1 00:00:00 --> 00:00:04 So today we're going to continue our focus on DNA which I'm personally 2 00:00:04 --> 00:00:09 enthusiastic about at least in terms of being such a fascinating molecule. 3 00:00:09 --> 00:00:14 And I told you the story last time of how we actually came to 4 00:00:14 --> 00:00:19 understand that DNA was the genetic material. And I still see comments 5 00:00:19 --> 00:00:24 that, oh, God, all this stuff is not relevant to 6 00:00:24 --> 00:00:29 the exam. We're trying to construct the exams in ways that test whether 7 00:00:29 --> 00:00:34 you got the concepts and not just whether you memorized every term 8 00:00:34 --> 00:00:39 that you ran into in the textbook. So I'm hoping that you will see some 9 00:00:39 --> 00:00:43 greater purpose in why I'm trying to talk about some of this. 10 00:00:43 --> 00:00:47 And also I'm sure some of you will forget the details of transformation, 11 00:00:47 --> 00:00:51 of DNA replication we're going to go into as we sort of burrow into it 12 00:00:51 --> 00:00:55 over the next lecture or so, but what I am hoping you may 13 00:00:55 --> 00:00:59 remember ten years from now, even those who don't go in biology, 14 00:00:59 --> 00:01:04 is how experiments are done, how real people do them. 15 00:01:04 --> 00:01:07 And that was partly what I was trying to tell you. 16 00:01:07 --> 00:01:11 And you guys are pretty good at figuring out the basic principle 17 00:01:11 --> 00:01:14 that someone had to somehow show that a DNA molecule in one organism 18 00:01:14 --> 00:01:18 could change some organism to have a new characteristic. 19 00:01:18 --> 00:01:21 And as I sort of told you with the work from Frederick Griffith. 20 00:01:21 --> 00:01:25 And then his initial stuff wasn't devoted to that at all. 21 00:01:25 --> 00:01:28 It was trying to solve a very pressing problem which is dealing 22 00:01:28 --> 00:01:32 with pneumonia in a pre-antibiotic era. 23 00:01:32 --> 00:01:36 And then the finding that he got, that this odd result that something 24 00:01:36 --> 00:01:41 in a heat-killed extract could be transferred to a live bacterium sort 25 00:01:41 --> 00:01:45 of set things up for Avery and his colleagues after a number of years 26 00:01:45 --> 00:01:50 of work to make a very powerful argument that DNA was the genetic 27 00:01:50 --> 00:01:54 material. But, as I said at the end of last lecture, 28 00:01:54 --> 00:01:59 that paper was published in the 1940s. 29 00:01:59 --> 00:02:04 And people didn't immediately say oh, wow, DNA is the genetic material. 30 00:02:04 --> 00:02:09 Often, and we'll see it again with genetics, there's sometimes sort of 31 00:02:09 --> 00:02:15 the body of science the average person thinks about. 32 00:02:15 --> 00:02:20 Science needs to reach to a certain state before an idea can take hold, 33 00:02:20 --> 00:02:26 even if there's evidence supporting it. Part of the problem was that 34 00:02:26 --> 00:02:31 chemists had isolated DNA. And the way they used to isolate DNA 35 00:02:31 --> 00:02:35 was really rough on it. Crack the cells open. And what 36 00:02:35 --> 00:02:39 happened, it would all get broken down into little pieces of DNA. 37 00:02:39 --> 00:02:43 And people had worked out the basic chemical structure that it was the 38 00:02:43 --> 00:02:47 deoxyribose and how the things were joined together, 39 00:02:47 --> 00:02:51 but nobody had ever seen anything more than just these little pieces 40 00:02:51 --> 00:02:55 of DNA. And there was a widely held conception that it was just an 41 00:02:55 --> 00:03:00 anonymous tetranucleotide of G, A, T and C. 42 00:03:00 --> 00:03:04 It wasn't clear why the cells made it, but it didn't look like anything 43 00:03:04 --> 00:03:08 that could encode information. Whereas, as I said, something like 44 00:03:08 --> 00:03:12 proteins, those seem to be very different. And so the world wasn't 45 00:03:12 --> 00:03:16 quite ready for it. Another thing, and this came from 46 00:03:16 --> 00:03:20 one of the comments here, was someone said they didn't know 47 00:03:20 --> 00:03:24 bacteria could take up DNA from the environment. And, 48 00:03:24 --> 00:03:28 in fact, most bacteria can. It happens that streptacoccucci and 49 00:03:28 --> 00:03:32 some other bacteria at certain phases in their lifetime develop 50 00:03:32 --> 00:03:36 this capacity to take up DNA from the outside. 51 00:03:36 --> 00:03:39 Given what I've told you about a membrane and how hard it is to get 52 00:03:39 --> 00:03:43 things across it, you could imagine it's not trivial 53 00:03:43 --> 00:03:46 to get a DNA molecule which is huge from one side to the other. 54 00:03:46 --> 00:03:50 So it doesn't normally happen. And what happens if you go into a 55 00:03:50 --> 00:03:53 lab and you're cloning something or other, and we'll talk about how to 56 00:03:53 --> 00:03:57 take a couple pieces of DNA and join them together in a test tube and 57 00:03:57 --> 00:04:01 then put them back into a bacterium. 58 00:04:01 --> 00:04:04 If we put it into E. coli that doesn't normally take up 59 00:04:04 --> 00:04:08 DNA you'll find that it's sort of basically black magic. 60 00:04:08 --> 00:04:11 You cook them up with some divalent cations at very high concentrations, 61 00:04:11 --> 00:04:15 you do temperature shifts and various things, 62 00:04:15 --> 00:04:18 or you give them a big jolt of electricity, and the next thing you 63 00:04:18 --> 00:04:22 know you get some DNA inside. And it's not a very efficient 64 00:04:22 --> 00:04:25 process, but all you need is one molecule to get in one bacterium and 65 00:04:25 --> 00:04:29 then you're in business. So that was another reason that 66 00:04:29 --> 00:04:33 this wasn't accepted right away. Because this was not a phenomenon 67 00:04:33 --> 00:04:38 that could easily be repeated with other bacteria. 68 00:04:38 --> 00:04:43 So it looked like it was something perhaps special to streptococcus. 