1 00:00:01 --> 00:00:06 Good morning. Yeah. All right. Good. 2 00:00:06 --> 00:00:08 Something to counteract the rainy 3 00:00:08 --> 00:00:12 days we have here. 4 00:00:12 --> 00:00:16 All right. Today we're going to make a very important transition. 5 00:00:16 --> 00:00:20 The transition goes back to this picture. 6 00:00:20 --> 00:00:24 Of course, what we want to do is 7 00:00:24 --> 00:00:28 understand biological function by taking our two favorite approaches. 8 00:00:28 --> 00:00:31 Understanding the organism minus an individual gene. 9 00:00:31 --> 00:00:34 Understanding an organism minus an individual component, 10 00:00:34 --> 00:00:37 and understanding the individual components minus the organism, 11 00:00:37 --> 00:00:40 genetics and biochemistry. And, as we know, the geneticist went off 12 00:00:40 --> 00:00:44 on their route finding mutants, doing mutant hunts, making crosses, 13 00:00:44 --> 00:00:47 making genetic maps, et cetera, didn't understand really what these 14 00:00:47 --> 00:00:50 genes had to do with anything specific in the organism, 15 00:00:50 --> 00:00:53 other than they produced phenotypes when they were mutated. 16 00:00:53 --> 00:00:56 And the biochemist went off purifying enzymes, 17 00:00:56 --> 00:01:00 working on biochemical pathways, et cetera, et cetera. 18 00:01:00 --> 00:01:04 We began to see some connection when we talked a bit about the mutants 19 00:01:04 --> 00:01:08 that affect the ability to make arginine and the fact that they 20 00:01:08 --> 00:01:12 could encode different enzymatic steps. And, in particular, 21 00:01:12 --> 00:01:16 I highlighted the work of Archibald Garrett who really, 22 00:01:16 --> 00:01:20 right at the start of the century, recognized that somehow genetic 23 00:01:20 --> 00:01:24 mutations were responsible for somehow affecting the production of 24 00:01:24 --> 00:01:29 enzymes in important biochemical pathways. 25 00:01:29 --> 00:01:33 So, that was sort of one connection of genetics into protein but still a 26 00:01:33 --> 00:01:37 rather tenuous one. The real next step to connect these 27 00:01:37 --> 00:01:42 two would be to do the biochemistry of genes. So, 28 00:01:42 --> 00:01:46 how would a biochemist approach heredity? A biochemist would grind 29 00:01:46 --> 00:01:51 up the organism, fractionate it in different 30 00:01:51 --> 00:01:55 components, and attempt to find heredity, purify heredity, 31 00:01:55 --> 00:02:00 get like a pure solution of heredity. And that's nuts, right? 32 00:02:00 --> 00:02:03 The notion that you could purify heredity as a biochemical entity 33 00:02:03 --> 00:02:06 because like how would you know you had it? But, of course, 34 00:02:06 --> 00:02:09 that's exactly what happened. That is what happened, was 35 00:02:09 --> 00:02:13 biochemistry developed sufficiently far that folks were actually able to 36 00:02:13 --> 00:02:16 purify substances, not just that could digest a 37 00:02:16 --> 00:02:19 particular sugar or substances that might, you know, 38 00:02:19 --> 00:02:23 like slide by each other like actinomycins of muscles, 39 00:02:23 --> 00:02:26 but substances that were actually heredity. And that began the real 40 00:02:26 --> 00:02:30 unification of those, and that's the point of today. 41 00:02:30 --> 00:02:39 And that is the field of molecular biology. And we're going to cover 42 00:02:39 --> 00:02:48 tremendous territory in unifying these two different fields. 43 00:02:48 --> 00:02:57 OK. So, let's dive right in. The discovery of the transforming 44 00:02:57 --> 00:03:04 principle. It's a wonderfully old-fashioned 45 00:03:04 --> 00:03:09 kind of word. Nobody would use language like this today. 46 00:03:09 --> 00:03:14 The discovery of the transforming principle. So, 47 00:03:14 --> 00:03:19 this starts, this shaggy dog story starts in about 1928 with the work 48 00:03:19 --> 00:03:24 of Griffiths. Griffiths had no particular interest in DNA or 49 00:03:24 --> 00:03:30 genetics or biochemistry for that matter. 50 00:03:30 --> 00:03:36 Griffith was interested in bacteria. He wanted to understand bacteria. 51 00:03:36 --> 00:03:42 And, in particular, he studied pneumococcus bacteria, 52 00:03:42 --> 00:03:48 which could infect and kill mice. And he was very interested in the 53 00:03:48 --> 00:03:54 mechanism by with these pneumococci could kill mice. 54 00:03:54 --> 00:04:00 Now, it turns out that the pneumococcus bacteria came in two 55 00:04:00 --> 00:04:05 different types. One, the bacteria, 56 00:04:05 --> 00:04:09 when they grew on a Petri plate, produced a glistening, smooth, shiny 57 00:04:09 --> 00:04:14 colony. We'll call them smooth bacteria here. 58 00:04:14 --> 00:04:19 And these bacteria, in addition to being smooth and 59 00:04:19 --> 00:04:23 glistening, were virulent. That is, if you inject into the 60 00:04:23 --> 00:04:28 mouse, these bacteria would kill the mouse. They are smooth because they 61 00:04:28 --> 00:04:33 have this encapsulating polysaccharide coat around them. 62 00:04:33 --> 00:04:36 And it's not necessarily the case that that's what makes them virulent, 63 00:04:36 --> 00:04:40 although it actually does have a role in that, but it is the case 64 00:04:40 --> 00:04:43 that they're smooth and they're virulent. So, 65 00:04:43 --> 00:04:47 you inject in a mouse, mouse dies because the mouse is not 66 00:04:47 --> 00:04:51 resistant to these bacteria. By contrast, there were strains of 67 00:04:51 --> 00:04:54 pneumococcus that were rough. They did not have the same kind of 68 00:04:54 --> 00:04:58 a polysaccharide coat, and therefore they had a very rough 69 00:04:58 --> 00:05:02 appearance. They didn't glisten. 70 00:05:02 --> 00:05:08 And these were non-virulent. If you inject these into the mouse, 71 00:05:08 --> 00:05:13 the mouse immune system was able to fight these particular rough 72 00:05:13 --> 00:05:19 bacteria. OK. So, Griffith did the obvious 73 00:05:19 --> 00:05:24 experiments. So, take some bacteria, 74 00:05:24 --> 00:05:30 we'll take some smooth virulent bacteria, we'll inject 75 00:05:30 --> 00:05:36 into a mouse. And what will happen? 76 00:05:36 --> 00:05:43 The mouse will die. This is one of the easier assays in the laboratory. 77 00:05:43 --> 00:05:50 It's the feet up, feet down assay, you have a dead mouse. 78 00:05:50 --> 00:05:57 OK. Number two. Then take the rough bacteria, 79 00:05:57 --> 00:06:05 inject it into a mouse, what happens? 