 We're the ministers for the invitation to speak, and I'm very privileged to give something that's out of the ordinary. My talk is not about the cutting edge research that you've been hearing about all day today. I was asked to sort of give a perspective on the best life to an extent over four decades as a biochemistry professor, and just to tell you what I had a view of the years. My name is Jim Dahlberg, and I'm currently the American Professor of Biochemical and Chemistry, and Associate Director of the Marvel Institute. I did a postdoc in Cambridge, England in the late 60s, and then joined the faculty here in 1969, which was definitely a couple of decades before the great majority of you were born. So what I'm going to be talking about is a number of careers that I've had along the pathway. Let me start off by saying the time that I spent in Cambridge in Fred Sanger's lab as a postdoc in his lab, that was really a great highlight in my life. Fred gave me a lot of insights on how to not only be a scientist, but to be a decent person, at least he's a decent person. I'm trying to do that. So many of the techniques that I'm going to be talking about were going to seem really antique to you both because you've been hearing a lot of cutting edge stuff. And back when I was a postdoc and joined the faculty here, we just didn't have that. So PCR had to wait another 20 years to be invented. Change shifts, likewise, maybe 25 years, 30 years, and so forth. And so there will be some kind of comic relief in the future of our time. Over the years I've had more than any hats. Teaching at the university, doing research, I co-founded a couple of biotech companies. I was an expert witness in a couple of litigation trials. I was a science advisor in the governor's oil, and most recently I served as the interim CBO of the Morgan Institute. Now I'm not going to talk about all these things. I'm going to emphasize three of these events, and I'll come back to each one individually. In terms of teaching, universities, I taught about half-time teaching medical students and half-undergrads. Of course, I had graduate students and postdocs in the lab. What I want to emphasize today is the research that went on in my lab during that period. I've always had a very small research group, five to eight people max. But having been here for 40 years or so, that translates into publishing people. So I'm not going to have time to name everybody who was involved in all these events and just take it from me right now. I didn't do it all. So I'm not taking better for that. My research has always focused on nucleic acids. These are some fairly old pictures that we've pulled out of the following camera here. That's Fred Blatner. This is shortly after I came here. Fred Vosov-Shavolsky. And he with a more hair and a different color. And actually I'm wearing a tie, which I was looking for that tie but I couldn't find it. So with these two guys, we worked on bacteria phase lambda initial sequences of the messenger RNAs that were made in vitro and then analyzed following a method that I had worked on as a postdoc. We also studied quite a bit on the mechanism of transcription. Collaborated with other several other groups here on campus. Bernie, Massiax and Julian, mainly on ribosomal RNAs. And with Goldman and Dick Burgess on transcription. And then most recently with Mike Sheets who's here and he's like a chemistry professor working in Zepis transcription. In the early days we developed some methods for looking at RNAs. This is a two-dimensional gel separation of small RNAs. In this case of E. coli, there's lots of different organisms as well. Each one of those spots represents an individual RNA molecule which at that time was quite an unusual thing to be able to study. And we could analyze each one of those RNAs by the fingerprinting methods that the sangria had developed and characterized what these molecules were. Using those methods and others related to it, we, over the years, about this didn't all happen in a week or anything, in a couple of decades, we worked on the fat-phage lambda, as I told you. We showed that the primers for DNA synthesis for reverse transcriptases are cellular tRNAs that are taken up by the virion during infection. We studied small RNAs in E. coli at the Schumacher. We looked at this three-mark medicine, 16S RNA, and amazingly, this was kind of revolutionary at the time because there were a lot of people at that time in the early 70s who actually thought that the function of ribosomal RNA was to hang proteins on them. And little did they know about ribosomes, of course, or the fact that that's a business, a big part of the business of ribosomes. And we showed the Julian that the three-prime-and, the 16S RNA is essential. We've also found that scionidase are encoded in ribosomal RNA genes and studied quite a bit of the kind of synthesis of small ribosomes, in transport. We had visitors to the lab. Joan Steis and John Nagelsen were posted in Cambridge at the same time. I was just happy to be passing through Madison and so on. We pulled the right picture of them. Also, Fred Sanger came in. He came for about a week. He wanted to switch physicians with the RNA. I had gone to his lab to learn the fingerprinting and analysis of RNAs and a few techniques that worked out here in Madison. And that was a lot of fun, too. Needless to say, we had lab parties. And this is my group at the time. In 1974, we were studying celebrating the identification of another primary tRNA or MLTP, I think it was. And at that time, you can see that the regulations for protocols for the history of the lab are a lot more less stringent than today. And of course, the champagne and the crisps and that sort of thing. However, you can see the ice buckets for the champagne bottles as well as the radiation bottles. Another visitor to the lab, we had another lab party is my brother. My brother, Al, is a professor at Brown