Greaser.M.1706_05.10.2017_Transcript This transcript is AI-generated and human reviewed: we utilize an AI software to generate the transcript, and it is then reviewed by Oral History Program (OHP) staff. As we review AI-generated transcripts, we cannot guarantee 100% accuracy and some inaccurate words and phrases will still exist. For these situations, words or phrases that are unclear are noted in brackets. Researchers should always refer to the original recording before quoting the text; they can also contact the Oral History Program if they cannot access the audio file for the document or for clarification about the text. Due to the scope of experiences encapsulated by the interviews in our collection, there may be offensive and/or distressing language present in both the transcripts and the audio recordings. The OHP stands against harmful and offensive language; at the same time, we do not censor such language when present in order to preserve the integrity of the interview as it was conducted. If not stated specifically here, funding for this transcript creation and editing was provided by either general OHP funds or specific gift of grant funds. 0:05 Joah Parrish: Okay, so I think, what's going on here? Okay, why don't you give me a little bit of- 0:13 Marion Greaser: Testing? Testing? Is this working? We don't know. 0:24 Joan Parrish: Yeah, I think it's okay, 0:30 Marion Greaser: All right. 0:36 Joan Parrish: So, now we need to think about what you want to talk about. 0:39 Marion Greaser: What do we want to talk about? So we haven't, I don't think we've talked about graduate school at this point. Well, I think we did talk about some of it. 0:57 Joan Parrish: Right. Because we talked about having to have carbon, or carbon paper to do your PhD thesis, and having to hire a good typist to do that, 1:05 Marion Greaser: Right, right. Yeah, there was no such thing as a Xerox machine, copy machine even, all. 1:21 Joan Parrish: Right, let's try this. I think I'm going to move up the, okay. So do you remember the title of your thesis? 1:37 Marion Greaser: Not off the top, but it was, the role of sarcoplasmic reticulum in post mortem muscle or something like that. But no, I don't remember the title of my thesis. That's one of those things that you don't have to remember. 2:02 Joan Parrish: But you published scientific papers from that? 2:05 Marion Greaser: Oh, yes, I think there were five. Interesting anecdote, the very first referee publication that I had was a jointly published paper where I was like the fourth author or something, but it was published in Nature, and this paper has never been cited. 2:42 Joan Parrish: No! You didn't manage to work it into any of your review papers. 2:48 Marion Greaser: It was about someone had made a claim that rigor mortis didn't occur in germ-free rats, and so both Cassins and Briskey, I had done a lot of stuff on on post mortem muscle and and developed this rigorometer. I don't know you were seen that machine or not, but what it does is it alternately puts a weight on a strip of muscle, and it stretches it out before muscle is in rigor mortis. You stretch, and you release the weight off of it while it shortens back up again. And you do this alternatively, until all of the ATPs do go on in muscle, and that's the definition of rigor mortis. Then when you try to stretch, it doesn't stretch very much, just trivial amount release. It doesn't go back. You know, it's essentially fixed length. Somebody made the claim that germ-free muscle didn't go into rigor mortis. So, we tested that and sure enough, it does go into rigor mortis. 4:03 Joan Parrish: But, well, that's interesting, yeah, but not very interesting to other people, enough to cite. 4:11 Marion Greaser: Nom but, but we got it published in Nature. So you can see the bar was a little bit lower back then. 4:23 Joan Parrish: So the meat science program here at the University of Wisconsin was very well known. 4:30 Marion Greaser: Yes, yes. Bob Bray was sort of the originator of the meat science program and one of his early students was Ernest Briskey, and they both were very, very gifted researchers, teachers. They were able to attract a lot of very good students to the program. They also, I think, were very forward-looking in terms of incorporating, you know, modern biology and science. And they collaborated extensively with people in biochemistry. Dr. Phillips, and then later Hoekstra collaborated with lots of people, not only in meat science, but in other areas in the department. And so there was always a very strong biological science component, more so than many other meat science programs around the country. They were more traditional, cut them up, weigh them, and determine how much fat there and so on, not mechanism, sort of things. And so a major impetus was that Ernie Briskey actually got a seven year NIH grant to study this pale, soft, exudative problem. So the PSE condition was originally discovered, I think, in Europe. And Briskey went over, I think to Denmark, or, I think it was Denmark or Norway, where the person that originated the description of the PSE condition, and did a post doc over there. And then he came back, you know, on the staff here. He was a very, very entrepreneurial scientist, build things bigger and bigger. He got a sort of a muscle biology group started on campus here, this guy is in meat science, but he recruited people from physiology and anatomy and so on, biochemistry, all in part of this sort of a core group of people. And he was the driving force to get the first, well, the second major addition to the building, the part that we're in now was built around 1970. Okay, so I was a grad student between '64 and '68, so this was built while I was away out in Boston on a post doc. And the laboratory conditions, and when we just had essentially the whole laboratory, I don't know whether I've told you about this, was in what's now Bucky's Butchery, no hood. Probably in the whole building, there was no hood. And I remember one time somebody dropped a bottle of glutaraldehyde. [JP: Oh, my. Oh my! So you just aired out, just aired it out. The whole thing cracked on the floor. But all the laboratory