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(0:02) Good morning. Yeah. All right. Good. Something to counteract the rainy days we have here. Alright. Today we are going to make a very important transition. The transition goes back to this picture. Of course, what we wanna do is understand biological function by taking our two favorite approaches -- understanding the organism minus an individual gene, understanding an organism minus an individual component, and understanding the individual components minus the organism, genetics and biochemistry. And as we know, the genetists went all find their route, finding mutants, through mutant hunts, making crosses, making genetics maps, et cetera, didn't understand really what these genes had to do with anything specific in the organism other than that they produced phenotypes when they are mutated, and the biochemists went off purifying enzymes, working out biochemical pathways, etc etc. (15:43) The third important part in building up DNA is to make the monomers that are used to produce DNA, we need triphosphate, we need to put on a triphosphate, and here we go, we'll take our sugar here, our base over here, and then, off this carbon (the 5' one) we have our phosphate. And we have a triphosphate. (16:38) There we go. So this is the monomer that is used to build up DNA, this guy here is called a NUCLEOSIDE, note the "s". This guy here, with the triphosphate on it, is called a NUCLEOTIDE. Ok. It's not usually written in such big capital letter, but nonetheless I point this out. (17:09) And obviously, what is this triphosphate gonna do for us? It's gonna provide the energy to allow us to make DNA polymer chains, we're going to do a dehydration synthesis, where we break two of those phosphates off and use it for the energy to be able to catalyze DNA chains to be made. Ok. Now, when you combine nucleotides into a DNA strand, you do so to create a SUGAR-PHOSPHATE BACKONE.
(17:53) Ok. You'll see for many molecules I don't care that you know their structures terribly well, but for the basic structure of DNA, including its sugar-phosphate backbone, it's gonna be important for all that we talk about. So what happens is, we have a chain of DNA, growing like this, (18:26) and we have our -OH here, we have our base here. And, which carbon is this? 5'. That's right. Ok. To the -- which carbon is this? This one? Great. That's the 3', (2', 1'…) great. Ok. To this 3' carbon, we add this triphosphate, breaking off two phosphates there, the diphosphate gets broken off, the pyrophosphate, and we get a single phosphate linkage… to the next subunit of the chain. (19:27) So here we go: phosphate, sugar, phosphate, sugar. And if we ignore these bases, which, you know, who cares about the bases anyway? What we have is just phosphate-sugar-phosphate-sugar-phosphate-sugar-phosphate-sugar. Ok? The… So it's a very simple structure. There's nothing hard to remember about this. The phosphate is always attached to the 3' carbon of the preceding sugar, and to the 5' carbon of the next sugar. (20:02) Ok? So, we often speak of chains of DNA growing from the 5' end to the 3' end. And I confuse non-molecular biologists, what we're talking about, 5' ends and 3' ends, this is what we're talking about, that the addition is catalyzed onto the 3' carbon of that sugar. It grows at its 3' end. So you have sugar phosphate sugar phosphate sugar phosphate. So that's it! We're all done. Um, well, there's the bases there I guess too. (20:34) So, we'll mention the bases. The bases, are, they come in two types. There are purines; adenines and guanines are purines, and they are six-membered rings with a five-membered ring. And there are two bases called pyrimidines, they're smaller, the thymine and the cytosine, and they're six-membered rings.
(21:15) And they have some carbons some nitrogens some oxygen and some hydrogen. But you gotta admit, that compared to proteins, this is pretty boring. It's just one long sugar-phosphate chain, and two purines, slightly bigger things, two pyrimidines, slightly smaller things, very similar structures for these two, I don't even bother to focus on the difference. And as compared to the richness of proteins, there's just no way anything interesting can happen with this. That was certainly the thinking at the time. You have to understand how important prior ideas, prior prejudices is to science. People look at it say, this must be some structural molecule, it's scaffolding, it's like the studs in the walls of the house you're building or something like that, not too interesting. (22:03) So uh, what happens? Well, you know, it takes time to sort things out. People come back to this problem, any thoughts? I mean, I've given you one reason why this did not make a huge impact, because it was, you know, DNA was kind of a boring molecule, and people weren't really sure this was right. It could be an artifact, right? Maybe, maybe some important protein had come along for the ride with the DNA fractions, right? What's another reason why people might not have paid tremendous attention to this result? Sorry? (Student answering) It was just bacteria.
