.

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