equipment and all the grad students worked in that area down there where Bucky's Butchery is, so obviously this addition here was tremendous impetus. But in regard to the PSE condition, we'll circle back. When Briskey came back to this country, and back to UW why he continued work on this area, and I think one of his first graduate students was Bob Cassens, so here we're getting into the lineage. And Cassens, first graduate student was myself, so the lineage is pretty tight here in terms of these people. Bob Kaufman was also a graduate student of Bob Bray's. 9:53 Joan Parrish: Can you describe PSE for people who don't know abot- 9:55 Marion Greaser: Yeah, PSE is an acronym for pale, soft, and exudative. The color of the meat is paler than normal pork would be. The texture is very soft. It's kind of mushy. I mean, you could actually just grab a piece of it and squeeze it, you know, and squeeze liquid out of it. 10:20 Joan Parrish: So not very attractive. 10:21 Marion Greaser: Not very attractive. And exudative, just means it gives off liquid. And you if you put it in a display case in the grocery store, you know there's usually a little pad that's in the bottom to soak up extra moisture. Well, that pad isn't sufficient enough for really bad cases of PSE to soak up all the liquid. So you look at a package of meat in the case and there's visible liquid all around the edge. People are going to probably try to choose something different, right? So it's a severe marketing problem. Also, when you cook it, the moisture comes out too easily, and so the meat may end up kind of dry. It's not, you know, inedible. There's no danger in terms of eating it, but it's a marketing problem. The other thing that happens is that that if you try to use it in processed meat, why, it doesn't retain the moisture very well. So, you know, you can, like, in a normal hot dog formulation, you may add 10 or 20% water to that, and it improves the texture of the hot dog. But water is cheap too. With PSE meat, why, you wouldn't be able to do that, it would start to break down, and the liquid had come out, and you get little pockets of liquid in the hot dog, which nobody wants to see, right? So, and hams, for example, are going to oftentimes you have some water added, because you insert the curing reagents into the ham, and you can't put a normal amount of liquid in there. Another thing that happens with hams is they get to be two toned. There's some of the muscles are more susceptible to PSE condition than others. And it's, it's the most valuable muscles in the whole pig. 12:38 Marion Greaser [cont.]: You know, it's Murphy's Law that developed the PSE condition. The two major muscles in the ham and the loin muscle, are the ones that develop the worst conditions. And scientifically, what's happening is that the pH drops down very rapidly post mortem, while the temperature of the muscle is still high, before you can get the muscle cooled down, and that causes denaturation of the myosin. And myosin is involved in holding a lot of the moisture in the meat. So that's, that's what's happened. So why does it really happen? Well, we were kind of on the track of this, but didn't get there. What we in my PhD thesis work, we found that that the calcium accumulating ability of fragments of sarcoplasmic reticulum was reduced at the time of death and rapidly declined post mortem due to the high temperature with low pH condition. And one of my papers, in fact, was a, you know, sort of in vitro testing the temperature pH effect on fragments of the sarcoplasmic reticulum. And it was clear that that you could abolish the calcium binding ability of those. The real mechanism of how PSE works is, there's a protein called the ryanodine receptor. And the ryanodine receptor is the calcium pump in the in the membrane of the sarcoplasmic reticulum, and normally during rest, why, that pump is pumping calcium out of the cytoplasm around the myofibril proteins, and that causes the muscle to relax. In PSE, that calcium pump has a mutation, and it leaks calcium when it shouldn't, and so you're kind of, a low grade leak of calcium all the time that partially activates the system, and the more contraction you get, there's heat generated when you break down ATP, and that heat goes up. The more the heat goes up, the more damage it does to the sarcoplasmic reticulum. And pretty soon, you're in a system where you can't control it. Happens in humans too. 15:24 Marion Greaser: It's referred to as malignant hyperthermia. There's nothing cancerous about it, but some people will react to anesthetic by their temperature going up, their muscles getting rigid, and they go into this sort of irreversible situation where they can never get control of their calcium anymore. And you burn up all the ATP and generate a lot of heat, and you denature proteins, and you've got a problem. So anyway, the idea that the sarcoplasmic reticulum was involved in this PSE condition was really a result of my work on my PhD thesis. 16:32 Joan Parrish: So were the pigs affected while they were alive? 16:35 Marion Greaser: There is, yes. There's a condition in the living pigs, uh... trying to, the name is... where pigs, they'll go into this rigidity situation. And oftentimes when pigs are loaded onto a truck, they're stressed under those conditions. This sort of brings on an episode of this of increased heat, muscle rigidity and so on, and the pigs will literally die. Now that's a problem for the farmer. You put 100 pigs on the truck, and you only get 95 of them that come off at the packing plant. The PSE condition is a problem for the packer, because he buys the pigs. They may look normal, but then they go into rapid post mortem pH decline. The meat gets soft and watery, and now he's got a problem with selling his products. So there are two related condition. So you can see some visible signs of the pigs having this. And now, of course, there are genetic tests that pick out the mutation. It's quite interesting. There's only one mutation that's been found in this ryanodine receptor. It's a giant protein, 2000 amino acids, something like that. And only one that's been found in pigs, but there's like 25 or 30 different ones that have been found in humans. They all cause the same problem, but many more have been found in humans than have been found in pigs. 18:45 Joan Parrish: So do you have any hypothesis on this? 18:47 Marion Greaser: I have no hypothesis. I mean, certainly there's been more more effort looking for it in in humans, but they're selecting against this one mutation in pigs, and have gotten a large degree of control over the situation with pigs. So if there was a different kind of mutation, that test wouldn't pick it out, so you wouldn't be able to eliminate it that easily. 