(22:45) Anything else? (Student answering) Couldn't imagine, right, how this DNA could encode the enzyme. Anything else? Date? It's in the middle of the Second World War. Maybe people had more important things to do. Right? So this is right in the middle of the Second World War II, which is worth noting, that these guys are working in the middle of the New York City at the Rockefeller Institute. It's in the middle of the Second World War. Anyway, the war is over, some more work continues on this. (23:18) And the work takes a somewhat different tact. Um, instead of working on bacteria, it now is, there's work here on certain BACTERIAL VIRUSES. So instead, you see bacteria get their own here, instead of using bacteria to infect mice, HERSHEY and CHASE and others at the time used viruses to infect bacteria. So here the bacteria is the victim. And people had found and had studied these amazingly interesting, really tiny things, that, could affect a bacteria and kill it. These particles, that have funny shapes, were called BACTERIOPHAGE. What does phage mean? To eat.
(24:17) Bacteria-eaters. Bacteriophage were these little viruses, they were incredibly tiny, you could filter them through very small filters. And yet when you add them to bacteria, they would kill the bacteria. These were very simple things. I'm reluctant to call them creatures. Are they alive? This is a favorite question people would like to debate. They say, are viruses alive? (24:40) And the answer is, who cares? I mean, it depends on what you wanna define alive to mean. To me it's not alive, in that it cannot replicate on its own without a host, so I won't call it alive. But anyway I'll refer to them loosely as these creatures that eat bacteria. They were very simple. And all they really had in them, was some DNA in their capsid, this capsid up here, and some protein. But they could attach to a bacteria, and after a certain amount of time cause the bacteria to burst open, and produce lots of daughter phage, lots of daughter bacteriophage. (25:22) It could replicate within this bacteria. So somehow this, while I may not wanna call it alive, certainly can reproduce itself, or at least with the help of a bacteria, can reproduce itself. When people first discovered these bacteriophage, what do you think they wanted to do with them? (Student answering) Sorry? (Student answering) Yeah, where? In humans! The first thought about what to do with bacteriophage were a whole bunch of interesting Russians who wanted to make up large quantities of bacteriophage and have people drink them, so they would kill all their bacteria, this was early ideas for antibiotics. (26:04) It didn't quite pan out that way. But you know, people have all these very exciting ideas of "Wow I got something to kill bacteria, let's pour it down a patient and see if that does something good for them." That's why there are institutional review boards, too, to make sure you can't just do that right off the bat. Somebody else's gotta think about it also. It turns not to be a great way to kill patient, uh, to kill, (laughter) sorry, you know it doesn't actually kill patients, but it doesn't also kill the bacteria so well in human beings. So anyways, so the question was, how is it, that these viruses kill the bacteria? Somehow they inject something into the bacteria, something causes something to happen, which causes virus particles to be made. (26:54) I don't put too fine a point on it, 'cause that's all you can really say at that point. Something goes in, and something comes out. So what goes in? How could we tell what goes in? Yep. (Student answering) By seeing what's left out. How can we see what's left out? Just being really practical. How are we gonna tell? Visually. Look. That turns out to be a terribly hard thing to do. You've got really good eyes to be able to say, "Oh, the proteins are still there but not the DNA, or the DNA…" (27:29) The thought was, if this thing is injecting its DNA, then the DNA must be carrying the instructions to make phage, and this would be hereditary material. So what we want to show is the protein stays out and the DNA goes in. But how's that gonna, how you do that? Practically? Radioactive labeling turns out to be the best way to do that. If we could label radioactively the DNA with one label and the protein with a different label, we could see which radioactive isotope goes into the bacteria. (28:01) Any candidates for an element that we can use to label DNA that won't be in protein? Sorry? Sorry? Oh. Who had one? Uranium! (laughter) Somebody's thinking World War II here. Right. Got spare uranium around? The problem with it is that the DNA does not actually have uranium in it. And so when you put uranium in it, it wouldn't still be DNA. We