69 00:04:43 --> 00:04:47 And what did really change people's understanding, 70 00:04:47 --> 00:04:52 or at least bring people to the understanding that DNA could 71 00:04:52 --> 00:04:57 possibly be the genetic material came about from the discovery of the 72 00:04:57 --> 00:05:02 actual structure of -- How the structure of DNA as a long 73 00:05:02 --> 00:05:06 molecule with complimentary strands and the double helix, 74 00:05:06 --> 00:05:10 the little pictures I showed you with the base pairs, 75 00:05:10 --> 00:05:14 which you know about, and how the two strands which now 76 00:05:14 --> 00:05:18 I'm going to start emphasizing run in opposite directions. 77 00:05:18 --> 00:05:22 We'll come back to that in a little bit, but the 5 prime to 3 prime 78 00:05:22 --> 00:05:26 direction is this way on one end and 5 to 3 on the other. 79 00:05:26 --> 00:05:30 And just remember back here that there's the 5 prime carbon and 80 00:05:30 --> 00:05:34 that's the 3 prime carbon. So this is the 5 to 3 prime 81 00:05:34 --> 00:05:38 direction of the strand. And then it twists up in 82 00:05:38 --> 00:05:43 3-dimensional space to form this double helix. And you've seen that 83 00:05:43 --> 00:05:48 movie several times. So once that structure was 84 00:05:48 --> 00:05:52 discovered then people began to see how these could possibly encode 85 00:05:52 --> 00:05:57 information. It was clearly not just a tetranucleotide 86 00:05:57 --> 00:06:02 of G, A, T, Cs. But we didn't move immediately to 87 00:06:02 --> 00:06:06 that understanding. And today, again sort of trying to 88 00:06:06 --> 00:06:10 show you how biological experiments are done and how they're done by 89 00:06:10 --> 00:06:14 real people, I want to just go on and tell you the key things that 90 00:06:14 --> 00:06:19 happened next. So someone who was very struck by 91 00:06:19 --> 00:06:23 the results of Avery when they came out was Erwin Chargaff who was at 92 00:06:23 --> 00:06:27 Columbia. And, in fact, my colleague Boris 93 00:06:27 --> 00:06:31 Magasanik whose office is next to mine was a post-doc 94 00:06:31 --> 00:06:35 in Chargaff's lab. So I've got a neighbor of mine who 95 00:06:35 --> 00:06:39 worked with Chargaff. And Chargaff was very struck by 96 00:06:39 --> 00:06:42 this result from Avery and his colleagues that you could take DNA 97 00:06:42 --> 00:06:46 and put it in another organism. And here are a couple of quotes 98 00:06:46 --> 00:06:50 from his writing. One that I'd liked. 99 00:06:50 --> 00:06:53 I've sort of had a sense of this in my own research career, 100 00:06:53 --> 00:06:57 this kind of thing. “I saw before me in dark contours the beginning of 101 00:06:57 --> 00:07:01 a grammar of biology.” He didn't really know quite how it 102 00:07:01 --> 00:07:05 worked but he sort of sensed that someone here where you could get 103 00:07:05 --> 00:07:09 down to the language that biology was written. So he started some 104 00:07:09 --> 00:07:14 experiments. And I started with the conviction that if different DNA 105 00:07:14 --> 00:07:18 species exhibited different biological activities there should 106 00:07:18 --> 00:07:23 exist chemically demonstrable differences between deoxyribonucleic 107 00:07:23 --> 00:07:27 acids. So he was able to start just doing some simple chemical 108 00:07:27 --> 00:07:31 experiments to try and look at DNAs from a whole variety of sources and 109 00:07:31 --> 00:07:37 see what he could learn. And this was not at the structural 110 00:07:37 --> 00:07:44 level. This was just at the chemical level. 111 00:07:44 --> 00:07:51 But one thing he learned was that the base content of DNA, 112 00:07:51 --> 00:07:58 that's the A, G, C, T part of it varied widely between organisms. 113 00:07:58 --> 00:08:07 So this was what Chargaff found in 114 00:08:07 --> 00:08:11 his lab, key findings. And that was important because if 115 00:08:11 --> 00:08:15 DNA was just a molecule of GATC, just a tetranucleotide that every 116 00:08:15 --> 00:08:20 organism made then you'd expect to find the same base composition in 117 00:08:20 --> 00:08:24 all organisms. He didn't, so that finding 118 00:08:24 --> 00:08:29 essentially buried the monotonous tetranucleotide hypothesis. 119 00:08:29 --> 00:08:38 Another thing he found was that DNA was the same in different 120 00:08:38 --> 00:08:49 tissues -- 121 00:08:49 --> 00:08:57 -- from the same organism -- 122 00:08:57 --> 00:09:05 -- but the proteins varied. 123 00:09:05 --> 00:09:08 And that's a characteristic you'd expect of something that was the 124 00:09:08 --> 00:09:12 genetic information from the cell that all cells have to have sort of 125 00:09:12 --> 00:09:16 the major blueprint. And if you had, even though 126 00:09:16 --> 00:09:19 proteins look like an attractive possibility for that because they 127 00:09:19 --> 00:09:23 had so much variation, this kind of finding wasn't 128 00:09:23 --> 00:09:27 consistent with it and it supported the idea that DNA was the 129 00:09:27 --> 00:09:31 genetic material. Well, the other thing he could do 130 00:09:31 --> 00:09:35 was he could measure the ATG and C content of all these different DNAs. 131 00:09:35 --> 00:09:40 And he noticed some similarities then. And he extracted out of that 132 00:09:40 --> 00:09:44 a couple of generalizations. One was that if you looked at the 133 00:09:44 --> 00:09:49 ratio of the purines, those are the ones with the two 134 00:09:49 --> 00:09:54 rings, adenosine and guanidine over the pyrimidines, 135 00:09:54 --> 00:09:58 those are the ones with the single ring which were C and T, 136 00:09:58 --> 00:10:03 there are about one. Another thing he noticed was that 137 00:10:03 --> 00:10:08 the ratio of A to T was about one and the ratio of G to C was about 138 00:10:08 --> 00:10:14 one. Now, that was an important clue but it didn't lead to any 139 00:10:14 --> 00:10:19 immediate breakthrough, even though maybe now that you know 140 00:10:19 --> 00:10:24 the structure you can see, gee, if I had been there maybe I 141 00:10:24 --> 00:10:30 would have been smart enough to jump on that number. 