19:18 Joan Parrish: Right. Maybe even at this point, this would have been in the early 60s, then? 19:23 Marion Greaser: The, well, I was in my PhD program between '64 and '68. [JP: So this would've been the in the 70s.] In the 70s. Yeah. 19:35 Joan Parrish: So by the 1970s, the swine industry had started to narrow their gene pool. [MG: Yeah.] And humans haven't narrowed their gene pool yet. 19:49 Marion Greaser: Right, right, yeah. But even so, the problem that persisted for so long is that typically, animals that were carrying this gene were lean, muscular, and fast growing, just exactly what you'd select for. And normal methods of selection, you were just increasing the frequency of the gene in the pool, and until you got a different way of trying to detect the problem. And it's very interesting. Poland China was a breed that had a large incidence of it, and there was a fairly big Poland China herd at the UW farms, and so we we could often get animals for further research without any trouble. Somebody was quoted as saying "Where does PSE occur?" And it says, "In and around, 50 miles within Madison, Wisconsin." That was somebody that didn't think it was as big a problem, I guess. 21:11 Joan Parrish: But it was throughout the swine industry. [MG: Yes.] Not here in America, but and in Europe also. 21:16 Marion Greaser: Oh, yes, right. 21:17 Joan Parrish: So it was a widespread problem. 21:27 Marion Greaser: Widespread problem, yeah. So again, you know, Briskey's NIH money, and things were much looser back then about NIH grants and what you could do with the money after you got it. You know, if you have some good hypothesis, why, you could go any off in any direction, and there wasn't somebody peering down your neck to see, did you spend it on the goals that you'd set it in your original proposal? Because very many people can set out seven years with goals for that length of time. 22:07 Joan Parrish: So this grant he got at the initial beginning was for seven years? 22:12 Marion Greaser: I mean, this was- He, I mean, there was quite a bit of stuff. I mean, this was, no, it was a, it was a seven year grant to start with, [JP: Wow.] Yeah. And it, I think, sort of overlapped the time that I was in school. I don't know, I was never supported by any NIH money myself, but in grad school, I'm sure the program was. 22:51 Joan Parrish: So you've gone off to Boston. 22:53 Marion Greaser: Gone off to Boston, and I changed my direction there to concentrate more on the muscle contractile proteins. And there was a new protein called troponin that had been discovered about three years before I went out there. And there were a large group of of people all over the world that were getting excited about this protein. It was involved also in calcium control of contraction. It's a calcium binding protein, and most of the people in Gurgly's [?] Lab, which was where I was working, were working on myosin and actin and tropomyosin, other well established proteins. But we decided, why not work on something new and different? So I started working on the troponin thing, and it proved to be very, very productive sort of area. I also was one of the first people in the whole troponin area to start using SDS gel electrophoresis. This was brand new at that time. There had been somebody that published a paper, I think, in '66, but there was a paper came out in '70 or '71 in the Journal of Biological Chemistry, which kind of showed that you could, in fact, separate and determine the molecular weights of sub units of proteins. The SDS would tear proteins apart so that they are down on the sub unit level, and most everything could be solubilized and so on. Well, contractile proteins are historically difficult to work with because of their solubility problems. They're either soluble in high salt. You have to use crazy with methods to purify them. You don't, just none of the proteins have been crystallized. 25:20 Marion Greaser [cont.] For example, you know, that was kind of the way you purified proteins back then. And you measured how much enzyme activity they had, and if you could purify them so they'd crystallized and recrystallized, and you didn't change the activity, why then you knew the protein was clean, pure. The other way was using analytical ultra centrifugation to look at the protein to see if there's a single peak or not. Well, most of these things didn't work very well with contractile proteins. So I started working with this SDS gel electrophoresis, and it was very illuminating what was going on. First of all, troponin wasn't just one protein, but there was several proteins, several different subunits, and nobody had come up with that. There was a paper that appeared while I was just starting that said there were two different fractions in troponin. Well, we were able to take the troponin complex apart, and it turns out that there's three different subunits. We were able to reconstitute them into an active protein, and this sort of set the troponin field on its ear, we're able to give them their names, the troponin I, troponin T and troponin C. And there were about five or six different nomenclature series about the same time. All these different groups are all struggling, trying to sort this thing out, but we were fortunate enough to get it done first and and the names, you know, are related to the fact that, the function of each protein we determined the function. The I was an inhibitor protein that did him an ATPase [?] activity. The T was the subunit of the troponin that bound to tropomyosin, and the C was the calcium binding one, so that it and C nomenclature has stuck, and all the others have fallen away, because it's so easy for people remember which, what's, which is, which by their functions. So I continue to do then the troponin work, coming back here to Madison and worked extensive with Rick Moss, up in physiology. He has done a lot of work with single muscle fibers that you can skin and measure the force of those things. Well, we were able to extract selectively the troponin out of those, the troponin C by itself, away from the rest of it, put it back and get it function back again. And so there's probably 20 or more papers that we've generated in our time working together. So it's been a very productive, collaborative effort. 