would like to be able to label it with an element that's actually in DNA, so the only difference is it's a radioisotope. (28:32) Phosphorus! Phosphorus, there's obviously phosphorus in that sugar-phosphate backbone. Is there phosphorus in a typical amino acid? Any of the 20 amino acids? No phosphorus. Great. So we can use a phosphorus isotope. We can P32 label the DNA. How do you -- but how do you make live bacteriaphage that are labeled with radioactive phosphorus? What kind of fancy chemistry would you need to do that? Yes? (Student answering) (29:09) Perfect! If you grow the bacteria in radioactively labeled, in medium, if you grow the virus and the bacteria in medium that has radioactive phosphate, the bacteria and the virus take care of it for you. The phosphate is automatically incorporated. So you don't have to do any chemistry, you just feed phosphate, radioactive phosphate, into the medium, and the phage produced are radioactively labeled, purify them and use them in your experiment. Similarly, what are we gonna label our proteins with? (29:36) Carbon? No. Hydrogen? No. Oxygen? Nitrogen? No, 'cause the bases have nitrogen. Sulfur, we only got sulfur. Where's sulfur gonna be? So for example, Cysteine, Methionines, right? We got sulfur. Here's S35. So we can take bacteria, and we can take a phage, and by growing them in presence of radioactive DNA, uh radioactive phosphorus, P32 and growing them in the presence of radioactive sulfur, S35, we're able to produce bacteriophage that are labeled. (30:16) Ok? So, P32, S35, now we infect bacteria with them. Let me take a big tube here, I'm gonna add bacteria. I've got the phage here. The phage particles are attached to the bacteria. And they're gonna inject whatever they inject. Now what do we have to do? We gotta knock off the viral, the bacteriophage particles from the bacteria. I wanna knock them off, and see what is staying with the viral particles and what goes into the bacteria. So how do I get in there, with tweezers and separate off, peel off each virus from the bacteria? (Student answering) (31:03) Washing turns out not to be strong enough to, you gotta be pretty violent, you get these things off. So you really needs some incredibly strong agitation, so specialized devices were used to create intense agitation. What specialized devices you're aware of that do that? Blenders, kitchen blenders. The Waring blender turns out to be the perfect laboratory device for this experiment, and this is actually known as the Waring blender experiment. (31:32) Um, you take the, you take the bacteria with the, with the phage attached to it, you let them, you let them attach and do whatever they're gonna do, inject their DNA as we know it turns out, it's the right answer. And then you press "Purée". And ZZZZZZZOOOM, and the viral particles fall off. So, it's important to know how things really happen. So then what happens is the bacteria are separated from these particles, and it turns out these particles are, the viral particles are much lighter, much less dense than the bacteria, so how do we separate them?
(32:07) Centrifuge them. We centrifuge them, the bacteria particles are there up in the supernatant, it turns out to be our phage capsids. And now what do we do? We take this stuff, we measure the radioactivity in the supernatant, that is, the material that stays above, and we measure the radioactivity in the pellet, and what do we end up seeing? Where does most of the P32, what shows up in the pellet? Mostly P32 shows up in the pellet. Is there no S35 in the pellet? (32:54) You know in the textbook story of course there's no S35 'cause they want to be nice and clean, but in reality, there's gonna be some S35. But it was you know it's less than one percent of the S35 ends up in the pellet, most of the S35 stays up here in the supernatant. Does all of the phosphorus go in? No of course not, some of the viruses didn't even attach, and not everything goes in, so there's still radioactive phosphorus up in the supernatant. But the striking thing is the pellet primarily has gotten the radioactive phosphorus, not the radioactive sulfate, sulfur, and therefore we can conclude that, what? Well, more DNA went in than protein. Are we therefore entitled to conclude that DNA is the hereditary material? Why? (Student answering)(33:59) Well I mean, but, you know, suppose that one percent sulfur, is tracking one minor protein that is the secret. You can't, it's very hard to rule out that there's no contaminant that's traveling along with the DNA. And if you really truly disbelieve DNA, you can be churlish and say, "Well, I just don't believe that you've so purified it that you can completely rule out that some minor protein component is really conferring heredity."