80 00:06:05 --> 00:06:09 Sorry? It lives. The mouse lives because these are 81 00:06:09 --> 00:06:14 non virulent. OK. Now, let's just do some simple 82 00:06:14 --> 00:06:18 controls. Let's take the smooth bacteria and autoclave them, 83 00:06:18 --> 00:06:23 heat them up to very high temperature to kill them. 84 00:06:23 --> 00:06:27 How will we know they're dead? You try plating them out. They 85 00:06:27 --> 00:06:32 don't grow anymore, so they're dead. 86 00:06:32 --> 00:06:37 So, take heat killed, and you can verify in the lab that 87 00:06:37 --> 00:06:43 they're killed, heat killed smooth, 88 00:06:43 --> 00:06:49 check that they really were heat killed, inject them into the mouse. 89 00:06:49 --> 00:06:54 And what happens? It lives because, I mean, they're bacteria, 90 00:06:54 --> 00:07:00 right? OK. Last of all we take the utterly harmless rough bacteria, 91 00:07:00 --> 00:07:06 plus the utterly harmless heat-killed smooth bacteria, 92 00:07:06 --> 00:07:12 we inject them into the mouse, and what happens? 93 00:07:12 --> 00:07:18 It dies. That is a notable result because the rough bacteria alone 94 00:07:18 --> 00:07:24 will not kill this mouse and the smooth bacteria that have been 95 00:07:24 --> 00:07:30 heat-killed will not kill this mouse, but together they killed the mouse. 96 00:07:30 --> 00:07:37 This is very puzzling. What was even more puzzling was 97 00:07:37 --> 00:07:44 when you autopsied the mouse, you can isolate from that mouse 98 00:07:44 --> 00:07:52 smooth, virulent, live bacteria, but you didn't put 99 00:07:52 --> 00:07:59 any in. Very strange. So, this actually yields live 100 00:07:59 --> 00:08:07 smooth virulent bacteria despite not having put any in there. 101 00:08:07 --> 00:08:12 Virulent bacteria. Somehow we were able to create 102 00:08:12 --> 00:08:17 smooth and virulent bacteria notwithstanding not having put any 103 00:08:17 --> 00:08:22 in here. So, of course, Griffith then attempted to say, 104 00:08:22 --> 00:08:28 well, what was it that allowed this to happen? 105 00:08:28 --> 00:08:31 So, he could try putting in dead rough bacteria with dead smooth 106 00:08:31 --> 00:08:35 bacteria. That doesn't do anything. You need to have something alive. 107 00:08:35 --> 00:08:39 So, you've got to have live rough bacteria. You can then say let me 108 00:08:39 --> 00:08:43 take the dead virulent bacteria and start fractionating it biochemically 109 00:08:43 --> 00:08:46 and asking what fraction of that material from the dead bacteria 110 00:08:46 --> 00:08:50 allows us to recover, to have this property of being able 111 00:08:50 --> 00:08:54 to now produce virulent bacteria that killed the mice? 112 00:08:54 --> 00:08:58 And do you realize how utterly tedious and painful that 113 00:08:58 --> 00:09:01 experiment is? You take the dead bacteria, 114 00:09:01 --> 00:09:05 you fractionate it into lots of different biochemical fractions. 115 00:09:05 --> 00:09:09 For each fraction, how do you test whether it has the property? 116 00:09:09 --> 00:09:13 You've got to shoot up a bunch of mice. This is a very tedious 117 00:09:13 --> 00:09:17 procedure. I mean it is, you know, you can't underestimate 118 00:09:17 --> 00:09:21 how important the assay is, how important it is to come up with 119 00:09:21 --> 00:09:25 easy ways to do things in order to be able to accelerate progress. 120 00:09:25 --> 00:09:28 Griffith tried hard and roughly began to purify fractions and get 121 00:09:28 --> 00:09:32 information about what the fractions were. But, in fact, 122 00:09:32 --> 00:09:36 this work really never did lead to a clear conclusion. 123 00:09:36 --> 00:09:40 But it did tell people that there was some material, 124 00:09:40 --> 00:09:44 which got named The Transforming Principle. This is almost like an 125 00:09:44 --> 00:09:48 old alchemical kind of word, a principle being a particular 126 00:09:48 --> 00:09:52 chemical composition of matter, which if you don't know what it is 127 00:09:52 --> 00:09:56 you call it The Living Principle or something like that. 128 00:09:56 --> 00:10:00 So, what was this transforming principle? 129 00:10:00 --> 00:10:07 Well, it really took work about 15 years later by Avery, 130 00:10:07 --> 00:10:15 McCarty and MacLeod to sort this out. What Avery, McCarty and MacLeod did 131 00:10:15 --> 00:10:23 was the same experiment basically, except minus the mice. 132 00:10:23 --> 00:10:30 What they found was you could take the dead bacteria, 133 00:10:30 --> 00:10:37 combine it, the dead virulent bacteria, the dead smooth bacteria, 134 00:10:37 --> 00:10:45 combine it with the live rough bacteria, and by combining it in the 135 00:10:45 --> 00:10:52 right way in a test tube you would be able to plate it out on a Petri 136 00:10:52 --> 00:11:00 plate and see smooth bacteria come out. 137 00:11:00 --> 00:11:03 Sans mouse. So, they didn't need the mouse. 138 00:11:03 --> 00:11:06 This dramatically accelerated work because if you're able to just take 139 00:11:06 --> 00:11:09 fractions of the dead bacteria, add it to the live bacteria and look 140 00:11:09 --> 00:11:13 for the presence of some smooth bacteria coming out of it, 141 00:11:13 --> 00:11:16 you would be able to work much more quickly. And they did. 142 00:11:16 --> 00:11:19 And they began purifying. And they began purifying and they 143 00:11:19 --> 00:11:23 tried to isolate the fraction that contained this new ability to make 144 00:11:23 --> 00:11:26 these bacteria acquire a new property. And they knew that they 145 00:11:26 --> 00:11:30 were transforming the heredity of this bacterium. 146 00:11:30 --> 00:11:34 They were transforming the traits of this bacterium. 147 00:11:34 --> 00:11:38 They were, in fact, transmitting heredity. 148 00:11:38 --> 00:11:42 And they purified and purified and purified. And eventually, 149 00:11:42 --> 00:11:46 testing many, many fractions and making them purer and purer and 150 00:11:46 --> 00:11:50 purer and purer, they found that consistently the 151 00:11:50 --> 00:11:54 fraction that contained heredity was the fraction that contained DNA. 152 00:11:54 --> 00:11:58 Now, it was a lot more work than that because no fraction is pure. 153 00:11:58 --> 00:12:02 DNA is in multiple fractions. But, you know, 154 00:12:02 --> 00:12:06 they kept trying to purify it. And it sure looked like the 155 00:12:06 --> 00:12:11 transforming principle. The property of being able to 156 00:12:11 --> 00:12:16 transform was co-purifying along with the DNA fraction. 157 00:12:16 --> 00:12:20 And you know what the reaction to that was? Well, 158 00:12:20 --> 00:12:25 mostly it was that they must have goofed because all smart right 159 00:12:25 --> 00:12:30 thinking people knew that DNA was an absolutely boring molecule. 160 00:12:30 --> 00:12:33 Because the interesting molecule at this time was proteins. 