28:37 Joan Parrish: So you were taking apart the system to measure how it worked. [MG: Right.] To ask questions on how it worked. 28:46 Marion Greaser: And you know, the individual parts have had functional properties that that were part of the function of the complex. But by using STS gel, we could, in fact, purify each individual component, then put them back, put pairs together, and so on and and see whether or not we could reconstitute what was considered the troponin activity. So it's worked out well, there's now over 2000 references to each of these subunits in the literature, you know. 29:31 Joan Parrish: So they're pretty important in contractile proteins, 29:34 Marion Greaser: Yes, right. And there are, of course, now quite a number of genetic mutations in the individual subunits that lead to various kinds of cardiomyopathy and muscle disease problems that all related to their function as well. So additional work that we've done on the troponin area included the crystallization of the troponin C in collaboration with Dr. Sunderlingen over in biochemistry, and that's led to a three dimensional structure of the protein. Again, you know, if you have good workers and people that you collaborate with, you can get a lot done. You don't have to do everything yourself. 30:36 Joan Parrish: And the right technology. 30:38 Marion Greaser: Yeah, right technology, yeah. Don't get hung up on technology that's not giving you the answer. You find something different if you can't. And there's still people working on troponin C and how its functions and so on. But you know, the major big questions have been answered long ago, you know, and some people stay on a protein forever. 31:09 Joan Parrish: And so now you were back, you were on faculty. [MG: Right, mhmm.] And did you have much teaching or? 31:18 Marion Greaser: No, I didn't. My appointment was 80% research, 20% teaching. And that appointment stayed throughout my career. And so, you know, a large part of my research success is related to the fact that I never had a heavy teaching load. I would help in the 305 class. I had a techniques, biochemical techniques class. The biochemistry department was kind of caught in quicksand with very old methodology and so on. And so nobody was teaching protein purification. So I taught a course called 645 animal science, which involved having students do a project where they would purify a protein and they'd have to use all these techniques. They'd have to use gel electrophoresis and ammonium sulfate purification, chromatography and so on. And so provided them, I think, a good, good education. I inherited the muscle biology 725 course from, from Dr. Cassens. This was one that had gotten started with, with Briskey when he was here, and he left, I think, right before I came back to go to Campbell Soup, and he wasn't at UW anymore after that point. So this was a jointly taught course between anatomy, physiology and animal science. And Cassens passed that one off on to me. But it was pretty well set up at that time already, because there were guest lectures. It was a two credit course taught, you know, the fall every other year, you know, not, not a heavy sort of teaching thing. And then I taught all the muscle structure and muscle, you know, muscle protein, parts of it, but you know, maybe a third of the course that I really face to face teaching on. So I had had time to devote to my research program that a lot of people in faculty positions don't have, you know, so I was blessed in that regard to have that opportunity and tenure. 34:13 Joan Parrish: How did, how did it go for tenure? 34:15 Marion Greaser: Tenure is kind of laughable, actually. You know, my post doctoral research had, had been very productive, and it had, you know, I'd had five papers published in refereed journals out of my graduate work, couple major papers in in Journal of Biological Chemistry. So I was promoted after two years as an assistant professor to associate professor. 34:56 Unknown Speaker: So that's pretty quick. That's pretty quick. 35:00 Marion Greaser: Right, and I think, and I was promoted to a full professor in another, another two years, I'll have to look back at that, but I know, you know, it was never an issue, and tenure wasn't as big of a thing back then. Most people that were making good progress got tenure. There wasn't, it wasn't as high a bar to jump over. And so I don't, don't remember much about the tenure process or the or the promotion process. I just went through, it was done. It was done. Yeah, not one of those things to worry about. 35:46 Joan Parrish: What was UW-Madison like when you when you came back here as a professor? 35:52 Marion Greaser: It was very, the protests were over. The Sterling Hall bombing had occurred in 1970, I think. I came back in June of '71, the protests had gone. There were no more protest activity on campus. So campus was calm, even when I was in graduate school, when there were some Dow Chemical things going on, most of those are the other end of campus. It didn't, it didn't reach this far down. Ag campus people weren't involved in those protests. So just went about your business. There wasn't any teardrop grass that drifted this far. I don't know if there was any tear gass when I was in grad school at all. 36:54 Joan Parrish: So it's very quiet. 