142 00:10:30 --> 00:10:35 So instead the work that led to the structure of DNA now introduces a 143 00:10:35 --> 00:10:40 couple of other characters who you've heard of a lot, 144 00:10:40 --> 00:10:45 Jim Watson and Francis Crick. At the time that Avery made his 145 00:10:45 --> 00:10:50 discovery reporting DNA was transformation, 146 00:10:50 --> 00:10:55 and Jim Watson described himself later as a precocious college boy in 147 00:10:55 --> 00:11:01 Chicago who was consumed by ornithology. 148 00:11:01 --> 00:11:06 So he was into bird watching. That's what he was excited about at 149 00:11:06 --> 00:11:12 the time Avery did his experiment. And Francis Crick at that point was 150 00:11:12 --> 00:11:17 a physicist, and he was in the British Navy designing Navel mines. 151 00:11:17 --> 00:11:23 So that's where those two players were at the time of Avery's results. 152 00:11:23 --> 00:11:29 So then both Francis Crick and Jim Watson ended up in Cambridge, 153 00:11:29 --> 00:11:34 England about 1950. I think Crick got there around 1949 154 00:11:34 --> 00:11:39 and Jim Watson got there in 1951. Francis Crick was a grad student, 155 00:11:39 --> 00:11:44 35 years old at the time. I'll show you pictures in a minute. 156 00:11:44 --> 00:11:49 35 years old at the time and still working on his PhD. 157 00:11:49 --> 00:11:54 So he was a pretty elderly grad student, if you want to think of it 158 00:11:54 --> 00:11:59 that way. And Jim Watson was a young hot-shot. 159 00:11:59 --> 00:12:03 He had done his PhD working with Salvador Luria who was at Indiana 160 00:12:03 --> 00:12:07 University at the time. Salvador Luria was one of the Nobel 161 00:12:07 --> 00:12:11 Laureates at MIT. He founded the Cancer Center, 162 00:12:11 --> 00:12:15 which is still here right across from the main biology building. 163 00:12:15 --> 00:12:19 And Jim was a very, very bright and brash young guy, 164 00:12:19 --> 00:12:23 and he had done his PhD with Salva and then he went to Cambridge as 165 00:12:23 --> 00:12:27 well. And the reason they both went to Cambridge was they were attracted 166 00:12:27 --> 00:12:31 by the power of x-ray crystallography. 167 00:12:31 --> 00:12:34 Now, I said a little work about that earlier, that if you take x-rays and 168 00:12:34 --> 00:12:37 you bounce them off a crystal and then measure the diffraction pattern 169 00:12:37 --> 00:12:41 you can work backwards by Fourier transforms and whatnot to figure out 170 00:12:41 --> 00:12:44 what the underlying crystal structure is. For the purposes of 171 00:12:44 --> 00:12:48 this course the mechanics of how that's done, we don't have to worry 172 00:12:48 --> 00:12:51 about that right now. You just need to know that you can 173 00:12:51 --> 00:12:55 work backwards from the diffraction pattern to figure out what the 174 00:12:55 --> 00:12:59 underlying structure was. And I told you, 175 00:12:59 --> 00:13:03 when I introduced to proteins, that the first clues that there were 176 00:13:03 --> 00:13:07 these regions of secondary structure, alpha helices and beta sheets came 177 00:13:07 --> 00:13:12 because people saw characteristic reflections in these diffraction 178 00:13:12 --> 00:13:16 patterns of certain proteins. And I also told you the story of 179 00:13:16 --> 00:13:21 how Linus Pauling had gone to Oxford, had gotten sick and tired of reading 180 00:13:21 --> 00:13:25 detective novels, started to try and explain the 181 00:13:25 --> 00:13:29 refractions in a certain class of proteins and came up with a model 182 00:13:29 --> 00:13:34 for the alpha helix. And so that was the sort of thing 183 00:13:34 --> 00:13:38 that inspired Watson and Crick. They were both interested in when 184 00:13:38 --> 00:13:43 one could get the structure of DNA. Now, Cambridge also had a very good 185 00:13:43 --> 00:13:48 x-ray crystallogram group. And just in passing it's 186 00:13:48 --> 00:13:52 interesting as to why they didn't come up with the structure of the 187 00:13:52 --> 00:13:57 alpha helix. There were two things. One was just lack of basic 188 00:13:57 --> 00:14:02 knowledge. I told you that the peptide bond, 189 00:14:02 --> 00:14:06 if you remember I emphasized that you cannot rotate it because the 190 00:14:06 --> 00:14:10 electrons are distributed. Pauling was an outstanding chemist. 191 00:14:10 --> 00:14:14 He knew that fact. And the folks at Cambridge who were doing that 192 00:14:14 --> 00:14:18 didn't learn this until later, so their models were far less 193 00:14:18 --> 00:14:22 constrained because they could have rotation around that bond. 194 00:14:22 --> 00:14:26 And the other one was just an experimental thing that the size of 195 00:14:26 --> 00:14:30 the photographic plates they used in the Cambridge lab were too small in 196 00:14:30 --> 00:14:34 the sense that they missed a key reflection that Pauling knew about 197 00:14:34 --> 00:14:39 and they didn't know about. So this combination led to them 198 00:14:39 --> 00:14:45 being scooped by the other group. But nevertheless the group at 199 00:14:45 --> 00:14:51 Cambridge was absolutely outstanding and at one of the top places in the 200 00:14:51 --> 00:14:57 world to do. And I showed you a couple of pictures when I was 201 00:14:57 --> 00:15:02 showing you the transition state. Sort of what you get out of working 202 00:15:02 --> 00:15:07 backwards from these diffraction patterns is they can measure regions 203 00:15:07 --> 00:15:12 of electron density, and then you fit atoms or fit 204 00:15:12 --> 00:15:16 molecules to the patterns that you see. And if it's all working you 205 00:15:16 --> 00:15:21 can explain why there are bumps here. There's an oxygen here and so on. 206 00:15:21 --> 00:15:26 There's another one. This is an ATP that's bound actually in a 207 00:15:26 --> 00:15:31 pocket in a protein. But you can sort of see how 208 00:15:31 --> 00:15:36 beautifully the patterns of electron density deduced from the x-ray 209 00:15:36 --> 00:15:41 crystallography will match the chemical structures that we put on 210 00:15:41 --> 00:15:47 the board. So that was the idea, they were going to work out the 211 00:15:47 --> 00:15:52 structure of DNA. Now, the thing about Watson and 212 00:15:52 --> 00:15:57 Crick, who at this point looked like this, they didn't look inordinately 213 00:15:57 --> 00:16:02 distinguished. In fact, Jim probably looked like, 214 00:16:02 --> 00:16:07 you've probably seen people who look approximately like that around MIT. 215 00:16:07 --> 00:16:12 He would have fit in right here and no one would have noticed. 