Oliver  Smithies (2014) - Where Do Ideas Come From?

Where do I begin? That’s the next question. And I thought that perhaps this is a good place to begin. It’s a combination of the beginning and the end because here I am at a school in England unveiling a plaque saying that Oliver Smithies was a student here from age 5 to 11. And here are all the 5 year olds helping me to unveil the plaque. It was a small school in a small place because it was a village in Yorkshire, Copley, with a population of only 1,500 people. So I wonder how many of you students here who are coming... How many of you are from a place as small as 1,500 population? Oh yeah, quite a few from 1,500. That’s very good. But this photograph is of where I lived. I lived here and this river is called the River Calder. And the school was about where the pointer is now. And I would walk to school down there. And that river at this time -I was living there- was very polluted. But now it’s recovering very well. But then my family moved to Halifax which was somewhere up here and had a bigger population. And there I went to school, a grammar school. These are the old... In fact this was founded in Elizabethan days, old schools of high quality. And I am their first Nobel Prize winner. But now let’s just go downstream a little way. And if we go downstream, we come to a place called Elland Yorkshire. And that’s about 5, less than 5 kilometres from here. And you can almost see it. And the students there went to another grammar school called Rastrick Grammar School. And you know, they also have a Nobel laureate. You just heard him talk. There he is, John Walker. But let’s pursue this and go upstream. So there’s Copley and you go upstream and you come to this place, population 15,000 and that’s Todmorden. And low and behold they have a grammar school too. And they have one, a Nobel Prize in 1951 in physics, and another Nobel Prize in chemistry in 1973. So what is the secret? But of course the water was actually rather poor. It had no fluoride in it and my teeth rotted with the candy I ate as a school child. But obviously it’s the teachers isn’t it. And in Todmorden that was the same teacher who taught them science. Both of those Nobel laureates were taught by the same person, 20 years apart. And I had some grand teachers of which I’ll mention perhaps only one in my high school, in the grammar school. And his name was Oddy Brown and he really was an awful man. Nobody liked him, I didn’t like him. And he was a cheat. But he loved mathematics. And he taught me calculus. And it was like, ah, this marvellous subject. So he could teach and you could learn from him. You could learn from him even if he wasn’t a nice person. You don’t have to learn necessarily from somebody who is nice. But I was lucky enough to get a scholarship to Oxford, to Balliol College. And my teacher there, my tutor Sandy Ogston, he was very nice. He was famous for a couple of things, one of which he’s a bit ashamed of because he said it only took him 15 minutes to think of it. And that was how enzymes can make levo or dextro rotary compound starting from something that is not optically active, the 3 point attachment hypothesis. But anyway Sandy was a good teacher obviously because he had a Nobel Prize winner in 1978 and he had another one in 2007. So you can be fortunate in your teachers and the way they teach. That’s where it comes from. So how was the teaching carried out in the university there and at that time? It was rather remarkable. You would be given a topic to write an essay on. For example he gave me this topic. And you were expected to go away and fiddle around and you weren’t expected to come back with something you got out of a text book or even a review. You were expected to come back with some work from original papers. And so here is then something that really was where ideas come from because this is a volume of Advances in Enzymology. And why on earth would I show you a picture of the volume. Well, the reason is that when I read the article, which I am going to tell you about in a moment in this journal, it was so revealing and so exciting that I remember where I was sitting, I remember what the colour of the journal was, I remember what the paper looks like. You can get so much inspiration from reading. Ideas come from reading and reading good things. And this was the article by Fritz Lipmann, who won the Nobel Prize in 1941. And it was about energy rich phosphate. And he invented this idea that some phosphate bonds have high energy and some have low energy. And I can give you an easy example from organic chemistry that some of you may be able to understand. If you take ethyl acetate, that’s a rather low energy ester between an alcohol and an acid. But if you take acetyl chloride, that is an acid and hydride, an acetic acid and hydrochloric acid. And that’s a very high energy compound and will acetylate many things. Whereas ethyl acetate hasn’t got much energy