36:56 Marion Greaser: Very quiet. Yeah, I think early on when there was the merger of the all the UW systems. I don't remember when that was. It was early 70s, I think. And you know, there's always been sort of fights between UW-Platteville and UW-whitewater and that group of campuses and and UW-Madison and UW-Milwaukee, which were the sort of the big ones, you know. But supposedly the field, playing field was leveled a little bit more 37:44 Joan Parrish: Right. So, within the department, though, at that time, there was the Department of Meat and Animal Sciences. Was that correct at the time. 37:52 Marion Greaser: Right. Department of Dairy Science. I mean, there was a separate poultry science department, you know, Milt Sunday was chairman for many, many years there, and then that got merged into Animal Science. And when Bray, Bray was chairman of the department, and he was involved in changing the name from Animal Husbandry to Meat and Animal science, I think. I don't know whether that was a two stage thing or not. It was Animal Science, I know, when I was in starting grad school. And I think it became Meat and Animal Science, and it was that for many years. And I think, think the meat part got dropped out at the time when poultry science merged into animal science. 38:56 Joan Parrish: Right, which would have been in the early 80s, I think. I was here. We came in 83. So, so lots of changes, in the animal departments. What about biochemistry? 39:17 Marion Greaser: Well, Houkstra, retired fairly early because of health reasons. I think the collaboration then with biochemistry declined a bit. I mean, I re established something with Sunderlingen then with a crystallography thing, but not as much in terms of collaboration as my graduate school years and maybe a few years post grad school time. 39:59 Joan Parrish: But then you were working with Rick Moss. 40:02 Marion Greaser: Yes, he's in Department of Physiology. So, you know, I've maintained, you know, close relationships with other departments throughout. And that was, I think, related to the way I'd seen how Briskey and Cassens had made very productive kind of interactions with other departments. The exercise physiology area, we've had several students who have kind of jointly worked with our program and so on. 40:53 Joan Parrish: So because you were mostly in research, did you feel any effect of changes by the deans? 41:10 Marion Greaser: No. There was very little top down pressure about what areas to work on and so on. Most of them had, if you can go get money somewhere, go for it. All right. So, you know, I've had a biomedical kind of relationship all along because of, you know, the interest in muscle proteins. They're found in skeletal muscle and heart. NIH is very interested in how those things function, maladies and so on. And so it was natural for me to consider going after funding from NIH. And I was successful in doing that. I've also had a number of grants from the American Heart Association. There's a Wisconsin Heart Association that funded research several times, both, both with the troponin stuff, as well as later on when I got into the titin work. 42:34 Joan Parrish: Okay. So what happened with Oscar Mayer? Were you ever involved with Oscar Mayer? 42:42 Marion Greaser: Yes, to some extent, not as great extent as some of the other people. Bob Cassens and Bob Kaufman have both worked quite closely with people in Oscar Mayer, I haven't done as much work with them. I was involved in a collaborative project with Larry Borchert. Likes to talk about all the time of, we did electron microscopy on wiener emulsion, and published paper about that. And he's very proud of that, that work, you know, and but I didn't work heavily with that. Andre Seisnicke [sp?], I don't know whether you know that name or not, but he worked at Oscar Mayer for a while. He was a post doc with Cassens, and then he got a position at Oscar Mayer, and continued to do some work with the university in that regard, but I've never had major funding with them. I've given a few talks out there and explained what work is about and so on. Some consultations, a few times about techniques and so on. 44:01 Joan Parrish: But you're more interested in basic science. 44:05 Marion Greaser: I'm more interested in basic science, yeah. 44:09 Joan Parrish: Which brings us back to titin. 44:10 Marion Greaser: Yes, very big project, protein. Yes. So after I considered I'd made my major contributions to the troponin field, I looked for a new challenge. And titin is definitely a new challenge. One of the things with titin that make it difficult is with a molecular weight around 3 million daltons, it doesn't move into a polyacrylamide gel very well. And so we found a number of different factors that that affect that. One of them is that that normally, you know, you solubilize protein in a very strong reducing condition, mercaptoethanol to break down any adult disulfide bonds. Well, it turns out that when you put a solution of proteins on the top of a gel and you start to electrofreeze them, that sulfhydryl reducing agent moves with the diaphragm, and so you're leaving your proteins behind in a condition where they're not being reduced fully. And that's a big problem with titin, because they're left at the start line almost immediately. 45:55 Marion Greaser [cont.] And one of the things that kind of showed, suggested that there might be something funny going on here, was, there are small format gels, which typically people use, and then there's big ones that are about this big. And the small format gels you can run, run a gel for about an hour, hour and a half. Big ones might take five or six hours. Well, the titin seems to move farther in the small format gels, than it did the large one. Relative, you know, relative to the diaphragm. And that didn't make any sense, because the exact same polychromide, you know, until we figured out that, in fact, we're leaving the leaving the protein behind. We're moving the sulfhydryl reducing agent down with the front. So if you put reducing agent in the upper reservoir buffer, now you're bathing the proteins with that, and you can move them on farther down. The other thing we found was that the stacking dye that you normally use to concentrate the protein before it goes into the separating gel was slowing down the protein the titin. You couldn't make, dilute enough stacking gel that you weren't sort of slowing the protein down. And so it didn't really all start at the beginning in a concentrated form like you expected it. So you get kind of smeary problem up at the top. Well, we found a different stacking gel that would do that, and that solved it. So, a major paper and analytical biochemistry that that described this sulfhydryl problem and the stacking problem. But even so, we're just moving the titin maybe couple millimeters into a 10 centimeter gel. That's not very, not a very good way of doing things. We found that we could use agarose instead of polyacrylamide, and that was one of those eureka moments in science. 