161 00:12:33 --> 00:12:36 Everybody knew there were zillions, there were 20 amino acids, they came 162 00:12:36 --> 00:12:39 in zillions of combinations, they had all sorts of different 163 00:12:39 --> 00:12:42 shapes and properties, and hydrophobic ones, hydrophilic 164 00:12:42 --> 00:12:45 ones that are enzymes. And, clearly, anything as important 165 00:12:45 --> 00:12:48 as heredity was not going to be encoded in some utterly boring 166 00:12:48 --> 00:12:51 structural molecule that was just a long polymer of four virtually 167 00:12:51 --> 00:12:54 identical units. And so, the sort of reaction was 168 00:12:54 --> 00:12:57 this is interesting but there must be some trick, 169 00:12:57 --> 00:13:00 something must be wrong in this experiment, give or take. 170 00:13:00 --> 00:13:03 Now, why did people think that DNA was so boring? 171 00:13:03 --> 00:13:07 Now, DNA had been known for a long time, since the 1860s. 172 00:13:07 --> 00:13:10 Lots of molecules were known, but why was DNA boring and why were 173 00:13:10 --> 00:13:14 proteins kind of exciting? So, for that, we really do have to 174 00:13:14 --> 00:13:17 look some more closely at the structure of DNA. 175 00:13:17 --> 00:13:21 I want to review the structure of DNA here because we're going to use 176 00:13:21 --> 00:13:25 it a lot. So, DNA has three components, 177 00:13:25 --> 00:13:32 as you undoubtedly know. It has first a sugar, 178 00:13:32 --> 00:13:43 or almost sugar, two prime deoxyribose, two prime deoxyribose, 179 00:13:43 --> 00:13:54 so it's a pentose, or almost the deoxypentose. And its structure, 180 00:13:54 --> 00:14:03 and this is an important structure. In order to be a true sugar, 181 00:14:03 --> 00:14:10 to be ribose you would have a hydroxyl. Deoxyribose just has a 182 00:14:10 --> 00:14:16 hydrogen there. And the way we number these carbons 183 00:14:16 --> 00:14:23 around this five carbon sugar are very important and we'll always talk 184 00:14:23 --> 00:14:30 about them, the one prime, two prime, three prime, four prime 185 00:14:30 --> 00:14:37 and five prime carbons of this deoxyribose. 186 00:14:37 --> 00:14:42 And you'll notice that it's the two prime carbon that is dioxi. 187 00:14:42 --> 00:14:48 So, that's the sugar. The next important component as we build up 188 00:14:48 --> 00:14:54 DNA is the base, OK? The base is put here. 189 00:14:54 --> 00:15:00 Now, I'm going to start simplifying our sugar. 190 00:15:00 --> 00:15:15 Base. So, there are four kinds of bases that can go here. 191 00:15:15 --> 00:15:30 And they are adenine, guanine, thymine, cytosine. 192 00:15:30 --> 00:15:38 So, that's the second important part of building up DNA. 193 00:15:38 --> 00:15:47 The third important part in building up DNA is to make the 194 00:15:47 --> 00:15:55 monomers that are used to produce DNA, we need to put on 195 00:15:55 --> 00:16:07 a triphosphate. And he we go. We'll take our sugar 196 00:16:07 --> 00:16:22 here, our base over here, and then off this carbon we have our 197 00:16:22 --> 00:16:35 phosphate. And we have a triphosphate. 198 00:16:35 --> 00:16:46 There we go. So, this is the monomer that is used to build up DNA. 199 00:16:46 --> 00:16:58 This guy here is called a nucleoside, note the S. 200 00:16:58 --> 00:17:02 This guy here with the triphosphate on it is called a nucleotide. 201 00:17:02 --> 00:17:07 It's not usually written with such a big capital letter, 202 00:17:07 --> 00:17:12 but nonetheless I point this out. And, obviously, what is this 203 00:17:12 --> 00:17:17 triphosphate going to do for us? It's going to provide the energy to 204 00:17:17 --> 00:17:22 allow us to make DNA polymer chains. We're going to do a dehydration 205 00:17:22 --> 00:17:27 synthesis where we break two of those phosphates off and use it for 206 00:17:27 --> 00:17:32 the energy to be able to catalyze DNA chains to be made. 207 00:17:32 --> 00:17:40 OK. Now, when you combine nucleotides into a DNA strand you do 208 00:17:40 --> 00:17:48 so to create a sugar phosphate backbone. And you'll see for many 209 00:17:48 --> 00:17:56 molecules, I don't care that you know their structures 210 00:17:56 --> 00:18:04 terribly well. But for the basic structure of DNA, 211 00:18:04 --> 00:18:12 including its sugar phosphate backbone, it's going to be important 212 00:18:12 --> 00:18:21 for all that we talk about. So, what happens is we have a chain 213 00:18:21 --> 00:18:30 of DNA growing like this, and we have our OH here. 214 00:18:30 --> 00:18:36 We have our base here. And which carbon is this? 215 00:18:36 --> 00:18:43 Five prime, that's right. OK. Which carbon is this? This one. 216 00:18:43 --> 00:18:49 Great. That's the three prime, two prime, one prime. 217 00:18:49 --> 00:18:56 Great. OK. To this three prime carbon we add this triphosphate 218 00:18:56 --> 00:19:05 breaking off two phosphates there. The diphosphate gets broken off, 219 00:19:05 --> 00:19:17 the pyrophosphate. And we get a single phosphate linkage to the next 220 00:19:17 --> 00:19:29 subunit of the chain. So, here we go phosphate, 221 00:19:29 --> 00:19:37 sugar, phosphate, sugar. And if we ignore these bases, 222 00:19:37 --> 00:19:41 which, you know, who cares about the bases anyway, what we have is just 223 00:19:41 --> 00:19:45 phosphate, sugar, phosphate, sugar, phosphate, 224 00:19:45 --> 00:19:50 sugar, phosphate, sugar. OK? So, it's a very simple structure. 225 00:19:50 --> 00:19:54 There's nothing hard to remember about this. And the phosphate is 226 00:19:54 --> 00:19:58 always attached to the three prime carbon of the preceding sugar and to 227 00:19:58 --> 00:20:03 the five prime carbon of the next sugar. 228 00:20:03 --> 00:20:06 OK? So, we often speak of chains of DNA growing from the five prime end 229 00:20:06 --> 00:20:10 to the three prime end. And that confuses non-molecular 230 00:20:10 --> 00:20:13 biologists to no end. What are we talking about, 231 00:20:13 --> 00:20:17 five prime ends and three prime ends? This is what we're talking about. 232 00:20:17 --> 00:20:20 But the additions are catalyzed onto the three prime carbon of that 233 00:20:20 --> 00:20:24 sugar. It grows at it three prime end. So, you have sugar, 234 00:20:24 --> 00:20:28 phosphate, sugar, phosphate, sugar, phosphate. So, that's it. 235 00:20:28 --> 00:20:33 We're all done. Well, there are the bases, 236 00:20:33 --> 00:20:41 I guess, too, right? So, we'll mention these bases. 237 00:20:41 --> 00:20:48 The bases are, they come in two types. There are purines. 238 00:20:48 --> 00:20:56 Adenine and guanine are purines. And there's a six member rings with 239 00:20:56 --> 00:21:02 a five member ring. And there are two bases that are 240 00:21:02 --> 00:21:07 called pyrimidines, they are smaller, the thymine and 241 00:21:07 --> 00:21:12 the cytosine. And there are six member rings. And they have some 242 00:21:12 --> 00:21:17 carbon, some nitrogen, some oxygen and some hydrogen. 