University in chemistry. And he came to work and did some experiments with Schemes for episomal RNAs. And this was the celebratory the celebration of our research. And here I'm doing my best to look like a cool surfer. And my brother is doing his best not to look like a surfer. Later on, we decided our intentions turned much more towards cell biology. And we wanted to relate RNAs to a more biological system. And for that, we focused on centipus latus. You can inject RNAs or DNAs into nuclei or in some case cytoplasm of these oocytes and study what happens. And using this methodology, we learned about mechanisms of transport of RNAs between the nucleus and cytoplasm. We showed that some T-RNAs can be in the nucleus. Not that they have to be every time, but at least at times. We also studied the control of micro-RNAs more recently in the biogenesis in oocytes and embryos. And very recently in collaboration with my sheets, we studied the RNAi in oocytes and embryos and discovered that oocytes and embryos of centipus cannot carry out RNAi. And then we traced this down to the lack of a protein that's essential, agotube, which has the nucleus that can be in RNAi. This is all kind of an RNA kind of experiment. We've also studied some DNAs, both in terms of gene structure, promoter mapping and that sort of thing. And when we were studying the small nuclear RNA genes, we discovered that there was an interesting structure in some of the SNR RNA genes that's a polyperinidine tract in the team, CT, CT, CT, in one strand aging in the other. The reason unusual is that you can form triple-stranded DNAs under super-helical conditions, negative super-helicy, and at low pH, and the region at low pH is required to see natural probe agencies, but the consequence is that you can form this triple strand here with a single strand moving out. And this is what the topology looks like in a model where this polyperinidine is going back on the helix. You have no idea whether this has a biological function, but it sure was fun to work out the topology. Now, at about that time, PCR was coming on the scene. We wanted to do a lot of PCR amplifications of DNAs for various reasons, and we found that some DNAs just did not amplify PCR. And we traced this down to the fact that a highly-folded DNAs are the ones that gave us the most trouble with PCR. And the question is, what is it about a highly-folded DNA, or a DNA-conform, the new mechanism? And that included this because David Gelfand in California had come up with the same observation that certain DNAs couldn't be amplified well, but this is using the taxilumnase, but David had a variation of the taxilumnase that lacks the phytochrimaxid nuclease domain, and so we thought that maybe the exo was really responsible and this shows that in fact that is the case. If you have a heroin or just to pull the DNA molecule then there's a perfect duplex out here but there's single strands down here because they're just not complementary just at the end of the classification. During PCR you would be elongating a primer running down the square and so that's what we've shown in here. This primer goes down here and pushes the end up to the bifurcation here, and it turns out that this is actually for the nucleus of taxilumnase. And it's been called the phytochrome exo-nuclease but in fact it's an endo-nuclease and the endo-nuclease in this case clats specifically right at this point here. The third is that it's a nucleus that recognizes the structure, and the structure is the plaque, the phytochrome brunteric region extended into a duplex as well as another duplex on the other side of the arm of the bifurcation. But we did this experiment we didn't know just where the end of that primer ended because we were carrying this reaction out with taxilumnase the intact taxilumnase and we didn't know just where the polymerase had stopped here. We now know that just to work the three main end of this so-called promised end has to overlap by one nucleotide at least this downstream duplex. So we say that that's invading this duplex. It's not fully invading it's just overlapping. We don't know that this is an actual displacement. When we saw that we realized that in fact what we're looking at is a structure-specific nucleates that see this kind of structure that doesn't care what sequence there is. So that is sort of an aha moment for us because meant that if we could synthesize biochemically synthesize any of these three components we could reform the substrate for the nucleates and that in fact is the case and so if we wanted to cut for example a molecule in a specific place we just synthesized the appropriate all of the nucleotides, put them all together, add the enzyme and then we cut the network. So we were very excited about that and this is our aha moment and we said we can cut any sequence we want. Nowadays it's what she would just synthesize the thing that it's not that way at the moment. So we have another celebration and this is now we've moved out of the lab into the offices. Victor the Amateur and Marianne Braugh are the people working in this that's also one in the back over there in this vehicle. Mike turns the postdoc and this young looking guy there is the next speaker who we have to collab right next to mine it's not that he was my graduate superman he was my best friend. So this was a patent that we found on nucleotide activity. We also wanted to take advantage of Victor's skills to generate an enzyme that did not have a nucleotide activity that was almost a specific nucleotide domain of TAC because the native TAC polymerase is two different enzymes. The polymerase are polymerizing in the nucleus. We want to get rid of the polymerase so we only rework with the nucleus and so by mutations we can do that that's fine. We applied for a patent on that variation of TAC polymerase and the title of the patent was something like a nucleus deficient DNA polymerase and