And what Lipmann was, to describe that difference. And this is where it comes into my sphere as it were because I wrote an essay for Sandy and came back with this scheme which I called the Smithies cycle. This was before Krebs cycle, before the tricarboxylic acid cycle. And I could take inorganic phosphate and through this series of reactions I could generate ATP. And that would require the loss of 2 hydrogen, 2 protons. And if I put the 2 protons back, I could go back here and I could keep going on round and round and round. I could make energy for nothing. Well, I knew this was wrong. But I didn’t know why it was wrong. And actually it took even Sandy a while to decipher it. But he did and he wrote an article as a result of this which was published in Physiological Reviews quite a while ago. But in it he showed that the system depended upon a gradient of chemical potential of the hydrogen ion or the hydrogen..., the proton moving along a gradient. Just as we’ve heard from John. And he also deduced that this would not be kinetically possible unless all the factors were in a complex because if they dissociated the rate of reaction would be too slow and they had to go in a complex. So he deduced that there should be a big complex and that the energy could come from the movement of a proton. So it’s very nice to have followed your talk John with Sandy. But anyway he published it and he was kind enough to put my name on it as a scholar of Oxford. But then somebody about probably 20 years ago came to me and said: “Are you that Smithies who wrote an article in Physiological Reviews in 1948?” And I said: “Well, yes as a matter of fact I am that Smithies.” He said: “Oh, I thought you were dead.” So anyway, let’s go on. And here’s my PhD thesis, my results. These are my experimental points. And those are the theoretical points. And you can see that the experimental points were so close together that you had to interrupt the line, the theoretical line, to let them show. I was very proud of this machine that I’d built. It was an osmometer. It doesn’t matter what that is. And I published it. And you know it has a record, this paper. And nobody ever quoted it. And nobody ever used the method. And I never used the method. So I ask the question: What was the point of it? And the answer is really rather revealing to you guys. Because the answer is that I enjoyed doing it and I learned to do good science. But it’s also obvious from this that it’s quite unimportant what you do, isn’t it? It doesn’t matter what you do to get a PhD. All that matters is that you learn to do good science. But there is a corollary. You have to enjoy it. If you don’t enjoy it, then go to your advisor and say: And then if your advisor won’t or can’t give you another problem, change your advisor. It sounds like a joke but really it’s the secret of life you might say, of scientific life certainly. Do something that you enjoy. Critical for your enjoyment in the future. And if you find you don’t like science go and play the guitar or go and write a book or go climbing or something. Do something you enjoy. It’s pointless doing something you don’t enjoy. It won’t work. Well, so my first job was in Toronto. And I went there and insulin was discovered in Toronto. And David Scott was important in that early work. And he said: “You can work on anything you like as long as it has something to do with insulin.” And so I did a very systematic literature search. But in those days there wasn’t Google and you couldn’t do anything. So you just went to a thing called a chemical abstract. And you looked for everything that had the word insulin in it. Which was tabulated and all of these were looked at. And then every now and then one was worth looking at a bit more. And in that way you could get some idea what to do. And I decided that I would look for a precursor of insulin for various reasons. I might say I never found it. But that’s what I set out to do. And so I was doing electrophoresis to try to see if I could see insulin because if I couldn’t see insulin, I certainly couldn’t see a precursor. And you might notice that I’m working on January first but that’s not work, I’m playing after all. And here was insulin unrolling like a carpet, it was very annoying. I couldn’t get any migration. And then I heard that people in the local hospital were using a method devised by Kunkel and Slater. And they’d done experiments with filter paper. And they showed that if they had a certain protein, it would stick to the paper and unroll. Just like my insulin. But if they used starch grains as a supporting medium, it didn’t stick. And the starch grains were made into a rather complicated apparatus. But it was really a moist bed of starch grains. But in order to find out where the protein was, you would have to cut it into many, many, many slices. And do a protein determination on every slice. So imagine. One electrophoresis would take you 50 protein determinations. Well