243 00:21:17 --> 00:21:22 But, you've got to admit that compared to proteins, 244 00:21:22 --> 00:21:27 this is pretty boring. It's just one long sugar phosphate 245 00:21:27 --> 00:21:31 chain. And two purines, 246 00:21:31 --> 00:21:34 slightly bigger things, two pyrimidines, slightly smaller 247 00:21:34 --> 00:21:38 things. Very similar structures for these two. I haven't even bothered 248 00:21:38 --> 00:21:41 to focus on the difference. And as compared to the richness of 249 00:21:41 --> 00:21:44 proteins, there's just no way anything interesting could happen 250 00:21:44 --> 00:21:48 with this. That was certainly the thinking at the time. 251 00:21:48 --> 00:21:51 You have to understand how important prior ideas, 252 00:21:51 --> 00:21:54 prior prejudice is to science. People look at it and say this must 253 00:21:54 --> 00:21:58 be some structural molecule. It is scaffolding. 254 00:21:58 --> 00:22:02 It's like the studs in the wall of the house you're building or 255 00:22:02 --> 00:22:07 something like that. Not too interesting. 256 00:22:07 --> 00:22:12 So, what happens? Well, you know, it takes time to sort 257 00:22:12 --> 00:22:17 things out. People come back to this problem. Any thoughts? 258 00:22:17 --> 00:22:22 I mean I've given you one reason why this did not make a huge impact, 259 00:22:22 --> 00:22:27 because it was, you know, DNA was kind of a boring molecule and people 260 00:22:27 --> 00:22:32 weren't really sure this was right. It could be an artifact, 261 00:22:32 --> 00:22:38 right? Maybe some important protein had come along for the ride with the 262 00:22:38 --> 00:22:43 DNA faction, right? What's another reason why people 263 00:22:43 --> 00:22:49 might not have paid tremendous attention to this result? 264 00:22:49 --> 00:22:54 Sorry? It was just bacteria. Anything else? Couldn't imagine, 265 00:22:54 --> 00:23:00 right, how this DNA could encode the enzyme. Anything else? Date. 266 00:23:00 --> 00:23:04 It's in the middle of the Second World War. Maybe people had more 267 00:23:04 --> 00:23:08 important things to do, right? So, this is right in the 268 00:23:08 --> 00:23:12 middle of the Second World War, too. It's just worth noting that 269 00:23:12 --> 00:23:16 these guys are working in the middle of New York City at the Rockefeller 270 00:23:16 --> 00:23:20 Institute and it's in the middle of the Second World War. 271 00:23:20 --> 00:23:24 Anyway, war is over and some work continues on this. 272 00:23:24 --> 00:23:28 And the work takes a somewhat different attack. 273 00:23:28 --> 00:23:32 Instead of working bacteria it now is, there's work here on certain 274 00:23:32 --> 00:23:36 bacterial viruses. So, instead, see, 275 00:23:36 --> 00:23:42 bacteria get their own here. Instead of using bacteria to infect 276 00:23:42 --> 00:23:47 mice, Hershey and Chase, and others at the time, used viruses 277 00:23:47 --> 00:23:53 to infect bacteria. So, here the bacterium is the 278 00:23:53 --> 00:23:58 victim. And people had found and had studied these amazingly 279 00:23:58 --> 00:24:04 interesting really tiny things that could affect a bacterium 280 00:24:04 --> 00:24:09 and kill it. These particles that had these funny 281 00:24:09 --> 00:24:13 shapes were called bacteriophage. What does phage mean? To eat. 282 00:24:13 --> 00:24:18 Bacteria eaters. Bacteriophages were these little 283 00:24:18 --> 00:24:22 viruses. They were incredibly tiny. You could filter them through very 284 00:24:22 --> 00:24:27 small filters. And, yet, when you added them to 285 00:24:27 --> 00:24:32 bacteria they would kill the bacteria. 286 00:24:32 --> 00:24:35 These were very simple things. I'm reluctant to call them 287 00:24:35 --> 00:24:38 creatures. Are they alive? This is a favorite question people 288 00:24:38 --> 00:24:42 would like to debate. They say are viruses alive? 289 00:24:42 --> 00:24:45 And the answer is who cares? I mean it depends on what you want 290 00:24:45 --> 00:24:49 to define alive to mean. To me it's not alive in that it 291 00:24:49 --> 00:24:52 cannot replicate on its own without a host, so I won't call it alive. 292 00:24:52 --> 00:24:56 But, anyway, I'll refer to them loosely as these creatures that eat 293 00:24:56 --> 00:24:59 bacteria. They were very simple. And all they really had in them was 294 00:24:59 --> 00:25:03 some DNA in their capsid, this capsid up here, and some 295 00:25:03 --> 00:25:08 protein. But they could attach to a bacterium 296 00:25:08 --> 00:25:15 and after a certain amount of time cause the bacteria to burst open and 297 00:25:15 --> 00:25:22 produce lots of daughter-phage, lots of daughter bacteriophage. It 298 00:25:22 --> 00:25:30 could replicate within this bacteria. 299 00:25:30 --> 00:25:33 So, somehow this, while I might not want to call it 300 00:25:33 --> 00:25:37 alive, certainly can reproduce itself, or at least with the help of 301 00:25:37 --> 00:25:41 a bacterium can reproduce itself. When people first discovered this 302 00:25:41 --> 00:25:45 bacteriophage, what do you think they wanted to do 303 00:25:45 --> 00:25:48 with them? Sorry? Yeah. Where? In humans. 304 00:25:48 --> 00:25:52 The first thought about what to do with bacteriophage were a whole 305 00:25:52 --> 00:25:56 bunch of interesting Russians who wanted to make up large quantities 306 00:25:56 --> 00:26:00 of bacteriophage and have people drink them. 307 00:26:00 --> 00:26:02 So, they would kill all their bacteria. And this was the early 308 00:26:02 --> 00:26:05 ideas for antibiotics. It didn't quite pan out that way. 309 00:26:05 --> 00:26:08 But, you know, people have all these very exciting ideas of, 310 00:26:08 --> 00:26:11 wow, I've got something to kill bacteria, let's pour it down a 311 00:26:11 --> 00:26:14 patient and see if that does something good for them. 312 00:26:14 --> 00:26:17 You know, that's why there are institutional review boards, 313 00:26:17 --> 00:26:20 too, to make sure that you can't just do that right off the bat. 314 00:26:20 --> 00:26:23 Somebody else has got to think about it also. 315 00:26:23 --> 00:26:26 It turns out not to be a great way to kill patients, to kill 316 00:26:26 --> 00:26:30 bacteria, sorry. No, it doesn't actually kill 317 00:26:30 --> 00:26:34 patients, but it doesn't also kill the bacteria so well in human beings. 