1 00:00:00,000 --> 00:00:04,000 Let's get started. So I'm going to finish up energy 2 00:00:04,000 --> 00:00:09,000 today. And then we're going to begin sort of section more or less 3 00:00:09,000 --> 00:00:14,000 we'll call it molecular biology but it's sort of dealing with the issues 4 00:00:14,000 --> 00:00:19,000 that revolve around the discovery that DNA was the genetic material 5 00:00:19,000 --> 00:00:24,000 and then working through how people understood how information got from 6 00:00:24,000 --> 00:00:29,000 the DNA into everything else, how things were regulated. 7 00:00:29,000 --> 00:00:32,000 There are an incredibly large number of important discoveries that form 8 00:00:32,000 --> 00:00:36,000 the foundation of how we think about biology that are going to come out 9 00:00:36,000 --> 00:00:40,000 in this next section, but before I do that I just want to 10 00:00:40,000 --> 00:00:44,000 finish up this section that I talked to you about, about energy. 11 00:00:44,000 --> 00:00:48,000 And I hope as we go along here you're going to see how some of 12 00:00:48,000 --> 00:00:52,000 these sort of disparate parts of the course begin to come together. 13 00:00:52,000 --> 00:00:56,000 Almost everything we're going to be talking about now is going to be 14 00:00:56,000 --> 00:01:00,000 needing energy such as replicating DNA or making proteins and 15 00:01:00,000 --> 00:01:04,000 all sorts of things. And those are driven ultimately by 16 00:01:04,000 --> 00:01:09,000 ATP. And what I've been trying to talk to about for the last lecture 17 00:01:09,000 --> 00:01:14,000 or two is how the cell gets the ATP, the energy money that it needs to 18 00:01:14,000 --> 00:01:19,000 make things. We talked through glycolysis, this ancient, 19 00:01:19,000 --> 00:01:24,000 ancient way of getting a couple of ATPs out of a molecule of sugar so 20 00:01:24,000 --> 00:01:29,000 well-imbedded in our genetic makeup it's in almost all organisms. 21 00:01:29,000 --> 00:01:33,000 And then I talked last lecture about this other principle which must have 22 00:01:33,000 --> 00:01:37,000 come up very, very early in evolution. Again, 23 00:01:37,000 --> 00:01:42,000 it's used by all organisms. And that's the principle of 24 00:01:42,000 --> 00:01:46,000 capturing the energy that's inherent in a proton gradient across a 25 00:01:46,000 --> 00:01:51,000 membrane. And I talked to you then about the idea then the way it 26 00:01:51,000 --> 00:01:55,000 worked was that the cell would have something in its membrane that would 27 00:01:55,000 --> 00:01:59,000 be a proton pump and it would pump the proton from one side of the 28 00:01:59,000 --> 00:02:04,000 membrane to the other. So it's working against the gradient. 29 00:02:04,000 --> 00:02:09,000 So it's doing energy. So there are a couple of different 30 00:02:09,000 --> 00:02:14,000 ways energy needs to be provided. It could be provided by some kind 31 00:02:14,000 --> 00:02:19,000 of light energy, and that's what drives 32 00:02:19,000 --> 00:02:24,000 photosynthesis. I'll say a few words about that. 33 00:02:24,000 --> 00:02:29,000 Or in the case of respiration with the oxidative phosphorylation, 34 00:02:29,000 --> 00:02:34,000 I showed you how it was the electrons sort of descend in 35 00:02:34,000 --> 00:02:39,000 stepwise fashion down from one state to another. 36 00:02:39,000 --> 00:02:44,000 There's energy given off, free energy is available, and that 37 00:02:44,000 --> 00:02:50,000 can be used to power the pump. And once the proton gradient is 38 00:02:50,000 --> 00:02:56,000 made then the cells can turn it around and use the energy that's in 39 00:02:56,000 --> 00:03:01,000 that proton gradient to make ATP. So in respiration remember the trick 40 00:03:01,000 --> 00:03:05,000 was then to take those two pyruvates, burn them all the way down to carbon 41 00:03:05,000 --> 00:03:10,000 dioxide and water, make as many ATPs and NADHs as you 42 00:03:10,000 --> 00:03:14,000 could, then take the NADHs, use them to make a proton gradient 43 00:03:14,000 --> 00:03:19,000 and eventually, if you will, convert everything into 44 00:03:19,000 --> 00:03:23,000 ATP so you've got it as energy. Our cells do it. That part, 45 00:03:23,000 --> 00:03:28,000 respiration and the oxidative phosphorylation is done in 46 00:03:28,000 --> 00:03:32,000 mitochondria, which I said sort of came from bacteria that were 47 00:03:32,000 --> 00:03:37,000 captured at some point. Here's a picture of a mitochondrion. 