48:06 Marion Greaser [cont.] The very first gel that we wrote with agarose, we moved the titin, like, three or four centimeters out of 10 centimeters into this, this agarose gel. And you could start to see, you know, multiple bands and so on. And it really, really helped us sort out things, and that that was related, of course, to later on, when we found this, this mutation in rats, because we had such a good gel system. Most everybody was using sort of dark ages gel systems, or using 2% acrylamide, you know, which was consistency of warm Jell-O to try to separate. And the bands would come like this, you know, inconsistently. But with the agarose gel system, why, you could get sharp bands weld separated from the start line, and you could see that, in fact, these are really different sizes. And some rats had large bands and some had smaller bands. And we, of course, sorted out that it was genetics. Have I told you that whole story or not? 49:27 Joan Parrish: Could you go back and tell us a little bit about what titin is and how it was first discovered? 49:30 Marion Greaser: Okay, sure. Titin was discovered back in 1979. So myosin was discovered in 1870, and actin in 1942, and tropomyosin in '47, and the troponin then was like '67. So now we're at, We've had SDS gels for almost 20 years, and they found, in fact, you know, everybody thought that this sort of wad of stuff up at the top was just cross linked protein that wasn't moving into the gel. But some man by the name of Quan Wong discovered that, in fact, it was really a separate protein. And it was named titin because it was after the Greek gods, which were titans, you know, giants. So this was this giant protein called titin. And a lot of work then has been done on it since then. Turns out that the protein extends the full length of the sarcomere, or half length of the sarcomere. You know, one thick filament has 300 myosins, and a myosin has a molecular weight of 500,000. So, you know, we're talking about something that's extremely extended, and nobody could believe that, until there were a series of monoclonal antibodies developed that showed that that all the monoclonals was buying sort of two bands per sarcomere. They were equally spaced from the middle of the sarcomere and the Z line in each case. 51:23 Marion Greaser [cont.]: And so that suggests there's two sets of molecules, and the antibody's picking up, you know, just one epitope on that site. All the molecules are lined up. So you get a line perpendicular to the myofibril where those antibodies are binding. So, then, there was a major discovery about, must have been '95 where the complete sequence of the titin was published, and it was clear that there were isoforms. There were some different sized ones. The protein was alternatively spliced, which means that it can, you can have a chunk either in or out, depending on the splicing pattern. And there are different kinds of titins and skeletal muscle and cardiac muscle and so on. But that the reason that we got into to looking at the titin with this agarose gel system was, we wanted to ask a simple question about, are there developmental changes in titin with age? Nobody's ever done a very complete job of this, because they'd used bad methodology before that. So we took rats at age. 1, 5, 10, 15, 20, 30, days of age and adult, and we looked at what the titin patterns were. Sure enough, there were larger ones at the beginning, at the younger age, and they got progressively smaller. So there's one major adult cardiac titin that's around 3 million daltons, but there were bigger ones up to three and a half million daltons early on. You know, at one day, we did this study right. There were six rats per group at each of these time periods, and all the patterns looked consistent, except a few rats. It looked like we mixed up the samples. So we looked at a 15 day sample, and maybe the major band was at 3.2 million daltons, but this one had a 3.5 million dalton band instead, didn't make sense. We were careful enough that we wouldn't have mixed up samples. And there were several of these, you know, at different ages, were they were off. We kept track of which litter each of these were from, and all the ones with the funny size were from the same litter. And the light bulb went off: this must be a genetic thing that's causing this. 54:47 Joan Parrish: Wow, so that's really good record keeping. 54:49 Marion Greaser: Yes, yes. We'd sacrificed the mother as the adult for all of these litters. The father still belonged to Sprague Dawley. We fortunately still had some litter mates from this same litter that's showing these funny bands left over, but the animal care people had mixed them up. The reason we knew that they mixed them up was there was a male and female that ended up in the same cage, and the female gave birth to three pups that were all dead. We were able to get samples from those dead pups and found out that they were carriers. So that mother was a carrier. So otherwise we'd have lost it. We just had a crazy observation, but no way to track it down. We got like, 20 males and 20 females from Sprague Dawley, and none of them showed the thing. So it's a rare mutation. And the father, you know, who knows what happened to the father, but there wasn't any others that we could ever find, you know, from from buying them from Sprague Dawley. But we had a litter, and so now we knew that that mother was a carrier, and we mated it with a wild type, and we got, half