needless to say we wanted to patent that but in fact that was a terrifically good patent because it gave us the options to work on a single purified domain without having to worry about the polymerase itself. So they turned the patent back to me and I turned it over to a company that I started to work with Floyd Smith which is Thirgu Wave Technology company here in Madison Thirgu Wave got a start using this technology in the biocrine nucleus it really started with a conversation I had with Floyd Smith industry professor when we were sitting next to each other and going down to the meeting in New Mexico Floyd had a friend Lance Ford's and that was more of a business person than a scientist but in both areas and he became the CBO of Thirgu Wave we founded the company just with our own savings as well as some angel investors venture capital investment and then we grant to the government NIST and FBI Harnes and then in various amounts from the different people of course we took the company off campus to rented space so our world headquarters of course was like Apple Lab and a small office it sounded good and to get started we used borrow equipment, scholarship with people throwing things away we did not have a great deal of money of course and so we actually licensed we got the license for this technology we got that license from Morph by giving Morphs some stock in our company and that was the first time Morph had done that and it's sort of served as a model since then it's worked out very well both for them and for us mainly for us we didn't have any money once the company got started we changed the goal from the original sequence cleavage of the sequence that started our observation to using that cleavage as a way of detecting sequence changes in DNA and I'll explain how this works in a separate way we call this technology the invader technology because as I said before you have to have a primer that sort of invades these overlaps the end of the bifurcate this is the bifurcated DNA and you want an invasion so the idea is to interrogate a sequence in a target this is a target DNA for the genome DNA of any sort or it could be RNA too so we take this target and synthesize two olivonucleotides what we call the invader and a probe olivonucleotide and these are both synthetic they're designed to be complementary to the target so you have to know the sequence of the target or at least part of the sequence and the probe will sit down at one end and then the invader olivonucleotide these are only about 15 nucleotides long the invader sits down right next to it and it's positioned such that the 3 prime n of this invader would overlap with the end of the duplex between the probe and the target and what we're doing is we're really interrogating this single nucleotide in this case it's a C and if that C is anything if this nucleotide is anything but a C the gene won't base pair with it and then you won't get an invasion and you just juxtaposition for the end of the molecule and this overlap is required for leakage and so we can carry out the reaction add the enzyme and the enzyme if this is really a C the enzyme will cleave the flap off of this it's an artificial flap and that will float off now we do this reaction at the melting temperature of this duplex and so once cleavage has occurred this duplex falls apart and this olivonucleotide comes off another olivonucleotide another probe olivonucleotide can sit down and be used again and so what we do is we can just saturate the system with lots of probes and this happens over and over again we get a lot of flap fragments so what does that mean well if we get these flap fragments it tells you something about that nucleotide right here now we have to be able to assay on this flap that's just the same thing we do that by second reaction from the same tube actually and this is just what we call signal generation where we have another olivonucleotide that folds on itself and has an open spot where the released flap can sit down and direct cleavage of the end of this olivonucleotide is a fresh olivonucleotide and the released flap will cause the cleavage of the end of this thing and at the end here we have a fluorescein dye which has been crunched by a nearby crunching dye but if you cleave this off then the fluorescein is released and you get white and so again you can do this at the melting temperature of that interaction between the flap and the bread olivonucleotide and you get signal amplification and what this does is in the original flap amplification you get about 10 to the fourth molecule's permanent target molecule then each one of those can be used to generate about 10 to the third and so you get a huge amplification signal and it's very very specific and so this works in it and we were very excited about it the characteristics are it's accurate and sensitive very inexpensive obviously it's not that easy to run procedure can be adapted to just about any sequence you want detection and doing nucleotide changes, snips or mutations that you want to see whether they exist we've used it to quantify gene copy number we used it in my old lab here at the University to quantify RNA this was one of the first papers to show that a particular microRNA level correlated severity of cancer in this case and it can also discriminate between various strains of viruses and we focused on HPV HPV has different strains that are very similar to each other some of them will cause cancer others are very benign there's just a few nucleotide changes and so this kind of acid could be very useful and it's a lot faster and cheaper than doing sequencing so this made us feel top of the world we had incorporated early on sold stock publicly in 2001 we got to have over 300 employees but it was like a kid in a candy shop we didn't know where to stop and so we lacked focus and we really were trying to do everything and our viability our cash flow was really going