I was alone in the lab. I didn’t even have anybody to do my dishes. I couldn’t possibly do an experiment like that. But I wanted the feature of not absorbing. And then I remembered helping my mother to do the laundry. And I think I ought to say “helping”. I was there. And she used to cook starch into a gooey mess and apply it to the shirt collars of my father’s shirts. And then iron it. That’s how you made stiff collars in those days. And in tidying up at the end it would set into a jelly. And I remembered that. And I thought that, well now, if I just cook the starch into a jelly, then I can stain it and I won’t have to do any of these protein measurements. And I went back that afternoon and found some starch and cooked it up. And sure enough there my insulin ran as a neat band in the system. I had gotten over the problem of absorption and begun to do gel electrophoresis. And here a little bit later, a couple of months later I tried serum just for fun and as a rough test. And I got some resolution and I set it up again at midnight, I was a bachelor. And a few days later this was the sort of result I was getting. And in those days people thought there were only 5 proteins in serum: albumin, alpha 1, alpha 2, beta and gamma globulin. And here I was seeing all these bands. But I couldn’t label them even because I didn’t know what was what. But it was pretty promising. So I asked my boss, David Scott, if I could change and work on serum proteins. And he was a good scientist and he said: “Yes, by all means you’ve got something very interesting.” So I began to do studies on serum proteins. And here is a couple of gels when I was about ready to publish. A couple of things are noticeable. First of all, that isn’t a photograph. And the reason is my lab didn’t own a camera. It was just me. So that’s why it’s a sketch. But I could go and arrange to have a photograph taken. The other thing is that these blood samples were from 2 of my friends. I got tired of bleeding myself. And I think... Didn’t you tell me John that you gave me some blood one time when you were passing through in Wisconsin? Did you? No it was somebody here, somebody else here. Some other member here passing through Wisconsin I bled. But anyway I was ready to publish and then just I want to stress what I had discovered. First of all, the critical thing is that starch only forms gels at high concentrations. And the concentrated gel impedes the movement of large molecules. And therefore starch gels separate molecules largely by size. And so quite accidentally I invented molecular sieving electrophoresis which of course you use these days with a polyacrylamide instead of starch. But ideas come from all sorts of places. And I was interested to find... I got a prize with Ed Southern. Some of you will have done Southern Blot for the discovery etc. of gel electrophoresis and so on. Both of us got our ideas from childhood memories. Mine, as I’ve told you, was from the laundry. And his was from a process called mimeographing. It was before the days of Xeroxing. And you would take a sheet of wax paper that had been typed on which had holes in it where the typewriter had hit it. And you would put dye through on to a gel below and then you would take imprints off the die which is what Southern blotting is. So ideas can come from many places. But just before publishing just by chance I ran another sample. And it was very strange. Most odd. Many extra components. All these extra bands. BW, what was different about BW? Well, BW was a woman. And this was the first time I’d run a sample from a woman. I thought I’d found a new method of telling men from women. And I called my pattern and the other guys pattern M and this, the F pattern. And I could do 2 a day. And I ran for the next 5 days, I ran the man and woman serum and it worked. The male, female, male, female. They fitted the pattern. And then about the 7th day they were switched. Well that’s alright, I’ve muddled the sample. Ran them again, no switching. So, oh, Casey Cock was the name of the guy and we went into him, Casey come on let’s have a look. But it turned out not to have anything to do with male femaleness. And this is my data file. But now although it looks crude, it’s a very good file because I can read it 60 years after it was written. Now your data files that you have in your computers I bet you can’t read them in 5 years from now. So make hard copies. Make hard copies of your data. Don’t rely entirely on an electronic copy. And this was... It turned out that the 2 types, the F and the M, had nothing to do with maleness and femaleness. And it turned out that there was a third type, a variation of the M type. This was worked out with the help of Norma Ford-Walker. And she and I together worked out that this was due to a difference in a single gene coding for a protein called haptoglobin. And then at that time Linus Pauling and Harvey Itano and Vernon Ingram just had worked out the first protein