the pups were carriers, and half of them not. We didn't know whether it was, you know, the fact that there were three dead pups, we didn't know whether that was just an anomaly or whether this mutation was causing problems too. You know, we don't know whether any of these are going to be old enough to breed. But it turns out that, yes, they are old enough to breed. They survive okay. And then we found that we could, in fact, breed two heterozygotes and get, you know, double carriers, heterozygotes and wild type offspring, and the homozygote carriers were able to reproduce. And so we can propagate the line, and that's where we ended up with it. 57:39 Joan Parrish: So when you were looking at these titin proteins, what kind of muscle were you looking at? It? 57:46 Marion Greaser: Cardiac muscle. [JP: Cardiac.] Yeah. So the mutation affects skeletal muscle also, but we didn't, we weren't working on that at the time. 57:55 Joan Parrish: So you were working on cardiac. [MG: Yeah.] And how were you able to tell about these little mice - [MG: Rats] -rats, whether they were carriers or not? 58:06 Marion Greaser: Just by crossing them and then taking the offspring and- 58:12 Joan Parrish: Did you have to sacrifice them to get cardiac tissue? 58:15 Marion Greaser: Yeah. 58:18 Joan Parrish: Okay. 58:27 Marion Greaser: But you know, if you, you can, for example, if you take, you take three heterozygotes, or you take two heterozygotes and cross them. You take those offspring. And if you have a homozygous carrier, mate it with a wild type, all the offspring will be heterozygous, right? And you know, you can get it from the offspring all the time. [JP: So what I'm trying to-] This is my 1965 level of genetics, 59:03 Joan Parrish: Well, I'm trying to, yeah, because people now have such expectations about being able to do genetic tests. and you really had to do the test meetings and look at the muscle to see what you had, and back track. 59:15 Marion Greaser: Right, right. Yeah. Se we didn't know- 59:18 Joan Parrish: So you were like Mendelssohn. 59:21 Marion Greaser: Mendel's peas. Yes, I like to joke that all these, these monks are sitting around the table and and he says, "Herr Mendel, we tire of peas." [laughs]. So, yes, I mean, we, we used the Mendelian genetics, and it was, it's almost, it's a single gene mutation, dominant, and we can figure it out just by doing offsprings, doing gel electrophoresis on the offspring, hearts of the offspring. And of course, then we were able to track down the gene for this, which was, you know, the part that that your daughter was involved with and collaboration with Michael Gotthardt from Germany, who got very excited when we presented some of this stuff about, you know, the titin mean being messed up with these, these rats. And says, you know, "I have some people in my institution that can help a lot in this, this sort of thing." And he's, he's a real outstanding molecular geneticist that that we got involved with the work. 1:01:00 Joan Parrish: So you had this mutation in this protein, was there a phenotype, then, did the muscle not work as well? 1:01:09 Marion Greaser: The muscle is, amazingly, it's still functional. Because here what we're doing is on these homozygous mutants, we're adding a million daltons to an existing protein that's normally 3 million daltons, and it's hooked in the sarcomere at both ends, and we've added an extra million daltons of of sequence in there. Why that thing still works is beyond me. There are single gene mutations in the titin, single point mutations that mess up the titin function more than this does. In a specific, you know, functional-. 1:02:05 Joan Parrish: In a functional sense. It just seems, how could you stuff so much more in that little space? 1:02:13 Marion Greaser: Well, the it's, it just, you know, zigzags and coils up and so on. 1:02:18 Joan Parrish: So it has to do with the three dimensional folding of it. You get it folded in there. 1:02:21 Marion Greaser: So, when you stretch muscle, the more you stretch it, the more resistance that that you have to stretch. And most of this extra resistance to stretch is due to the titin molecule. You're sort of like extending, you got a rubber band that's kind of wadded up a little bit, and now you can straighten it out, and there's not much resistance to get it straight. But now when you start to stretch the rubber, why, it's resisting you more. And you get more and more force developed, and it's like a door spring to kind of protect the muscle from being stretched out too far. The thick and thin filaments overlap with each other, but they're not attached to each other. So if you didn't have some protection, and you did this to it might not go back right. But the titin prevents it from being over stretched. 1:03:16 Joan Parrish: So it really just, like, keeps things aligned. 1:03:18 Marion Greaser: Yeah, it keeps everything aligned. 1:03:20 Joan Parrish: Okay, well, that was pretty exciting research. 1:03:24 Marion Greaser: It was very exciting, very exciting indeed. And you know, to figure out, you know, that we, in fact, had recovered the mutation, and it has no great functional effect on how much stretch ability the muscle has. This has got to have some biomedical kind of relevance to it, and that we had a, you know, an animal model for for this is important. Now independently, people had found that there was a mutation in this protein called RBM 20 that causes dilated cardiomyopathy in humans. They didn't know anything about the titin side of it. This was independently going on. They map the mutation, you know. So we were able to, then, you know, map the mutation in the rat and find out, you know, there's a big segment of the RBM 20 gene that's missing in these in these rats. And so we know exactly where the mutation is and what it's doing, and that was major emphasis of that Nature Medicine paper. 