216 00:16:12 --> 00:16:18 They were not actually x-ray crystallographers. 217 00:16:18 --> 00:16:23 They were just trying to model other people's data. 218 00:16:23 --> 00:16:28 And the best DNA crystallography data was a young woman Roselyn 219 00:16:28 --> 00:16:33 Franklin who was working in London. A very somewhat uneasy alliance with 220 00:16:33 --> 00:16:37 Maurice Wilkins. And in trying to read the history 221 00:16:37 --> 00:16:41 it's a bit complicated because, at least some of what I've read, 222 00:16:41 --> 00:16:46 I think that when Roslyn Franklin arrived at the lab she was told this 223 00:16:46 --> 00:16:50 DNA structure problem was hers. And Maurice Wilkins in whose lab 224 00:16:50 --> 00:16:54 she was working was told that he was sort of working for her. 225 00:16:54 --> 00:16:59 So there was a bunch of confusion in this. 226 00:16:59 --> 00:17:03 But, in any case, Roslyn Franklin was collecting 227 00:17:03 --> 00:17:08 crystallographic data. And Watson and Crick located some 228 00:17:08 --> 00:17:13 distance away in Cambridge were trying to come up with models that 229 00:17:13 --> 00:17:18 could explain the structure of DNA. And they learned about Roslyn's 230 00:17:18 --> 00:17:22 data. And it was here data that they used to work out the basis, 231 00:17:22 --> 00:17:27 her crystallographic data that they used when they put together 232 00:17:27 --> 00:17:32 their structure. So if it hadn't been for her they 233 00:17:32 --> 00:17:36 wouldn't have been able to make their discovery. 234 00:17:36 --> 00:17:40 So part of the reason I'm dwelling on this is I think their discovery 235 00:17:40 --> 00:17:44 of the structure of DNA was arguably one of the great intellectual 236 00:17:44 --> 00:17:48 advances of our time. It just opened doors. 237 00:17:48 --> 00:17:52 The whole field of molecular biology became possible once people 238 00:17:52 --> 00:17:56 suddenly saw that DNA was complimentary strands. 239 00:17:56 --> 00:18:00 You could almost immediately see how you could copy genetic 240 00:18:00 --> 00:18:03 information. It laid the groundwork for what 241 00:18:03 --> 00:18:07 later turned out to be, you know, recombinant DNA and 242 00:18:07 --> 00:18:11 everything else. So much of this pivots around this 243 00:18:11 --> 00:18:15 one discovery. And I think I wouldn't be doing 244 00:18:15 --> 00:18:19 justice to this finding, which you all have heard about for 245 00:18:19 --> 00:18:23 years and years, if I had let you walk away from here 246 00:18:23 --> 00:18:27 thinking this was too young geniuses who sat down in a room with some 247 00:18:27 --> 00:18:31 crystallographic data and emerged with a structure that sort of 248 00:18:31 --> 00:18:35 changed the course of the study of biology. 249 00:18:35 --> 00:18:38 And, as you can see, changes our society and everything 250 00:18:38 --> 00:18:42 else. There are a couple of accounts of this, 251 00:18:42 --> 00:18:45 there are numerous accounts. One that I found pretty interesting 252 00:18:45 --> 00:18:49 is called “The Eighth Day of Creation,” if you ever want to read 253 00:18:49 --> 00:18:52 an interesting book on science. This was Horace Judson's effort to 254 00:18:52 --> 00:18:56 try and put together a history of this happening. 255 00:18:56 --> 00:19:00 And with all history he's ultimately -- 256 00:19:00 --> 00:19:03 You know, there are some judgment calls by the historian, 257 00:19:03 --> 00:19:07 but this one certainly he tried to be pretty fair-handed and 258 00:19:07 --> 00:19:11 even-handed and he tried to get at the heart of what was going on. 259 00:19:11 --> 00:19:14 Watson wrote a book called “The Double Helix”. 260 00:19:14 --> 00:19:18 Jim Watson's a very colorful character, quite brash particularly 261 00:19:18 --> 00:19:22 when he was younger, and that's reflected in this book. 262 00:19:22 --> 00:19:26 It's an interesting read. Probably more balanced point of view for sure 263 00:19:26 --> 00:19:30 in “The Eighth Day of Creation”. And there are now a lot of other 264 00:19:30 --> 00:19:34 books. But what I did, just to try and do this in about a 265 00:19:34 --> 00:19:38 minute or two, was I took a couple of the key 266 00:19:38 --> 00:19:42 things that happened during their adventure of trying to work out the 267 00:19:42 --> 00:19:47 structure of DNA and just kind of ran some of their missteps together, 268 00:19:47 --> 00:19:51 because even though this was a marvelous discovery it just didn't 269 00:19:51 --> 00:19:55 happen. So they started out, they were inspired by Linus 270 00:19:55 --> 00:20:00 Pauling's discovery of the alpha helix. 271 00:20:00 --> 00:20:04 And I don't know if you can remember the story, but what Pauling decided 272 00:20:04 --> 00:20:08 to do when he was lying in bed and with a strip of paper trying to work 273 00:20:08 --> 00:20:12 out the structure that was giving these reflections in the crystal 274 00:20:12 --> 00:20:16 structure, he said I'm going to start by ignoring the side chains. 