difference, the globin gene with a G to V mutation, a glutamic acid to valine mutation. So this was an inspiration. And so George Connell and Gordon Dickson, my 2 friends, came back to Toronto and together we worked out what the difference was in haptoglobin. And it turned out that there was a simple difference of 1 amino acid and that was... I call it the F to S. But one of the types, the one that gave the multiple band, was due to the rather unusual form of crossing over, made a duplication. And the crossing over is called non-homologous because you see GH and BC have nothing to do with each other. They are not homologous sequences. But once you had the duplication you could get homologous crossing over because of an unusual thought. Because you could get CDE of the second copy of the gene, in the duplication. Could line up with CDE in the first half of the duplication. And you could get a crossover here which would produce a triplication. And this was predictable and we found it in the population. So this gave me the feeling that homologous recombination was a predictable event. It might be rare but it’s predictable. And it goes into the mind. So the time came, time marched on and we were able to clone genes and our lab looked at the G gamma and the A gamma of the globin genes. But then I was teaching and I was teaching molecular biology. And this was a very striking paper there by Terry Orr-Weaver in Rodney Rothstein’s lab. And she showed that in yeast you could get recombination between an incoming plasmid and the yeast chromosome due to homology. And this crossing over could lead to the insertion of a sequence. So a teaching, I knew about this and I taught it. And I began to think maybe it would be possible to do that with a globin gene and corrector gene, a homologous crossing over where this is a mutant gene. Here is the normal sequence. Line up, cross over and correct the gene. But I didn’t know how to do it. And I wasn’t even sure that it would be possible of course. But then teaching, I had to teach my course. And this paper came out in April of ’82. And I’m not going to go into the method except to say it required a thing called gene rescue in which they found a blue piece of DNA next to a red piece of DNA. And I thought I can use that for my gene targeting test. I can make an assay for gene placement. And the aim is to place the correcting DNA in the correct place. And I had this idea that this would be the blue piece of DNA, the same piece that those guys had used. And here’s a red piece of DNA on the globin gene. This is on the target. This is on the incoming DNA. If those 2 things come together, I have proven homologous recombination. And I did calculations here which led me to believe that the method was sensitive enough that even if it was random, I could find something that had a frequency of less than 1 over 3 times 10^9th the size of the human genome. So on with the work and make a targeting construct. We didn’t have DNA sequencing or we didn’t have DNA synthesising. And it was complicated. And there’s a page, a couple of pages from my notebook. They’re pretty aren’t they? I don’t know what they mean. But anyway the time came and I collaborated with Raju Kucherlapati and I sent my DNA to him. He would do the cellular work and send me back the pooled DNA and I would look for this recombinant fragment with a horrible assay. And here is the first test. The cosmos, the real thing. This was not an artifactual thing now from Raju. And here it is. And then I noticed the date. It won’t mean anything to you, this is my birthday. And it was my 58th birthday. So here I am doing some of my best experiments on my 58th birthday. And it didn’t work. You don’t get special dispensations on your birthday. But you can do experiments on them. Anyway, then we simplified the system. It looks more like Terry Orr-Weaver’s system now. And we could get recombination. And I’ll show you the end result of it where we could expect that if we hadn’t modified the gene, there would be a fragment of DNA, 11 kilobases. And if we had modified it, the fragment would be 8 kilobases. And here is the gel that showed... In fact one of the columns that we had, had the 8 kilobase fragment instead of the 20. And so as it says here: Number 20 is it! And very exciting, 3 years and 1 month after starting. Rather quick I think. We published. This is the important sentence. That although the frequency of success which is actually 10^-6 of the number of cells treated. It’s at present modest. This showed unequivocally that gene targeting was possible. And so on with the march. And then we heard of Martin Evans’s lovely work. And here EK cell system. That’s what he called it at that time and he brought personally into the lab. He came into the lab one day, visiting from the UK. He came in and here they are Oliver. So that’s what science is also about. It’s about sharing. Share willingly. And he shared willingly. And he shared