down fast and so in 2005 we hired a suit he said no Kevin Conroy is a great guy he came on the CEO he focused the company on HPV assays and in 2008 a company in Boston bought the company all the stock in a third way so we were Kevin came to the rescue but this tells us that focus is important it's crucial that you're going to start a company and there's another factor in this too though and that is I want to point out the dates the IPO was in 2001 and it was like a week before the dot com bubble so we just got in under the wire we were bought out by whole logic in 2008 that was about a month before the stock market crashed so not only is focus crucial so is luck so with that in mind I'll give you a few ideas here you want to stay focused on your goals if you're starting a company I didn't get into this but you do want to be very careful about conflict of interest especially if you're working at the university Mike Cox was very generous and he ran it served on a conflict of interest committee that I set up this was before there were only one so we had to fill out we were the first people that sort of brought up the potential problem of having a conflict so we overdid it we had the associate dean of research of the medical school and the associate dean of research of the graduate school on this committee so we really had a conflict another thing you want to do is keep everybody informed your co-workers, the chair of the dean I actually went over to talk to Tom who's the kind of chancellor to discuss whether or not everybody knew what we were doing you want to hire good people Marianne Victor was spectacular and so were several other people from bio and white chemistry and then finally you have to know what people will take over people who know what they're doing and with that in mind I thought it was time to ride off into the sunset and this is the problem in Montana but even though we had cut back from my participation it's a great deal I still have a small lab here at the university and we continue to work around a few other people in the lab so I thought that was it and then I said many has from one last app and that is the Mortimer's Institute of Research Mortimer's Institute is the private part of this discovery building down here on Johnson Street University of Johnson and I was asked to join the Board of Directors of the Mortimer's Institute and then in 2012 I was asked last year I was asked to become the information CEO and currency good stuff down they asked me because I was familiar with the Mortimer's Institute I knew how the university operates on the scientists living in Madison and had some time that I could give up so I agreed to do it but the problem was I had no experience as an administrator but I was able to learn and stop up the task I changed the mission of the Mortimer's Institute so now it's much more focused on helping the university and promoting collaboration with the university and the most important job that I had was to find my replacement and that is Grant Schwartz who is the new CEO and he's also a professor in biochemistry it gives me time to go back to my day job which is the professor so looking back I'm pleased to have this great experience wonderful time in science I've been able to stay keeping close contact with my friends and colleagues and I've also been fortunate to work in a field where they're actually taking me to do what I enjoy I was also very lucky to be coming into molecular biology when it was in its infancy so there were a lot of weak questions to ask I've been very fortunate to have a professor who took me into his lab and I've had wonderful students, flourators and postdocs especially those of us here and I've great support from the department of chemistry and I want to thank them for their support and I thank you for your attention thank you how did you decide you were a professor as a postdoc? how did I decide you were a professor as a postdoc? I had heard that he was just a wonderful person I knew that he purchased food I had heard that he had this method for studying RNAs in fact the RNA was a big commission of people who just didn't know what they were working with so I thought it was a great way to get in on the ground floor so this was joining his lab when he was between Nobel Prizes he'd gotten one for proteins he was on his way to DNA and I'd say it would help in that process and it was just a wonderful conversation and it was just an exciting time a wonderful postdoc colleague service what prompted you to form an oversight I guess committee for interfacing between your lab and third wave technologies since you said you were the first one to what prompted me to start an oversight committee for the conflict actually I thought to give credit to the chairman of my department who didn't say I shouldn't do this but Harry was very knowledgeable he said you don't want people to suspect anything and I also looked at the regulations of the university already they said that you cannot use university facilities for private enterprise and I wanted to make sure that it was clear that there was a paper trail that we weren't doing anything about it do you have a question if you were starting out today what field of biology would you go into if I were starting today what field of biology would I go into it depends on you mean with the talents or lack of talents that I have now biochemistry I think everybody has to choose their own way I just love to do research in the lab and have a group a small group I mean Fred's group involved four people plus him and I just think that is a good way of running a group staying close touch with your colleagues or people who are actually in there and I I just wanted to follow my nose and do what's exciting ask the right questions I think the important thing in my system is not what the answer is but what's the question don't just ask something because you can't well with that let's say you