(34:24) In fact, when we look really closely, Avery, McCarty, and McLeod's biochemistry, I believe, was purer than the purity of this experiment. But, by this point thinking has begun to shift toward DNA being a reasonable hereditary molecule. In addition, it was a second line of proof, different from the pneumococcus, using a different system, both pointing to the same answer. And the intellectual tide shifted to recognizing that this probably was right, and the reason these experiments were pointing to DNA was DNA had to be the right answer. But of course, how was it the right answer? What was it about DNA that can confer these properties? This was still unclear in 1953. But not for that long. It became clarified relatively soon thereafter. (35:21) And of course it became clarified with the understanding of the DNA's structure, THE DOUBLE HELIX. Nobody here has not heard of the double helix, probably there's nobody, no grown-up who doesn't know about double helix and all that, but nonetheless, I wanna stop and take a little a bit about -- and also I'll say on a personal note, this is the first year I've taught this class after, uh, first time I've taught this class, when Crick and Watson have not both been alive. Some of you may know that Francis Crick died just this past summer, which is very sad. He was an incredible person. And you know, as I've said, Mendel was one of my heroes, Francis Crick was also one of my heroes. He's just an extraordinary person. But Jim Watson is still alive and kicking, and still quite active. And so in any case, you're not far removed, so I tell you a little about this stuff is history, but this history here I'm telling you about, these people are for the most part, Francis is passing notwithstanding, alive and kicking. Jim Watson is still quite alive, actually, McCarty is still alive.(36:28) It's really, uh, anyway. So 1953, just a year later, Jim Watson and Francis Crick are working in England. Watson is a student from Indiana, former ornithologist, had his interest in ornithology originally, and then studied more biology and came to England, 'cause he wanted to study the gene. Francis Crick, a physicist who worked in the Admiralty during World War II. (37:03) And of course, what they did, was on the basis of an awful lot of modeling, and getting to see experimental X-ray diffraction pictures of Rosalin Franklin from London, made a model, and the model is this beautiful, and I haven't drawn it to its proper proportions, but this this beautiful double helical structure, 5', one chain of DNA running in one direction, 5' to 3', an anti-parallel chain of DNA, going in this direction, 5' to 3'. (37:47) It was a beautiful structure. Um, Jim Watson has written a whole book about the discovery of double helical structure, and we're only 51 years past that. It was, anybody who hasn't read the Double Helix, this book, he really should. It's one of the great books of science literature and actually is on many people's lists of some of the great books of the 20th century. It's a wonderful competitive story of Crick and Watson, racing against Linus Pauling, it's you know. (38:21) Someone came along, and had lunch in Cambridge with Crick and Watson, and they came away and said, this is before they discovered the structure, about a year or so, and said "These guys are idiots! They can't even memorize the structure of A and T and C and G, and they're trying to find, you know, the structure of DNA! These guys are never gonna get anywhere." So this person, whom we'll come back to in a moment, was wrong, about this particular point. (38:49) Because what Crick and Watson did was they played around the models, and what they ended up noticing was a couple of things. First off, from Rosalin Franklin's pictures, that this was helical. The X-ray diffraction pictures could tell you at a glance that the structure was helical. They saw that. They then tried to make helices. Now there were other people. Linus Pauling knew something that DNA probably had to be helical, and somehow he just got it totally wrong. He made a, just a nutty model of DNA. Linus Pauling, smartest chemist of the century, made a crazy model of DNA, where he took the sugar-phosphate backbones, and he put all the sugar-phosphate backbones in the middle, and he had three of them. He had a triple helical model with sugar-phosphates in the middle. (39:32) And what can you tell me about the charge on these sugar-phosphate backbones? Very negative, you're gonna stick a whole bunch of negative charges near each other in the middle? No way! Anybody could've known. This was bushly mistake. But so Crick and Watson: "Whew, Pauling's got it wrong." They put together this model, and the key to the model was the recognition of base-pairing. The recognition of base-pairing that, if I take a thymine here, and I take an adenine here, that, (40:41) these two groups, would be pointing at each other in such ways as to make two hydrogen bonds with a certain characteristic distance. And, not just that, but cytosine and guanine could also be fit into that same distance, and they would have three hydrogen bonds.