also with Mario Capecchi. And both of us then went on and started to work on this. And I used [name inaudible 00:30:01] method instead of the complicated method eventually to detect the recombinant. And here’s the apparatus that it was done with. Still around in the lab. You wouldn’t believe it but this is the one that did homologous recombination in our lab in ES cells the first time. It should have this label on it. That was the label they used, that one of my graduate student friends when I was a graduate student, used to put on things, that were around about in the lab. NBGBOKFO it’s pronounced. And that’s translated as “no bloody good but ok for Oliver”. Which means that this is all junk that’s put together to make this machine. I didn’t have to buy anything. I’d just go around and scrounge. So that’s how you do science. But anyway, the gene targeting worked beautifully and my wife Nobuyo Maeda made a marvellous model of atherosclerosis with it. I’m not an author on that paper. It was the method we were using but it was all her idea. Oh, and there’s Sandy again. Well I’m at the end of my time but I’ll take a couple of minutes extra here to say that now I’ve got the date of his death and life because he asked me to write his scientific memoires when he died. If you had your advisor ask you to do that, you would probably say yes too. And then I had to read it and here was an equation he’d written. And this was an equation talking about gels and the space available in gels. And he was showing that he could calculate that big molecules could find only a small space and little molecules a large space. And I got exposed to the kidney. And there I thought that this idea might work in the kidney. And I began to test this idea. This is just a quick view of a high electron micrograph of the kidney. And here is the plasma and a red cell would be about as big as where I’m talking now. And these are albumin molecules. And I had the idea that this is a gel and here is the urine and that molecule could go through the gel and be separated according to their size using Ogston’s equation as it were. And to do that what do you have to do? Well you have to have something that you can see in the electron microscope. And Michael Faraday has a lovely paper. He had many discoveries. But he has a 50 page paper on making gold nanoparticle. And there are some made with the type of method that he used. And I still have one with me, I rather like these particles. And I don’t know whether I can get this out anymore but, ah yeah, here we are. Here they are, maybe you can just have some idea. That’s tube number 4X there and these gold nanoparticles are stable for long periods of time. And I’ve been putting them into kidneys and making them in different sizes as you can see there. Some of which are approaching molecular sizes of a immunoglobulin molecule and so on. And here are the smallest we can make, an image taken by my postdoc Marlon Lawrence very recently. And that’s got about 1,000 gold atoms in it. You’re seeing the individual gold atom in the crystal. And they assemble into various sizes. And here’s an example, a large particle which is mainly confined to plasma but can cross into the basement membrane. Rather like the diagram I showed you. Oh, what’s this? 2014. Still working at the weekends, Smithies. Why do you do it? Because you enjoy it. One last thing that I will take time for. I have a hobby and that’s flying. And this man here, Field Moray, taught me to fly. And like all pilots when they’re learning, you’re scared. At least you’re certainly nervous and I used to sweat terribly. I mean it would pour off me. So that I remember saying to him one day after a lesson: And I learned to teach flying too. And I taught some of these guys. And John Cooper used to get so sweaty that his shirt would be absolutely sodden through after he’d had a lesson. And when he went by himself the first time, he came back from going by himself and he walked across to the office and said: It’s a lesson in science too. If you want to do something and you’re frightened of it, learn about it, take lessons, go and teach, have somebody to teach you. You can do anything you want. You have to overcome your fear with knowledge. Very important part of learning to do science. Don’t be frightened of something new. Go and learn. And then you might have an airplane. This is my airplane and it’s a grand thing to have a companion with whom you’re happy. And that’s where I’ll end. Applause.

Oliver Smithies (2014)

Where Do Ideas Come From?

Oliver Smithies (2014)

Where Do Ideas Come From?

Abstract

At many times in your scientific lives, you will hopefully have ideas – good or not so good – that will determine what you will do next. Where do ideas come from? My intention is to tell you where some of mine came from, and particularly those when I was as young as you. Some useful principles emerge.

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