275 00:20:16 --> 00:20:20 So that was a brilliant move in the case of the alpha helix because he 276 00:20:20 --> 00:20:24 was then able to figure out that that hydrogen bond between the 277 00:20:24 --> 00:20:28 carbonyl and the amino group, you could see how if you got helix 278 00:20:28 --> 00:20:32 going it would repeat at exactly the way that would give the reflections 279 00:20:32 --> 00:20:36 that were observed in the crystallography. 280 00:20:36 --> 00:20:40 So that was how Watson and Crick sort of did it. 281 00:20:40 --> 00:20:44 Linus Pauling had shown the way. So they decided they would ignore 282 00:20:44 --> 00:20:48 the side chains of DNA. So they started out by saying we 283 00:20:48 --> 00:20:52 won't consider the ATs, the Gs and the Cs. Well, 284 00:20:52 --> 00:20:56 given what you know about the structure of DNA that was not a 285 00:20:56 --> 00:21:01 helpful move in trying to work out the structure of DNA. 286 00:21:01 --> 00:21:05 Another thing, for example, that happened was that 287 00:21:05 --> 00:21:09 Jim Watson has no lack of self-confidence. 288 00:21:09 --> 00:21:13 And so it turned out when he went to hear scientific talks he didn't 289 00:21:13 --> 00:21:18 take notes. And so he went to hear a talk on x-ray crystallography 290 00:21:18 --> 00:21:22 given by Roslyn Franklin, but he didn't quite remember the 291 00:21:22 --> 00:21:26 numbers right. He got the facts a little jumbled, 292 00:21:26 --> 00:21:30 and he and Francis spent a while trying to design models to data that 293 00:21:30 --> 00:21:35 wasn't the right data. It was just not quite remembered 294 00:21:35 --> 00:21:41 right, so there was kind of an inefficiency there. 295 00:21:41 --> 00:21:46 And then Jim had a bias almost to the end that the phosphate backbones 296 00:21:46 --> 00:21:51 they knew would somehow be on the inside and the bases would be on the 297 00:21:51 --> 00:21:57 outside of the structure. So if that's your sort of starting 298 00:21:57 --> 00:22:02 place then it's sort of hard. So Watson, excuse me, 299 00:22:02 --> 00:22:07 Francis Crick was beginning to suspect that maybe the bases were 300 00:22:07 --> 00:22:12 important. So he hired a young mathematician. 301 00:22:12 --> 00:22:16 And he said, “Can you see if you could work out whether there would 302 00:22:16 --> 00:22:21 be any chemical attraction between any pairs of bases? 303 00:22:21 --> 00:22:26 And the young mathematician came back and said that he thought G 304 00:22:26 --> 00:22:31 might go with C and A with T. And given what happened here you 305 00:22:31 --> 00:22:36 might have thought that a light bulb would have gone off, 306 00:22:36 --> 00:22:41 but it didn't. And, in fact, Chargaff visited them and the light 307 00:22:41 --> 00:22:46 bulb went off for nobody. And, in fact, Chargaff wasn't a 308 00:22:46 --> 00:22:51 terribly big fan of what Watson and Crick were trying to do. 309 00:22:51 --> 00:22:56 So the pieces are piling up but still not there. 310 00:22:56 --> 00:23:01 Then a big experimental advance came from Roslyn Franklin. 311 00:23:01 --> 00:23:05 And that was she discovered that the DNA that they had been diffracting 312 00:23:05 --> 00:23:10 was actually a mixture of two forms. So there were actually two 313 00:23:10 --> 00:23:15 structures in the mix that were contributing to the diffractions. 314 00:23:15 --> 00:23:20 She was able to separate out the two kinds of DNA, 315 00:23:20 --> 00:23:25 DNA-A and DNA-B she called it. And so now this gave a much clearer 316 00:23:25 --> 00:23:30 diffraction pattern, and that's the diffraction pattern 317 00:23:30 --> 00:23:35 that she saw. And Watson and Crick managed to get 318 00:23:35 --> 00:23:41 a look at this data. And it's a little complicated how 319 00:23:41 --> 00:23:46 that happened, but Crick realized almost right away 320 00:23:46 --> 00:23:51 that there were two strands running in opposite directions. 321 00:23:51 --> 00:23:57 So he know knew it was 5 to 3 in one direction and 5 to 3 in the 322 00:23:57 --> 00:24:02 other direction like that. So you might have thought they were 323 00:24:02 --> 00:24:07 home-free, but no. Jim Watson immediately built a 324 00:24:07 --> 00:24:12 model that paired like with like, A with A, T with T, G with G. They 325 00:24:12 --> 00:24:16 wrote it up and they were ready to submit the paper. 326 00:24:16 --> 00:24:21 And they gave a presentation to their colleagues at the lab in 327 00:24:21 --> 00:24:26 Cambridge. And they were shot down. And one of the key things was they 328 00:24:26 --> 00:24:31 learned the chemical fact that most of the textbooks were wrong at that 329 00:24:31 --> 00:24:36 time in the way that they depicted the structure of guanine. 330 00:24:36 --> 00:24:46 If you look in your textbook, 331 00:24:46 --> 00:24:58 excuse me, here. 332 00:24:58 --> 00:25:03 So if you were to look in a textbook today you'd see guanine like this, 333 00:25:03 --> 00:25:09 but there is another way you could draw this. 334 00:25:09 --> 00:25:23 So this you may remember when we 335 00:25:23 --> 00:25:30 were talking about phosphoenolpyruvate that this is an 336 00:25:30 --> 00:25:36 enol form and this is a keto form. And this is the way most of the 337 00:25:36 --> 00:25:40 textbooks were showing guanine at the time. So they were looking at 338 00:25:40 --> 00:25:44 the structure of guanine in textbooks. And if you were trying 339 00:25:44 --> 00:25:48 to work out schemes for putting bases together you can see what's 340 00:25:48 --> 00:25:52 going on up here would be very different. And if we have a 341 00:25:52 --> 00:25:56 hydrogen here versus if we have an oxygen, if you're trying to say make 342 00:25:56 --> 00:26:00 hydrogen bonds at that particular position, I think all of you 343 00:26:00 --> 00:26:04 understand hydrogen bonds well enough to see how that 344 00:26:04 --> 00:26:09 would throw you off. So once that