48 00:03:37,000 --> 00:03:41,000 It still looks more or less like a bacterium. And I showed you the 49 00:03:41,000 --> 00:03:46,000 little parts that are in there. It was funny. Right after lecture 50 00:03:46,000 --> 00:03:50,000 I went back to my lab. I picked up a recent issue of 51 00:03:50,000 --> 00:03:55,000 Science. And I opened up to a page that said something about rats that 52 00:03:55,000 --> 00:04:00,000 had been bred to be very poor at aerobic exercise. 53 00:04:00,000 --> 00:04:03,000 And it went on to talk about the health problems they had. 54 00:04:03,000 --> 00:04:07,000 And there was a sentence in there that they think the underlying cause 55 00:04:07,000 --> 00:04:10,000 is by breeding these rats and selection four rats that are poor at 56 00:04:10,000 --> 00:04:14,000 doing aerobic exercise. What they think it all stems from 57 00:04:14,000 --> 00:04:17,000 is having very inefficient mitochondria that don't work nearly 58 00:04:17,000 --> 00:04:21,000 as well. And that would make a lot of sense. I was going to scan that 59 00:04:21,000 --> 00:04:24,000 article but we had some technical issues this morning. 60 00:04:24,000 --> 00:04:28,000 Maybe I can show it to you next lecture. OK. 61 00:04:28,000 --> 00:04:32,000 So the one last thing then that I want to do is I want to say a few 62 00:04:32,000 --> 00:04:37,000 words about photosynthesis because that actually preceded respiration. 63 00:04:37,000 --> 00:04:41,000 Respiration couldn't evolve until there was oxygen in the atmosphere. 64 00:04:41,000 --> 00:04:46,000 So probably the first use or certainly one of the first uses of 65 00:04:46,000 --> 00:04:51,000 the proton gradient happened in this scale of evolution and put here 66 00:04:51,000 --> 00:04:55,000 somewhere maybe 3. billion years ago or so when what I 67 00:04:55,000 --> 00:05:04,000 had called photosynthesis -- 68 00:05:04,000 --> 00:05:09,000 -- release I on day one in the sort of trivial fashion. This 69 00:05:09,000 --> 00:05:16,000 is known as cyclic -- 70 00:05:16,000 --> 00:05:22,000 -- photo phosphorylation. And the principle is relatively 71 00:05:22,000 --> 00:05:29,000 simple. It's to capture the energy in sunlight -- 72 00:05:29,000 --> 00:05:37,000 -- to make a proton gradient. 73 00:05:37,000 --> 00:05:43,000 And that then can be used, as you now know, to make ATP. 74 00:05:43,000 --> 00:05:50,000 So in order to capture energy from sunlight nature had to evolve 75 00:05:50,000 --> 00:05:56,000 molecules that are able to observe in the appropriate wavelength 76 00:05:56,000 --> 00:06:01,000 range. You know the names of those 77 00:06:01,000 --> 00:06:05,000 molecules. Chlorophyll, the come in two principle species. 78 00:06:05,000 --> 00:06:08,000 You don't have to remember the structure. What you can see is a 79 00:06:08,000 --> 00:06:12,000 lot of conjugated double-bonds. That's how you sort of tune the 80 00:06:12,000 --> 00:06:15,000 absorption of a molecule. If you want to make it absorb a 81 00:06:15,000 --> 00:06:19,000 longer and longer wavelength you start hooking together double bonds. 82 00:06:19,000 --> 00:06:22,000 And you can set it up sort of you can get a molecule to absorb at just 83 00:06:22,000 --> 00:06:26,000 about any maximum absorption, any wavelength you want. So 84 00:06:26,000 --> 00:06:30,000 chlorophyll is able to absorb this energy. 85 00:06:30,000 --> 00:06:34,000 And the principle of this is what happens. You have this chlorophyll 86 00:06:34,000 --> 00:06:39,000 and it absorbs a photon. And an electron gets excited so it 87 00:06:39,000 --> 00:06:44,000 basically moves to another orbital. It's farther away from the nucleus. 88 00:06:44,000 --> 00:06:49,000 It's easier now for that electron to get lost than it was before. 89 00:06:49,000 --> 00:06:54,000 Now, if nothing was happening that electron would eventually just fall 90 00:06:54,000 --> 00:06:59,000 down to its ground state and you'd lose all the energy as heat. 91 00:06:59,000 --> 00:07:05,000 So what happens in photosynthesis, though, is that the electron falls 92 00:07:05,000 --> 00:07:11,000 back down to the ground state again in a series of steps. 