(41:45) And here, and H …. H, three hydrogen bonds. And they'd fit the same -- what? (Student questioning) Oops! Thank you. Good point. That's the problem. Yup. Well, it's a little messy, but anyway, the business end here is three hydrogen bonds and two hydrogen bonds, and they both fit into the same distance perfectly. So this double helix here, could have either A's and T's, or G's and C's, or C's and G's, or T's and A's. And they'd all fit perfectly with each other. (42:36) Now, there was an old observation. Not that old, there was an observation floating around at the time, that said, when you analyze the amount of A's and T's in DNA, you always found out that the amount of A tend to be very close to the amount of T, the amount of C tend to be very close to the amount of G. Although these amounts could be different. This was due to a biochemist called Chargaff. And they're called, this was called CHARGAFF'S RULE, or Chargaff's Law, or Chargaff's Observation. Chargaff noted that the percentage of these amounts tended to be equal, but didn't know what to make of it.(43:27) This, perfectly explained it. It was very good. Remember I said somebody came through Cambridge and said these guys were turkeys, Crick and Watson were turkeys just 'cause they couldn't remember the structures and all that? This was a very distinguished chemist who said this about Crick and Watson, it was Chargaff. Chargaff came through and said these guys were turkeys but it was Chargaff's Rule that Chargaff had missed the importance of. He was quite bitter about this through much of his life. And there's a very wonderful, biting quote that Chargaff says when Crick and Watson became famous for the double, the DNA double helix? Um, see if I can get it right. He says, "That such pygmies should cast such giant shadows (referring to Crick and Watson), that such pygmies should cast such giant shadows only shows how late in the day it is." (44:19) Woo! Anyway, he was not happy. So uh, all right. Now, this was a big deal thing. Crick and Watson knew this was very important, they raced to publish the paper about this, they sent it off to Nature. It's a gem of the papers. It's a page roughly in text, it's very short, very clear, has this beautiful picture drawn by Francis Crick's wife Odile, and it's, it is just a charming paper. They know that they've cracked the secret of life. Why'd they know they've cracked the secret of life? Because the most important thing about this model here, is not its structure per se, but that it explains how it is that a DNA molecule can be replicated. (45:06) That somehow, all it takes is for those two strands to come apart, who knows how, and when they come apart, each can serve as a template for the other, because since A's always match T's, and C's always match G's, each strand has enough information for the other. That's how replication happens. You have two strands, each of which has sufficient information to encode the other, they somehow come apart, they each serve as a template for the other, and that's that. That's the secret of life, how life replicates itself. Not just that, we've explained replication, what about mutation? What's mutation? Sometimes it gets it wrong! Sometimes it screws up. So, for one little biochemical model we've explained replication and mutation. That's pretty good.
(45:51) Now, the thing is, in writing this paper and getting this off to the journal, this was not an easy thing to get done quickly. You couldn't, you just didn't have enough time to explain all these details. They (48:20) This model of the strands actually serving as a template would predicate each new DNA double helix was composed of one old and one new strand. If you could prove that, then you'll have real confirmation of this Crick and Watson model of semiconservative replication. And so a young student, two young students, Matt Meselson, who's still working and is down the road at Harvard, a wonderful person, and Frank Stahl, who's still working in Oregon, proved that the new DNAs that were made after each generation were in fact composed of one old strand and one new strand. But how could you possibly do that? (Student answering) Sorry? Radioactive labeling. But how do you radioactively label that so you can see that you got a double helix that's half one and half the other? Old ones labeled with (student answering) well with one isotope -- it actually turns out to be nitrogen – the new one with a new isotope, say N14, you can do heavy nitrogen and ordinary nitrogen. (49:35) And if what you can do is grow up your DNA, when you first grow it in normal nitrogen, then you shift to N15, heavy nitrogen, you could make DNA molecules that were half old half new, and therefore half labeled with normal nitrogen, half labeled with heavy nitrogen, and how would I prove that these DNA molecules were fifty-fifty hybrid? What would be the property that I would be able to test? (Student answering) But radioactivity turns out to be really hard to… Wait, density, it turns out density. If I could just measure the density of the DNA, I would show that if the semiconservative model was true, the molecules would now have intermediate density between all heavy and all light nitrogen. (50:21) They had to work out a centrifugation technique so sensitive, a salt gradient centrifugation where you can put DNA on it, you got a really fine salt gradient spun in a centrifuge, and depending on where the DNA migrated, you can measure the density of the DNA, and they were able to show that in fact, newly replicated DNA strands had this intermediate density that would be expected from the semiconservative model. And so in fact, by that point, the semiconservative model, I think, is well established. In some sense, you would say the beauty of the double helix was almost, one of these very rare scientific results where when you look at it, you say, "It can't possibly be wrong, that it explains too much. It's too beautiful." But as we've discussed before, that's not enough. You need some proof so that it's real, and this Meselson-Stahl experiment provided a real confirmation of that. Onto next time!
Last Modified 3/19/07 2:46 AM |