insight came, 345 00:26:09 --> 00:26:13 once they learned that then the rest of the structure came pretty fast. 346 00:26:13 --> 00:26:18 And there's a movie about this. One of the nice things in it was 347 00:26:18 --> 00:26:22 sort of trying to recreate the experience where I think it was 348 00:26:22 --> 00:26:26 Watson who was shuffling these base pairs around. And he suddenly 349 00:26:26 --> 00:26:31 realized that you could set up base pairs with A and T and with G and C, 350 00:26:31 --> 00:26:35 and when you looked at them you could see they were geometrically 351 00:26:35 --> 00:26:40 exactly the same shape. You could just take the shape of the 352 00:26:40 --> 00:26:44 G and C pair and lay it right down on the A and T pair. 353 00:26:44 --> 00:26:49 And then you could see how you could build either a G-C or an A-T 354 00:26:49 --> 00:26:53 pair into the repeating structure of this DNA and it would be compatible. 355 00:26:53 --> 00:26:57 So they built a model and they thought, we can just hit the lights 356 00:26:57 --> 00:27:05 for a second here maybe. 357 00:27:05 --> 00:27:08 I just want you to see what that first model looked like. 358 00:27:08 --> 00:27:12 It looks like something you could hack together in a chemistry lab. 359 00:27:12 --> 00:27:16 They had the bases cut out of metal. And you can see just, 360 00:27:16 --> 00:27:20 you know, here the retort sort of stands using chemistry and various 361 00:27:20 --> 00:27:24 clamps that you would use for clamping a flask or something if 362 00:27:24 --> 00:27:28 you're doing a chemical lab. That's the stuff that they were 363 00:27:28 --> 00:27:32 using to put the model together. And they published then a paper in 364 00:27:32 --> 00:27:38 Nature that told about this result. That's the entire paper reporting 365 00:27:38 --> 00:27:43 the structure of DNA. And maybe you can see there's a 366 00:27:43 --> 00:27:49 little hand-drawn double helix right there that captures the elements. 367 00:27:49 --> 00:27:54 That is the paper, and that was in the journal Nature. 368 00:27:54 --> 00:28:00 And it had in it, right near the end, one of the coyest sentences in 369 00:28:00 --> 00:28:05 the scientific literature. They didn't want to go into all the 370 00:28:05 --> 00:28:10 details that if you had an A paired with G and G paired with a C and you 371 00:28:10 --> 00:28:16 pulled them apart then you could replicate the molecule by redoing it. 372 00:28:16 --> 00:28:21 So all they said was, “It has not escaped our notice that 373 00:28:21 --> 00:28:26 the specific pairings we expostulated immediately suggests a 374 00:28:26 --> 00:28:32 copying mechanism for DNA. So this is a picture of Jim Watson 375 00:28:32 --> 00:28:37 wearing short pants at Cold Spring Harbor in 1953 reporting 376 00:28:37 --> 00:28:42 this structure of DNA. Cold Spring Harbor is on Long Island. 377 00:28:42 --> 00:28:47 It's been one of the Meccas for molecule biology since the 1940s. 378 00:28:47 --> 00:28:52 They have a famous symposium once a year. The topic changes every year 379 00:28:52 --> 00:28:57 and rarely repeats. And it was at one of those symposia 380 00:28:57 --> 00:29:02 -- This was the year that they 381 00:29:02 --> 00:29:07 discovered the structure of DNA. And there was Watson. So two years 382 00:29:07 --> 00:29:12 ago they had another meeting, a special meeting just exactly this 383 00:29:12 --> 00:29:17 time of year. It was in February within a couple of days of right now. 384 00:29:17 --> 00:29:22 So I gave this lecture and I showed the student in the class that this 385 00:29:22 --> 00:29:27 year, I said here's a picture of Jim Watson displaying the structure. 386 00:29:27 --> 00:29:32 They're having a meeting 50 years later in 2003. 387 00:29:32 --> 00:29:35 And I'm going down there. I'm asked to give a talk. 388 00:29:35 --> 00:29:38 And I'll come back and I'll tell you what it was like. 389 00:29:38 --> 00:29:41 So I gave my lecture. I dashed out to the airport. 390 00:29:41 --> 00:29:45 I hoped on the plane. I went down and I registered. 391 00:29:45 --> 00:29:48 They gave me, you know, the stuff to get into my room, 392 00:29:48 --> 00:29:51 a little envelope with the key card and things. And I went up to my 393 00:29:51 --> 00:29:55 room. And I took out the key card. And what did I find myself looking 394 00:29:55 --> 00:29:58 at? The same picture I had shown to the class just a couple 395 00:29:58 --> 00:30:01 of hours earlier. Here's another picture of Jim the 396 00:30:01 --> 00:30:05 way he looked at the time when he made this amazing discovery. 397 00:30:05 --> 00:30:09 That's Salvador Luria who I mentioned. I tell you about him in 398 00:30:09 --> 00:30:12 a subsequent lecture. I was at another meeting a few 399 00:30:12 --> 00:30:16 years earlier where some of the old-timers were razzing each other, 400 00:30:16 --> 00:30:20 and someone showed this picture. And then they got up and they gave 401 00:30:20 --> 00:30:23 it a title. And that was “Picture of a Man Picking His Own Pockets”. 402 00:30:23 --> 00:30:27 So they would tease each other a lot. And I'm hoping maybe you'll get a 403 00:30:27 --> 00:30:31 chance to hear a little bit more about that soon. 404 00:30:31 --> 00:30:35 This is what Jim Watson looks like now. I asked to get a picture taken 405 00:30:35 --> 00:30:39 just so you could see he's still around and is very active and still 406 00:30:39 --> 00:30:43 very controversial. This doesn't make much of a 407 00:30:43 --> 00:30:48 difference. Here's a picture of Watson and Crick a little bit later 408 00:30:48 --> 00:30:52 just sitting out on a porch in Cold Spring Harbor. 409 00:30:52 --> 00:30:56 It's sort of right on the edge of a bay down there in a very relaxed 410 00:30:56 --> 00:31:00 kind of atmosphere that still permeates molecule biology 411 00:31:00 --> 00:31:04 research to this day. Francis Crick just died last July at 412 00:31:04 --> 00:31:06 the age of 88, so we've just lost the link to one 413 00:31:06 --> 00:31:09 of the two people who did this amazing experiment. 