318 00:26:34 --> 00:26:38 So, anyway. So, the question was how is it that 319 00:26:38 --> 00:26:42 these viruses kill the bacteria? Somehow they inject something into 320 00:26:42 --> 00:26:47 the bacteria, something causes something to happen, 321 00:26:47 --> 00:26:51 which causes virus particles to be made. I don't put too fine a point 322 00:26:51 --> 00:26:55 on it because that's all you could really say at that point, 323 00:26:55 --> 00:27:00 something goes in and something comes out. 324 00:27:00 --> 00:27:05 So, what goes in? How could we tell what goes in? 325 00:27:05 --> 00:27:10 Yup. By seeing what's left out. How could we see what's left out? 326 00:27:10 --> 00:27:15 Just being really practical, how are we going to tell? 327 00:27:15 --> 00:27:20 Visually look, but that turns out to be terribly hard thing to do. 328 00:27:20 --> 00:27:25 You've got to have really good eyes to be able to say the protein is 329 00:27:25 --> 00:27:30 still there but not the DNA, or the DNA. 330 00:27:30 --> 00:27:33 Because the thought was if this thing is injecting its DNA then the 331 00:27:33 --> 00:27:37 DNA must be carrying the instructions to make phage, 332 00:27:37 --> 00:27:40 and this would be hereditary material. So, 333 00:27:40 --> 00:27:44 what we want to show is that the protein stays out and the DNA goes 334 00:27:44 --> 00:27:47 in. But how is that going to, how do you do that practically? 335 00:27:47 --> 00:27:51 Radioactive labeling turns out to be the best way to do that. 336 00:27:51 --> 00:27:54 If we could label radioactively the DNA with one label and the protein 337 00:27:54 --> 00:27:58 with a different label, we could see which radioactive 338 00:27:58 --> 00:28:02 isotope goes into the bacteria. Any candidates for an element that 339 00:28:02 --> 00:28:06 we could use to label DNA that won't be in protein? 340 00:28:06 --> 00:28:10 Sorry? I'm sorry? Oh, who had one? Uranium. 341 00:28:10 --> 00:28:14 Somebody's thinking World War II here, right, there would be some 342 00:28:14 --> 00:28:19 spare uranium around. The problem with that is that DNA 343 00:28:19 --> 00:28:23 does not actually have uranium in it, and so when you put uranium in it 344 00:28:23 --> 00:28:27 wouldn't still be DNA. We would like to label it with an 345 00:28:27 --> 00:28:31 element that's actually in DNA. So the only difference is that it's 346 00:28:31 --> 00:28:35 a radioisotope. Phosphorus. Phosphorus. 347 00:28:35 --> 00:28:39 Well, there's obviously phosphorus in that sugar phosphate backbone. 348 00:28:39 --> 00:28:43 Is there phosphorus in a typical amino acid? Any of the 20 amino 349 00:28:43 --> 00:28:46 acids? No phosphorus. Great. So, we could use a 350 00:28:46 --> 00:28:50 phosphorus isotope. We could P32 label the DNA. 351 00:28:50 --> 00:28:54 But how do make live bacteriophages that are labeled with radioactive 352 00:28:54 --> 00:28:58 phosphorus? I mean what kind of fancy chemistry do you 353 00:28:58 --> 00:29:02 need to do that? Yes? Perfect if you grow the 354 00:29:02 --> 00:29:06 bacteria in radioactively, in medium. If you grow the virus 355 00:29:06 --> 00:29:10 and the bacteria in medium that has radioactive phosphate, 356 00:29:10 --> 00:29:14 the bacteria and the virus take care of it for you. 357 00:29:14 --> 00:29:18 The phosphate is automatically incorporated. So, 358 00:29:18 --> 00:29:22 you don't have to do any chemistry. You just feed phosphate, 359 00:29:22 --> 00:29:26 radioactive phosphate into the medium. And the phage that are 360 00:29:26 --> 00:29:30 produced will be radioactively labeled. 361 00:29:30 --> 00:29:34 Purify them and use them in your experiment. Similarly, 362 00:29:34 --> 00:29:39 what are we going to label our proteins with? 363 00:29:39 --> 00:29:43 Carbon? No. Hydrogen? No. Oxygen? Nitrogen? No, 364 00:29:43 --> 00:29:48 because the bases have nitrogen. Sulfa. We've only got sulfa. 365 00:29:48 --> 00:29:53 Where is sulfa going to be? So, for example, cysteines with 366 00:29:53 --> 00:29:58 thiamines, right, we've got sulfa. Here's S35. 367 00:29:58 --> 00:30:03 So, we can take bacteria and we could, we can take phage, 368 00:30:03 --> 00:30:08 and by growing them in the presence of radioactive DNA, 369 00:30:08 --> 00:30:13 no, radioactive phosphorus, P32 and growing them in the presence 370 00:30:13 --> 00:30:19 of radioactive sulfa, S35, we are about to produce 371 00:30:19 --> 00:30:24 bacteriophage that are labeled. OK? So, P32, S35. Now we infect 372 00:30:24 --> 00:30:30 bacteria with them. Let me take a big tube here. 373 00:30:30 --> 00:30:33 I'm going to add bacteria. I've got the phage here. The phage 374 00:30:33 --> 00:30:37 particles are attached to the bacteria and they are going to 375 00:30:37 --> 00:30:41 inject whatever they inject. Now what do we have to do? We've 376 00:30:41 --> 00:30:45 got to knock off the bacteriophage particles from the bacteria. 377 00:30:45 --> 00:30:48 I want to knock them off and see what is staying with the viral 378 00:30:48 --> 00:30:52 particles and what goes into the bacteria. So, 379 00:30:52 --> 00:30:56 how do I get in there with tweezers and separate off, 380 00:30:56 --> 00:31:00 peel off each virus from the bacteria? 381 00:31:00 --> 00:31:04 Washing turns out not to be strong enough. You've got to previal to 382 00:31:04 --> 00:31:09 get these things off, so you really need some incredibly 383 00:31:09 --> 00:31:13 strong agitation. So, specialized devices were used 384 00:31:13 --> 00:31:18 to create intense agitation. What specialized devices you're 385 00:31:18 --> 00:31:23 aware of that do that? Blenders. Kitchen blenders. 386 00:31:23 --> 00:31:27 The Waring blender turns out to be the perfect laboratory device for 387 00:31:27 --> 00:31:32 this experiment. And this is actually known as the 388 00:31:32 --> 00:31:36 Waring Blender Experiment. You take the bacteria with the 389 00:31:36 --> 00:31:40 phage attached to it, you let them attach and do whatever 390 00:31:40 --> 00:31:44 they're going to do, inject their DNA, as we know turns 391 00:31:44 --> 00:31:48 out is the right answer. And then you press puree and then 392 00:31:48 --> 00:31:52 vrrrr, and the viral particles fall off. So, it's important to know how 393 00:31:52 --> 00:31:56 things really happen. So then what happens is the 394 00:31:56 --> 00:32:00 bacteria are separated from these particles. 395 00:32:00 --> 00:32:04 And it turns out these particles are, the viral particles are much lighter, 396 00:32:04 --> 00:32:08 much less dense than the bacteria. So, how do we separate them? 397 00:32:08 --> 00:32:13 Centrifuge them. We centrifuge them. 398 00:32:13 --> 00:32:17 The bacterial particles are there up in the supernatant turn out to be 399 00:32:17 --> 00:32:21 our phage capsids. And now what do we do? 400 00:32:21 --> 00:32:26 We take this stuff, we measure the radioactivity in the supernatant, 401 00:32:26 --> 00:32:30 that is the material that stays above, and we measure the 402 00:32:30 --> 00:32:36 radioactivity in the pellet. And what do we end up seeing? 403 00:32:36 --> 00:32:43 Where does most of the P32, what shows up in the pellet? 