93 00:07:11,000 --> 00:07:17,000 And how this happens, electrons are basically getting 94 00:07:17,000 --> 00:07:23,000 passed from one carrier to another. And the same principle as we saw in 95 00:07:23,000 --> 00:07:29,000 respiration applies in that at each phase in here a proton is pumped. 96 00:07:29,000 --> 00:07:35,000 Now, the out and the in are reversed from what it says in respiration. 97 00:07:35,000 --> 00:07:41,000 You have notes on respiration, or should anyway. 98 00:07:41,000 --> 00:07:48,000 But the in and the out, as you'll see, it's sort of an 99 00:07:48,000 --> 00:07:54,000 arbitrary. You sort of take a frame of reference and then something's in 100 00:07:54,000 --> 00:07:59,000 and something's out. The point is that in both of them 101 00:07:59,000 --> 00:08:03,000 protons go from one side of the membrane to the other and you get 102 00:08:03,000 --> 00:08:07,000 more on one side, you pump them in one direction and 103 00:08:07,000 --> 00:08:11,000 they flow back in the other. And the in and the out is sort of 104 00:08:11,000 --> 00:08:15,000 an arbitrary way of describing what's happening, 105 00:08:15,000 --> 00:08:19,000 but in both cases the key thing is that you're pumping electrons out. 106 00:08:19,000 --> 00:08:23,000 And then these can be used to make ATP, as we talked about with ATP 107 00:08:23,000 --> 00:08:27,000 synthesis. In this case the electrons eventually end up back on 108 00:08:27,000 --> 00:08:30,000 the chlorophyll. And so that's why it's called cyclic 109 00:08:30,000 --> 00:08:34,000 phosphorylation. What you get out of this, 110 00:08:34,000 --> 00:08:37,000 as you can see, is ATP. So this was probably a really big deal in 111 00:08:37,000 --> 00:08:41,000 evolution because the current thinking is perhaps there was an RNA 112 00:08:41,000 --> 00:08:44,000 world that's still sort of being debated. At some point it's clear 113 00:08:44,000 --> 00:08:48,000 that somewhere around 3. billion years or so something that 114 00:08:48,000 --> 00:08:51,000 looks sort of like a present-day bacterium arose, 115 00:08:51,000 --> 00:08:55,000 probably eight molecules that had already been made in the sort of 116 00:08:55,000 --> 00:08:58,000 primordial soup, but when those started to run out 117 00:08:58,000 --> 00:09:02,000 then it needed other ways of making energy. 118 00:09:02,000 --> 00:09:07,000 It needed other ways of making carbon. Here's a way of getting 119 00:09:07,000 --> 00:09:12,000 energy, but what's available to make more organic molecules is only 120 00:09:12,000 --> 00:09:17,000 carbon dioxide. And if you remember that little 121 00:09:17,000 --> 00:09:22,000 thing I showed you of if we're going from a methyl to a hydroxyl to an 122 00:09:22,000 --> 00:09:29,000 aldehyde to an acid -- 123 00:09:29,000 --> 00:09:33,000 -- to CO2, that direction is oxidation and that direction is 124 00:09:33,000 --> 00:09:38,000 reduction. So if we go in that direction and we end up generating 125 00:09:38,000 --> 00:09:42,000 NADH because we're taking electrons and giving them to something else, 126 00:09:42,000 --> 00:09:47,000 if we want to go the other way if we're starting with C02 what we need 127 00:09:47,000 --> 00:09:52,000 to do is we need to have a supply of reducing power so we can take the 128 00:09:52,000 --> 00:09:56,000 C02 and get it down to all the less-oxidized states that are 129 00:09:56,000 --> 00:10:01,000 necessary for building all the molecules that we've 130 00:10:01,000 --> 00:10:06,000 been talking about. So making ATP was a great idea, 131 00:10:06,000 --> 00:10:10,000 but the cell still needed to have some form of reducing agent. 132 00:10:10,000 --> 00:10:15,000 And what they used was they used hydrogen sulfide. 133 00:10:15,000 --> 00:10:19,000 This is at least one of the major ways that it was done. 134 00:10:19,000 --> 00:10:24,000 And so there is a very slight twist here. This is NADP. 135 00:10:24,000 --> 00:10:28,000 It's the same molecule as NAD except there's an extra 136 00:10:28,000 --> 00:10:33,000 phosphorylation. And the one with the phosphate on it 137 00:10:33,000 --> 00:10:37,000 tends to be used in biosynthetic reactions, but otherwise it's 138 00:10:37,000 --> 00:10:41,000 exactly the same thing. It's an electron banking thing. 