414 00:31:09 --> 00:31:11 OK. So I want to then set things up for the details of DNA 415 00:31:11 --> 00:31:14 replication. So there was a basic principle that came across from this 416 00:31:14 --> 00:31:16 that you could see how this could work, that DNA was sort of like 417 00:31:16 --> 00:31:18 having a photograph and a negative. And so the information is actually 418 00:31:18 --> 00:31:21 in there twice. It's just in different forms. 419 00:31:21 --> 00:31:23 And when I tell you about DNA repair in another lecture you can 420 00:31:23 --> 00:31:26 maybe see already how useful that is because if you damage one strand 421 00:31:26 --> 00:31:28 you're not really out of luck because you've still got the 422 00:31:28 --> 00:31:33 information in the other strand. And you could probably, 423 00:31:33 --> 00:31:41 on the basis of that, device a repair strategy if you thought about 424 00:31:41 --> 00:31:48 it. But more importantly for DNA replication finally gave an insight 425 00:31:48 --> 00:31:55 to this thing that had been vexing people forever. 426 00:31:55 --> 00:32:03 If you had to have all this information for making a cell, 427 00:32:03 --> 00:32:10 and every time a cell divided and you saw how it can happen pretty 428 00:32:10 --> 00:32:17 quickly with something like a bacterium of yeast, 429 00:32:17 --> 00:32:25 how could you accurately copy all that DNA, excuse me, 430 00:32:25 --> 00:32:29 all that genetic information? How is it stored? 431 00:32:29 --> 00:32:29 How could it be done? And once you saw ah, it's just a 432 00:32:29 --> 00:32:29 matter of separating the strands, and if there's an A there put a T 433 00:32:29 --> 00:32:29 there, if there's a C you put a G and so on, was a huge breakthrough. 434 00:32:29 --> 00:32:30 But that then didn't tell people how DNA replicated or even if this 435 00:32:30 --> 00:32:30 is the mechanism. You can actually come up with all 436 00:32:30 --> 00:32:30 kinds of models for how you could replicate things based on this 437 00:32:30 --> 00:32:30 principle, including crisscrossing between strands and all 438 00:32:30 --> 00:32:43 sorts of things. The predominant model and perhaps 439 00:32:43 --> 00:33:07 the simplest one was called semi-conservative. 440 00:33:07 --> 00:33:15 And it thought of the problem in 441 00:33:15 --> 00:33:21 this kind of way, that if you had two strands of the 442 00:33:21 --> 00:33:26 original DNA molecule and then you pulled them apart that one of the 443 00:33:26 --> 00:33:32 strands here would become one of the strands of the daughter, 444 00:33:32 --> 00:33:38 and then the new one would be here and the same thing would happen on 445 00:33:38 --> 00:33:44 the other side. And then if you did it again this 446 00:33:44 --> 00:33:50 thing would happen again with a new strand. This time the skinny strand 447 00:33:50 --> 00:33:56 here would be like this, the skinny strand here would be like 448 00:33:56 --> 00:34:02 this, and then this one again. We'd have one that was nearly 449 00:34:02 --> 00:34:08 synthesized plus one of the originals. So this model was one of 450 00:34:08 --> 00:34:14 the simplest because it kept this strand intact throughout the whole 451 00:34:14 --> 00:34:19 process while some of the other models had them being patched back 452 00:34:19 --> 00:34:25 together, all based on the idea that A pairs with T and G pairs with C. 453 00:34:25 --> 00:34:31 But proving that this was the correct model was then another 454 00:34:31 --> 00:34:36 important advance. And that was done by Frank Stahl and 455 00:34:36 --> 00:34:40 Matt Meselson. Actually, I think I'll skip this 456 00:34:40 --> 00:34:44 for right now. Matt is a professor up at Harvard, 457 00:34:44 --> 00:34:48 just up at Harvard Square not very far from here, 458 00:34:48 --> 00:34:52 still very active. Frank Stahl is a professor out in 459 00:34:52 --> 00:34:56 Oregon. He's still active. So one of the differences about 460 00:34:56 --> 00:35:01 this course is a lot of the things I'm telling you about -- 461 00:35:01 --> 00:35:06 And this is pretty old stuff right now, right, molecule biology. 462 00:35:06 --> 00:35:11 The people who did these are still around and very active. 463 00:35:11 --> 00:35:16 This is most of modern biology is a pretty young scientist, 464 00:35:16 --> 00:35:22 and many of the major characters are still running around and with us 465 00:35:22 --> 00:35:27 today. So, anyway, what Matt and Frank were at Caltech. 466 00:35:27 --> 00:35:32 And they with a bunch of other 467 00:35:32 --> 00:35:37 students had an apartment. And they were sitting around trying 468 00:35:37 --> 00:35:42 to work out a way to figure out this model. And they came up with an 469 00:35:42 --> 00:35:47 idea, and that was to see if you could differentially label what we 470 00:35:47 --> 00:35:52 might call “old DNA” and the “new DNA” here. And since it's 471 00:35:52 --> 00:35:57 chemically the same stuff it's a bit of a trick. How do you tell old DNA 472 00:35:57 --> 00:36:03 from new DNA? So their idea was since nitrogen 473 00:36:03 --> 00:36:09 comes in two different isotopes, N14 which is the common one and N15 474 00:36:09 --> 00:36:16 with is one mass heavier, that maybe you could start out with 475 00:36:16 --> 00:36:22 the DNA, for example, grown in N15. And then when you 476 00:36:22 --> 00:36:28 started replication switch to N14. And then you'd be able to tell, 477 00:36:28 --> 00:36:32 if you could separate these molecules on the basis of their 478 00:36:32 --> 00:36:36 density since the one with the N15 would be heavier than the one with 479 00:36:36 --> 00:36:41 the N14, then maybe you could work this out. And the story goes, 480 00:36:41 --> 00:36:45 this has been written, they were sitting arguing about this, 481 00:36:45 --> 00:36:50 or talking about this idea at the table. And it was a good idea but 482 00:36:50 --> 00:36:54 there was a problem. And that was how could you separate 483 00:36:54 --> 00:36:59 the two kinds of DNA based on their density? 