1:04:49 Joan Parrish: Right, right. But your little rats didn't have the same disease that the humans have- [MG: No.] -it's a slightly different 1:04:55 Marion Greaser: Well,I mean humans and rats are different, obviously, in many ways. But one of them is that humans typically live 30, 40, 50, 80, years. A rat lives maybe a year and a half, two years, three years max, I think. Other things that show up only later in life in humans are major, major health related things. The first 40 years are pretty healthy. You don't see any problem, but it shows up later on or under certain kind of stress conditions. And with these animals, you know, you stress them. Why, you can start to see some things that are funny happening, right? 1:05:50 Joan Parrish: So, you had an animal model, 1:05:51 Marion Greaser: We have an animal model. Yes, yes. 1:05:55 Joan Parrish: That's exciting. 1:05:59 Marion Greaser: Yes, yes. I'm sure that I would have retired earlier if we hadn't had this exciting thing to chase down and so on. 1:06:12 Joan Parrish: And so you retired. [MG: Yes, mmhmm. ] And you're still active coming to work. 1:06:25 Marion Greaser: Mmhmm. I still review papers. From time to time, people look to me for expertise, and so on. So I still can share that. I've written a couple reviews, or helped write a couple reviews. And the work with the RBM 20 is being carried on. By Wei Goo who's out in University of Wyoming now, and he has the rat model. We don't have any more rats on campus anymore. 1:06:59 Joan Parrish: So I should have asked you about your graduate students. 1:07:08 Marion Greaser: Yes, I had quite a number of them. I think I won't be able to name you all of them. I do know when I first arrived, probably the one of the first ones I had was Clark Brecky, who did some work with cardiac troponin. He went on to a faculty position and eventually became came a dean. Mamoru Yamaguchi did all the work on the electron microscopy of, we did some electron microscopy of the troponin interactions that were very well received. He worked at a faculty position at Ohio State University. Let's see. Jeff Fritz was a student. He was the one that that worked on development, had that analytical biochem paper on electrophoresis. The agarose gel stuff was done by, names are getting away from me. 1:08:42 Joan Parrish: Well, that's okay. We can look up in the papers. [MG: Yeah, right.] And then the titin person is...? 1:08:51 Marion Greaser: Wei Guo is a post doc. Another grad student that finished up was Lee. Shijun Lee. He's got a major paper that appeared in Nucleic Acids Research, and that paper is going to be widely cited. It's already been cited pretty heavily. I think that the Nature Medicine paper has been cited 200 times now. [JP: Wow, that's something.] That paper, the original troponin reconstitution paper, is somewhere above 600 citations for that one. 1:09:58 Joan Parrish: Right. So, just to sort of wrap it up, it seems to me that you believe in the Wisconsin idea. [MG: Yeah, mmhmm.] because you're using your knowledge broadly and collaborating with people all over the world for the good of all of humankind, even though you're doing really basic kind of questions. 1:10:23 Marion Greaser: Yeah, right, yeah, I believe in the Wisconsin Idea. 1:10:30 Joan Parrish: And is there anything else you want to add to for posterity? 1:10:37 Marion Greaser: Well, thankful for all the people that have worked with me through the years. I've had really good associations, very diligent people, and any success I've had really is related to the people that I've worked With, because you can't do it all yourself. And usually, if you you're willing to provide resources and help to people, it'll come back sooner or later. It isn't just all out-go, you know. And giving credit to people, students and post docs and so on, they're going to feel good about that, and that'll help them advance their careers. And I've helped a lot of people outside of UW as well with advice about techniques and so on. So never, never felt like that was an imposition on me to do that. 1:11:47 Joan Parrish: And this leads me to a question. The last 20 years, it seems in science that we've been really worried about patents. 1:11:59 Marion Greaser: Yes, uh, I have a couple patents. One was with Bob Kaufman on putting baking soda in meat to prevent the PSE problem. And it's an effective way of correcting the PSE problem, and it's been used by Hormel and company. They purchased the rights, and there's still a little bit of royalty money trickles in every six months or so from that. I think the patent is just about run out now. And we've also patented some things related to the RBM 20, although that hasn't yielded anything productive at this point, but for detection of the mutation in humans, right. 1:13:05 Joan Parrish: That's still maybe pretty young kind of technology. 1:13:12 Marion Greaser: Right. But I haven't done a lot of pursuit of patented kind of things. But you know, sometimes when things, but the baking soda and meat, you know, very simple technology. This is not high science. 1:13:36 Joan Parrish: So this is intriguing. Was it just in a solution you sprayed on the meat? Did you inject it-? 1:13:40 Marion Greaser: No, no, you inject it. You inject it into the meat. Yeah, right. Well, it's a simple matter. It's going to raise the pH. So low pH is part of the problem with the water holding capacity of meat and so, so if you raise the pH- 1:14:01 Joan Parrish: And decrease the temperature, because you could be putting in colder fluids. 1:14:05 Marion Greaser: Yeah. Although some of this is done, you know, after, even after the meat is in the PSE condition, you can inject it in the meat and raise the pH of that meat, and it'll now bind the water better. [JP: Oh, okay.] So you don't necessarily have to do it really early on. That's what we were doing. We started out doing that, but it turns out that you don't have to just do it early on. 1:14:32 Joan Parrish: So if you're just worried about water retention in the meats. 1:14:35 Marion Greaser: Water retention, yeah. 1:14:39 Joan Parrish: Well that is interesting. Well, thank you very much. [MG: Okay. Good.] We can do another session. If you think of anything you need to have in archives. 1:14:47 Marion Greaser: maybe I'll take a look through these things and see where there's other ideas about things that I might have forgotten to mention that I could share. 1:15:09 Joan Parrish: Alright. There we go.