404 00:32:43 --> 00:32:49 Mostly P32 shows up in the pellet. Is there no S35 in the pellet? You 405 00:32:49 --> 00:32:56 know, in the textbook story, of course, there's no S35 because 406 00:32:56 --> 00:33:02 they want it to be nice and clean. But in reality there's going to be 407 00:33:02 --> 00:33:06 some S35. But it was, you know, less than 1% of the S35 408 00:33:06 --> 00:33:11 ends up in the pellet. Most of the S35 stays up here in 409 00:33:11 --> 00:33:16 the supernatant. Does all of the phosphorus go in? 410 00:33:16 --> 00:33:20 No, of course not. Some of the viruses didn't even attach and not 411 00:33:20 --> 00:33:25 everything goes in. So, there's still radioactive 412 00:33:25 --> 00:33:31 phosphate up in the supernatant. But the striking this is that the 413 00:33:31 --> 00:33:38 pellet primarily has gotten the radioactive phosphorus, 414 00:33:38 --> 00:33:45 not the radioactive sulfa, and therefore we can conclude that 415 00:33:45 --> 00:33:52 what? Well, more DNA went in than protein. Are we therefore entitled 416 00:33:52 --> 00:34:00 to include that DNA is the hereditary material? Why? 417 00:34:00 --> 00:34:03 Well, I mean suppose that 1% sulfate is tracking one minor protein that 418 00:34:03 --> 00:34:07 is the secret. You can't, it's very hard to rule 419 00:34:07 --> 00:34:11 out that there's no contaminants traveling along with the DNA. 420 00:34:11 --> 00:34:15 And if you really truly disbelieve DNA, you could be churlish and say, 421 00:34:15 --> 00:34:18 well, I just don't believe that you've so purified it that you can 422 00:34:18 --> 00:34:22 completely rule out that some minor protein component is really 423 00:34:22 --> 00:34:26 conferring heredity. In fact, when you really look 424 00:34:26 --> 00:34:30 closely, Avery, McCarty and McLeod's biochemistry, 425 00:34:30 --> 00:34:34 I believe, was purer than the purity of this experiment. 426 00:34:34 --> 00:34:38 But by this point thinking had begun to shift toward DNA being a 427 00:34:38 --> 00:34:42 reasonable hereditary molecule. In addition, it was the second line 428 00:34:42 --> 00:34:46 of proof, different from the pneumococcus, using a different 429 00:34:46 --> 00:34:50 system, both pointing to the same answer. And the intellectual tide 430 00:34:50 --> 00:34:54 shifted to recognizing that this probably was right, 431 00:34:54 --> 00:34:58 and the reason these experiments were pointing to DNA was DNA had to 432 00:34:58 --> 00:35:02 be the right answer. But, of course, 433 00:35:02 --> 00:35:08 how was it the right answer? What was it about DNA that could 434 00:35:08 --> 00:35:13 confer these properties? This was still unclear in 1953, 435 00:35:13 --> 00:35:19 but not for that long. It became clarified relatively soon thereafter. 436 00:35:19 --> 00:35:24 And, of course, it became clarified with the 437 00:35:24 --> 00:35:30 understanding of DNA structure, the double helix. 438 00:35:30 --> 00:35:34 Nobody here has not heard of the double helix. Probably there's 439 00:35:34 --> 00:35:38 nobody, no grownup who doesn't know about the double helix and all that, 440 00:35:38 --> 00:35:42 but nonetheless I want to stop and think a little bit also, 441 00:35:42 --> 00:35:47 I'll say on a personal note, this is the first year I've taught 442 00:35:47 --> 00:35:51 this class after, the first time I've taught this 443 00:35:51 --> 00:35:55 class when Crick and Watson have not both been alive. 444 00:35:55 --> 00:36:00 Some of you may know that Frances Crick dies just this past summer. 445 00:36:00 --> 00:36:03 Which was very sad. He was an incredible person and, 446 00:36:03 --> 00:36:06 you know, as I've said, Mendel was one of my heroes. 447 00:36:06 --> 00:36:09 Francis Crick was also one of my heroes. He was just an 448 00:36:09 --> 00:36:12 extraordinary person. But Jim Watson is still alive and 449 00:36:12 --> 00:36:15 kicking and still quite active. And so, in any case, you're not far 450 00:36:15 --> 00:36:18 removed. So, I tell you a little bit about this stuff as history, 451 00:36:18 --> 00:36:21 but this history I'm telling you about, these people are, 452 00:36:21 --> 00:36:24 for the most part, Francis' passing notwithstanding, 453 00:36:24 --> 00:36:27 alive and kicking. Jim Watson is still quite alive. 454 00:36:27 --> 00:36:30 Actually, McCarty is still alive. It's really, anyway. 455 00:36:30 --> 00:36:36 So, 1953, just a year later, Jim Watson and Francis Crick are 456 00:36:36 --> 00:36:42 working in England. Watson is a student for Indiana, 457 00:36:42 --> 00:36:48 a former ornithologist, had his interest in ornithology originally, 458 00:36:48 --> 00:36:54 and then studies more biology and came to England because he wanted to 459 00:36:54 --> 00:37:00 study the gene. Francis Crick, 460 00:37:00 --> 00:37:05 a physicist who worked in the admiralty during World War II. 461 00:37:05 --> 00:37:11 And, of course, what they did was on the basis of an awful lot of 462 00:37:11 --> 00:37:16 modeling and getting to see experimental x-ray diffraction 463 00:37:16 --> 00:37:21 pictures of Roselyn Franklin from London who made a model. 464 00:37:21 --> 00:37:26 And the model is this beautiful, and I haven't drawn it to its proper 465 00:37:26 --> 00:37:32 proportions, but this beautiful double helical structure. 466 00:37:32 --> 00:37:37 Five prime, one chain of DNA running in one direction, 467 00:37:37 --> 00:37:42 five prime to three prime. An anti-parallel chain of DNA going 468 00:37:42 --> 00:37:47 in this direction, five prime to three prime. 469 00:37:47 --> 00:37:52 It was a beautiful structure. Jim Watson has written a whole book 470 00:37:52 --> 00:37:57 about the discovery of the double helix structure, 471 00:37:57 --> 00:38:02 and we are only 51 years past that. It was, anybody who hasn't read The 472 00:38:02 --> 00:38:06 Double Helix, this book, really should. It's one of the 473 00:38:06 --> 00:38:10 treat books of science literature, and actually is on many people's 474 00:38:10 --> 00:38:15 lists of some of the great books of the 20th century. 475 00:38:15 --> 00:38:19 It's a wonderful competitive story of Crick and Watson racing against 476 00:38:19 --> 00:38:23 Linus Pauling. It's, you know, 477 00:38:23 --> 00:38:28 someone came along and had lunch in Cambridge with Crick and Watson. 478 00:38:28 --> 00:38:31 And they came away and said, this was before they discovered the 479 00:38:31 --> 00:38:34 structure, about a year or so before, and said these guys are idiots. 