484 00:36:59 --> 00:37:02 So they had a piece of fingernail and they were trying to see whether 485 00:37:02 --> 00:37:06 they could get it to float by dissolving more and more sugar. 486 00:37:06 --> 00:37:09 And they figured if they added more and more sugar the water would get 487 00:37:09 --> 00:37:13 denser and denser so the could float the fingernail. 488 00:37:13 --> 00:37:16 And they weren't able to do it. But all chemists made a periodic, 489 00:37:16 --> 00:37:20 probably some places here at MIT, they had a periodic chart right in 490 00:37:20 --> 00:37:23 their living room. So they went and they looked. 491 00:37:23 --> 00:37:27 And then they looked at sodium. And they went down the periodic 492 00:37:27 --> 00:37:31 table and then they saw cesium. And thought maybe, 493 00:37:31 --> 00:37:35 you know, if you took a solution of cesium chloride and you put it a 494 00:37:35 --> 00:37:39 centrifuge and you spun really hard then you'd get a gradient of varying 495 00:37:39 --> 00:37:43 concentrations, of slightly different concentrations 496 00:37:43 --> 00:37:47 of cesium chloride. And that they could tune that to a 497 00:37:47 --> 00:37:51 range that would discriminate between the heavy and the lighter 498 00:37:51 --> 00:37:55 forms of DNA. So the experiment they did is known as the 499 00:37:55 --> 00:37:59 Meselson-Stahl experiment. But, as I say, these are names that 500 00:37:59 --> 00:38:05 come from real people. And the idea was pretty simple. 501 00:38:05 --> 00:38:13 They grew the bacteria for many generations -- 502 00:38:13 --> 00:38:22 -- in N15 medium. 503 00:38:22 --> 00:38:30 This is the so-called heavy 504 00:38:30 --> 00:38:37 or H isotope -- 505 00:38:37 --> 00:38:45 -- of nitrogen. And then that time equals zero in 506 00:38:45 --> 00:38:53 their experiment, when they're ready to start the 507 00:38:53 --> 00:39:01 experiment they switched to medium with N14, which we'll think of as 508 00:39:01 --> 00:39:09 the light or the L isotope. And then they isolated DNA -- 509 00:39:09 --> 00:39:21 -- after let's say increasing rounds 510 00:39:21 --> 00:39:31 of replication that you could tell simply by measuring how much DNA was 511 00:39:31 --> 00:39:41 in your bacterial culture when the bacteria had doubled their DNA. 512 00:39:41 --> 00:39:47 And this is the data they got which looks something like this. 513 00:39:47 --> 00:39:53 In fact, in this case the blackboard representation is pretty 514 00:39:53 --> 00:39:59 close. So this is cesium chloride. And it has been centrifuged very 515 00:39:59 --> 00:40:05 hard so that there's a gradient now that's light at the top and a little 516 00:40:05 --> 00:40:11 heavier at the bottom of the gradient. 517 00:40:11 --> 00:40:14 There's a little more cesium chloride per mil here then there is 518 00:40:14 --> 00:40:18 there in the tube. And I'll just give us three little 519 00:40:18 --> 00:40:22 sort of reference marks here. So what they found when they 520 00:40:22 --> 00:40:26 started was that all of the DNA was at that position down 521 00:40:26 --> 00:40:32 at the heavy end. And then this is after one 522 00:40:32 --> 00:40:40 generation. So the DNA has now doubled. What they found was that 523 00:40:40 --> 00:40:49 all the DNA was now at this intermediate position. 524 00:40:49 --> 00:40:58 And after two generations or two DNA 525 00:40:58 --> 00:41:04 replications they now found that some of the DNA was here, 526 00:41:04 --> 00:41:10 some of the DNA was there. And if they went to three or more 527 00:41:10 --> 00:41:16 what they saw was they began to pile stuff up there. 528 00:41:16 --> 00:41:21 And I think most of you could probably make the connection between 529 00:41:21 --> 00:41:27 that data and that picture that I've got up there. This is 530 00:41:27 --> 00:41:32 the heavy-heavy DNA. This is the heavy-light. 531 00:41:32 --> 00:41:38 So this would be heavy-heavy, heavy-light, light-heavy. After one 532 00:41:38 --> 00:41:44 round it will all be here. After two we have heavy-light, 533 00:41:44 --> 00:41:50 but this one is light-light, light-light, light-heavy. 534 00:41:50 --> 00:41:56 And so now we've got light-light, the heavy-light, no heavy-heavy is 535 00:41:56 --> 00:42:01 ever going to show up again. And the longer you do this the more 536 00:42:01 --> 00:42:06 you'll get the light accumulating. A very simple experiment done by 537 00:42:06 --> 00:42:11 real people but enormously powerful because now it showed that this 538 00:42:11 --> 00:42:15 basic idea, you have the photograph and negative, you pull them apart 539 00:42:15 --> 00:42:20 and copy them was right. So at this point you begin to see 540 00:42:20 --> 00:42:25 why data of Avery's that before people had trouble accepting, 541 00:42:25 --> 00:42:30 all of a sudden now it was really you needed a CYD and A was 542 00:42:30 --> 00:42:34 the genetic material. And this is what sort of ushered in 543 00:42:34 --> 00:42:38 this great burst of molecular biology. So in the next lecture 544 00:42:38 --> 00:42:42 what we're going to start doing now is once you, this is all great, 545 00:42:42 --> 00:42:45 but once we start figuring out how to replicate it we're going to have 546 00:42:45 --> 00:42:49 to get down to enzymes and biochemical steps. 547 00:42:49 --> 00:42:52 And there are some formidable challenges to replicating DNA, 548 00:42:52 --> 00:42:56 and it's also awesome. I'll tell you at the beginning of next lecture 549 00:42:56 --> 00:43:00 how much DNA we have and just how accurate it is. 550 00:43:00 --> 00:43:03 It always blows me away. I'll see you then. Take care.