480 00:38:34 --> 00:38:38 They can't even memorize the structure of A and T and C and G, 481 00:38:38 --> 00:38:41 and they're trying to find, you know, the structure of DNA. 482 00:38:41 --> 00:38:44 These guys are never going to get anywhere. So, 483 00:38:44 --> 00:38:48 this person, who we'll come back to in a moment, was wrong about this 484 00:38:48 --> 00:38:51 particular point. Because what Crick and Watson did 485 00:38:51 --> 00:38:54 was they played around with the models, and what they ended up 486 00:38:54 --> 00:38:58 noticing was a couple of things. First off, from Rosalyn Franklin's 487 00:38:58 --> 00:39:01 pictures, that this was helical. The x-ray to fraction pictures could 488 00:39:01 --> 00:39:05 tell you at a glance that the structure was helical. 489 00:39:05 --> 00:39:09 They saw that. They then tried to make helices. Now, 490 00:39:09 --> 00:39:12 other people, Linus Pauling knew something that DNA probably had to 491 00:39:12 --> 00:39:16 be helical, and somehow he just got it totally wrong. 492 00:39:16 --> 00:39:19 He made just a nutty model of DNA. Linus Pauling, the smartest chemist 493 00:39:19 --> 00:39:23 of the century made a crazy model of DNA where he took the sugar 494 00:39:23 --> 00:39:27 phosphate backbones and put all the sugar phosphate backbones in the 495 00:39:27 --> 00:39:30 middle, and had three of them. He had a triple helical model with 496 00:39:30 --> 00:39:34 sugar phosphates in the middle. And what can you tell me about the 497 00:39:34 --> 00:39:38 charge on these sugar phosphate backbones? Very negative. 498 00:39:38 --> 00:39:42 You're going to stick a whole bunch of negative charges near each other 499 00:39:42 --> 00:39:46 in the middle? No way. Anybody could have known. 500 00:39:46 --> 00:39:50 This was a bush-league mistake, so Crick and Watson said phew, 501 00:39:50 --> 00:39:54 Pauling has got it wrong. They put together this model. 502 00:39:54 --> 00:39:58 And the key to the model was the recognition of base pairing, 503 00:39:58 --> 00:40:15 the recognition of base paring. That if I take a thiamine here and I 504 00:40:15 --> 00:40:36 take an adenine here. That these two groups would be 505 00:40:36 --> 00:40:48 pointing at each other in such a way as to make two hydrogen bonds with a 506 00:40:48 --> 00:41:01 certain characteristic distance. And, not just that, but cytosine 507 00:41:01 --> 00:41:14 and guanine could also be fit into that same distance. 508 00:41:14 --> 00:41:30 And they would have three hydrogen bonds. 509 00:41:30 --> 00:41:40 And here NH, H, doo, doo, doo, doo, 510 00:41:40 --> 00:41:50 doo, doo, doo, three hydrogen bonds. And they would fit the, what do I 511 00:41:50 --> 00:42:00 got? Oops. Thank you. Good point. 512 00:42:00 --> 00:42:06 That's the problem. Yup. Well, it's a little messy but 513 00:42:06 --> 00:42:13 anyway. The business end here is three hydrogen bonds and two 514 00:42:13 --> 00:42:19 hydrogen bonds, and they both fit into the same 515 00:42:19 --> 00:42:26 distance perfectly. So, this double helix here could 516 00:42:26 --> 00:42:33 have either As and Ts or Gs and Cs or Cs and Gs or Ts and As. 517 00:42:33 --> 00:42:37 And they would all fit perfectly with each other. 518 00:42:37 --> 00:42:42 Now, there was an old observation, not that old, there was an 519 00:42:42 --> 00:42:46 observation floating around at the time that said when you analyze the 520 00:42:46 --> 00:42:51 amount of As and Ts in DNA, you always found out that the amount 521 00:42:51 --> 00:42:55 of A tended to be very close to the amount of T. The amount of C tended 522 00:42:55 --> 00:43:00 to be very close to the amount of G. 523 00:43:00 --> 00:43:06 Although, these amounts could be different. This was due to a 524 00:43:06 --> 00:43:12 biochemist called Chargaff. And they're called, this was called 525 00:43:12 --> 00:43:18 Chargaff's Rule or Chargaff's Law or Chargaff's Observation. 526 00:43:18 --> 00:43:24 Chargaff noted that the percentage of these amounts tended to be equal 527 00:43:24 --> 00:43:30 but didn't know what to make of it. This perfectly explained that. 528 00:43:30 --> 00:43:33 It was very good. Remember I said somebody came 529 00:43:33 --> 00:43:36 through Cambridge and said these guys were turkeys, 530 00:43:36 --> 00:43:39 Crick and Watson were turkeys because they couldn't even remember 531 00:43:39 --> 00:43:43 the structure and all that? This was a very distinguished 532 00:43:43 --> 00:43:46 chemist who said this about Crick and Watson. It was Chargaff. 533 00:43:46 --> 00:43:49 Chargaff came through and said these guys are turkeys. 534 00:43:49 --> 00:43:53 But it was Chargaff's Rule that Chargaff had missed the importance 535 00:43:53 --> 00:43:56 of. He was quite bitter about this through much of his life. 536 00:43:56 --> 00:43:59 And there's a very wonderful biting quote that Chargaff says when Crick 537 00:43:59 --> 00:44:03 and Watson become famous for the DNA double helix. 538 00:44:03 --> 00:44:09 Let's see if I can get it right. He says that such pigmies should 539 00:44:09 --> 00:44:15 cast such giant shadows, referring to Crick and Watson, 540 00:44:15 --> 00:44:21 that such pigmies should cast such giant shadows only shows how late in 541 00:44:21 --> 00:44:27 the day it is. Anyway, he was not happy. 542 00:44:27 --> 00:44:31 So, all right. Now, this was a big deal thing. 543 00:44:31 --> 00:44:34 Crick and Watson knew this was very important. They raced to publish a 544 00:44:34 --> 00:44:37 paper about this. They sent it off to Nature. 545 00:44:37 --> 00:44:41 It's a gem of a paper. It's a page roughly in text. 546 00:44:41 --> 00:44:44 It's very short, very clear, has this beautiful 547 00:44:44 --> 00:44:47 picture drawn from Francis Crick's wife, Odile. And it is just a 548 00:44:47 --> 00:44:50 charming paper. They know that they've cracked the 549 00:44:50 --> 00:44:53 secret of life. Why do they know they've cracked 550 00:44:53 --> 00:44:56 the secret of life? Because the most important thing 551 00:44:56 --> 00:45:00 about this model here is not its structure per se. 552 00:45:00 --> 00:45:04 But that it explains how it is that a DNA molecule can be replicated, 553 00:45:04 --> 00:45:09 that somehow all it takes is for those two strands to come apart, 554 00:45:09 --> 00:45:13 who knows how, and that when they come apart each can serve as a 555 00:45:13 --> 00:45:18 template for the other because since As always match Ts and Cs always 556 00:45:18 --> 00:45:22 match Gs, each strand has enough information for the other. 557 00:45:22 --> 00:45:27 That's how replication happens. You have two strands, each of which 558 00:45:27 --> 00:45:32 has sufficient information to encode the other. 559 00:45:32 --> 00:45:35 They somehow come apart. They each serve as a template for 560 00:45:35 --> 00:45:38 the other. And that's that. That is the secret of life, how 561 00:45:38 --> 00:45:41 life replicates itself. Not just that. We've explained 562 00:45:41 --> 00:45:45 replication. What about mutation. What's mutation? It sometimes gets 563 00:45:45 --> 00:45:48 it wrong. It sometimes screws up. So, for one little biochemical 564 00:45:48 --> 00:45:51 model we've explained replication and mutation. That's pretty good. 565 00:45:51 --> 00:45:54 Now, the thing is, in writing this paper and getting this off to the 566 00:45:54 --> 00:45:58 Journal, this was not an easy thing to get done quickly. 567 00:45:58 --> 00:46:01 You couldn't, you just didn't have time to explain all these details. 568 00:46:01 --> 00:46:05 They wanted to stake their claim about this, so they wrote up the 569 00:46:05 --> 00:46:09 structure. And instead of going through a long about how this 570 00:46:09 --> 00:46:13 explains replication and dah, dah, dah, dah, dah, dah, dah, dah, 571 00:46:13 --> 00:46:16 there's one sentence in the paper, the last sentence of the paper in 572 00:46:16 --> 00:46:20 which they just say it has not escaped our notice that this model 573 00:46:20 --> 00:46:24 explains replication and everything else. Basically the last sentence 574 00:46:24 --> 00:46:28 says, oh, and by the way, it has not escaped our notice that 575 00:46:28 --> 00:46:31 this explains the secret of life. Although, it didn't say it like that. 576 00:46:31 --> 00:46:35 It's the coyest sentence in the scientific literature. 577 00:46:35 --> 00:46:38 It's really just an amazing sentence there. 578 00:46:38 --> 00:46:42 And then they come back a couple minutes later and write a paper 579 00:46:42 --> 00:46:45 explaining what they mean and all that, but it's just a great sentence. 580 00:46:45 --> 00:46:49 So, you will hear molecular biologists make reference, 581 00:46:49 --> 00:46:52 use in their speech the phrase, it has not escaped our notice that. 582 00:46:52 --> 00:46:56 And it's always a homage back to this particular sentence in this 583 00:46:56 --> 00:46:59 paper of Crick and Watson. OK, now, last thing. Yes? 584 00:46:59 --> 00:47:03 Jim Watson was 25 and Francis was 35 when he did this. 585 00:47:03 --> 00:47:09 Yes. He was a 25-year-old kid when he did this. That's right. 586 00:47:09 --> 00:47:16 Pretty amazing stuff. So, last point I want to touch on, 587 00:47:16 --> 00:47:23 and I'm not sure I'll get to, get all the way there, but this 588 00:47:23 --> 00:47:29 model, this model here of DNA coming apart and each strand serving as a 589 00:47:29 --> 00:47:36 template for the other strand is called Semi-Conservative 590 00:47:36 --> 00:47:43 Replication. That is one strand is used as the 591 00:47:43 --> 00:47:50 template for the other strand, so there's an old strand and a new 592 00:47:50 --> 00:47:57 strand that's made. In theory, you could imagine that 593 00:47:57 --> 00:48:05 DNA replication occurred not like this. 594 00:48:05 --> 00:48:09 But instead somehow, I can't imagine how, but you could 595 00:48:09 --> 00:48:14 imagine, and people were willing to imagine it, that the old strands 596 00:48:14 --> 00:48:18 stayed together but somehow became a template for making a new double 597 00:48:18 --> 00:48:23 helix without actually using them. This model of the strands actually 598 00:48:23 --> 00:48:27 serving as a template would predict that each new DNA double helix was, 599 00:48:27 --> 00:48:32 in fact, composed of one old and one new strand. 600 00:48:32 --> 00:48:36 If you could prove that then you'd have real confirmation of this Crick 601 00:48:36 --> 00:48:40 Watson model, semi-conservative replication. And so, 602 00:48:40 --> 00:48:44 a young student, two young students, Matt Meselson, who's still working 603 00:48:44 --> 00:48:48 and is down the road at Harvard and a wonderful person, 604 00:48:48 --> 00:48:52 and Frank Stahl, who's still working in Oregon, proved that the new DNAs 605 00:48:52 --> 00:48:56 that were made after each generation were, in fact, 606 00:48:56 --> 00:49:00 composed of one old strand and one new strand. 607 00:49:00 --> 00:49:06 How could you possibly do that? Sorry? Radioactive labeling. But 608 00:49:06 --> 00:49:12 how do you radioactively label it so that you can see that it's, 609 00:49:12 --> 00:49:18 that you've got a double helix that's half one and half the other? 610 00:49:18 --> 00:49:24 Old ones labeled with, well, with one isotope. It actually turns out 611 00:49:24 --> 00:49:30 to be nitrogen. The new one with a new isotope. 612 00:49:30 --> 00:49:34 Say N14. You can do heavy nitrogen and ordinary nitrogen. 613 00:49:34 --> 00:49:38 And if what you could do is grow up your DNA, when you first grow it in 614 00:49:38 --> 00:49:42 normal nitrogen, then you shift to N15, 615 00:49:42 --> 00:49:46 heavy nitrogen, you could make DNA molecules that were half old, 616 00:49:46 --> 00:49:50 half new, and therefore half labeled with normal nitrogen, 617 00:49:50 --> 00:49:54 half labeled with heavy nitrogen. And how would I prove that these 618 00:49:54 --> 00:49:58 DNA molecules were a 50/50 hybrid? What would be the property that I 619 00:49:58 --> 00:50:02 would be able to test? Well, radioactivity turns out to be 620 00:50:02 --> 00:50:06 really hard to weight density. It turns out density, if I could 621 00:50:06 --> 00:50:10 just measure the density of the DNA, I would show that if the 622 00:50:10 --> 00:50:14 semi-conservative model is true, the molecules will now have 623 00:50:14 --> 00:50:19 intermediate density between all heavy and all light nitrogen. 624 00:50:19 --> 00:50:23 They had to work out a centrifugation technique so 625 00:50:23 --> 00:50:27 sensitive, a salt gradient centrifugation where you could put 626 00:50:27 --> 00:50:32 DNA on it. You had a really fine salt gradient spun in a centrifuge. 627 00:50:32 --> 00:50:35 And depending on where the DNA migrated, you could measure the 628 00:50:35 --> 00:50:38 density of the DNA. And they were able to show that, 629 00:50:38 --> 00:50:41 in fact, newly replicated DNA strands had this intermediate 630 00:50:41 --> 00:50:45 density that would be expected from the semi-conservative model. 631 00:50:45 --> 00:50:48 And so, in fact, by that point the semi-conservative model, 632 00:50:48 --> 00:50:51 I think, is well established. In some sense you would say the 633 00:50:51 --> 00:50:55 beauty of the double helix was almost one of these very rare 634 00:50:55 --> 00:50:58 scientific results where when you look at it you say it can't 635 00:50:58 --> 00:51:02 possibly be wrong. It explains too much. 636 00:51:02 --> 00:51:06 It's too beautiful. But, as we've discussed before, that's 637 00:51:06 --> 00:51:11 not enough. You need some proofs that it's real. 638 00:51:11 --> 00:51:15 And this Meselson-Stahl experiment provided a real confirmation of that. 639 00:51:15 --> 51:20 Onto next time.