139 00:10:41,000 --> 00:10:45,000 And what this gave was NADPH plus sulfur plus a hydrogen. 140 00:10:45,000 --> 00:10:50,000 So sulfur is a waste product. Here's the reducing power. Here's 141 00:10:50,000 --> 00:10:54,000 the ATP. That's what those organisms need to be able to 142 00:10:54,000 --> 00:10:58,000 synthesize new organic material without having to have 143 00:10:58,000 --> 00:11:03,000 pre-made molecules. A really big deal in evolution. 144 00:11:03,000 --> 00:11:07,000 And the idea for making ATP is based on this use of establishing a 145 00:11:07,000 --> 00:11:12,000 proton gradient, the same principle we've seen again. 146 00:11:12,000 --> 00:11:17,000 Now, there's another possible source of reducing power, 147 00:11:17,000 --> 00:11:21,000 and that would be to use water as the source of the reducing power. 148 00:11:21,000 --> 00:11:26,000 But in order to do that you've got to put more energy into it. 149 00:11:26,000 --> 00:11:31,000 And this system wasn't able to handle it. 150 00:11:31,000 --> 00:11:43,000 But that happened soon enough with the development of what I called on 151 00:11:43,000 --> 00:11:55,000 the first day photosynthesis release II, which is technically known as 152 00:11:55,000 --> 00:12:04,000 noncyclic photophosphorylation. Again, it uses the energy of 153 00:12:04,000 --> 00:12:08,000 sunlight. But the twist this time, it not only makes ATP, it also makes 154 00:12:08,000 --> 00:12:13,000 NADPH, it makes reducing power at the same time. 155 00:12:13,000 --> 00:12:17,000 So you can see that is a really major advance. 156 00:12:17,000 --> 00:12:22,000 If you can use sunlight to make both of them now you're really 157 00:12:22,000 --> 00:12:26,000 efficient. So this is how this one works. It's related 158 00:12:26,000 --> 00:12:31,000 to the other one. And the first part is more or less 159 00:12:31,000 --> 00:12:36,000 the same idea. A photon is absorbed by a molecule 160 00:12:36,000 --> 00:12:42,000 of chlorophyll. It kicks the chlorophyll up to an 161 00:12:42,000 --> 00:12:47,000 activated state where the electrons are at a higher orbital far away. 162 00:12:47,000 --> 00:12:52,000 It wants to come back down. Energy is going to be released. 163 00:12:52,000 --> 00:12:58,000 So electrons gets passed, protons get pumped from one side of 164 00:12:58,000 --> 00:13:04,000 a membrane to another. Except this time, 165 00:13:04,000 --> 00:13:10,000 instead of coming back this lands in a different chlorophyll that has 166 00:13:10,000 --> 00:13:16,000 just recently lost a pair of electrons. But there's a new energy 167 00:13:16,000 --> 00:13:22,000 input here that kicks this chlorophyll up to an even higher 168 00:13:22,000 --> 00:13:28,000 energy state than this one. And as these electrons start to 169 00:13:28,000 --> 00:13:34,000 come down the energy hill there's enough energy here to take a 170 00:13:34,000 --> 00:13:40,000 molecule of NADP+ plus a hydrogen ion and give NADPH. 171 00:13:40,000 --> 00:13:46,000 There's one thing that this isn't going to work like a cycle or a 172 00:13:46,000 --> 00:13:53,000 machine yet. Anybody see what hasn't been taken care of yet? 173 00:13:53,000 --> 00:14:00,000 Say again. Send the electrons back to this chlorophyll, exactly. 174 00:14:00,000 --> 00:14:05,000 However, the way the energetics are structured now the cells were able 175 00:14:05,000 --> 00:14:10,000 to take reducing power from here and generate 2H+ plus a half of an 176 00:14:10,000 --> 00:14:16,000 oxygen molecule. And this would really be two waters 177 00:14:16,000 --> 00:14:21,000 giving four hydrogens and one oxygen molecule. So what you can see here 178 00:14:21,000 --> 00:14:27,000 now, there are a couple of really important things about this. 179 00:14:27,000 --> 00:14:33,000 It needs more energy. It makes ATP and NADPH which leaves 180 00:14:33,000 --> 00:14:41,000 the cell able to carry out biosynthesis. And the third thing, 181 00:14:41,000 --> 00:14:48,000 which is an incredible influence on our planet, it started to generate 182 00:14:48,000 --> 00:14:56,000 oxygen as a waste product. And it's really a mixed blessing. 183 00:14:56,000 --> 00:15:01,000 I mean oxygen is very reactive. It damages our DNA. 184 00:15:01,000 --> 00:15:05,000 It damages our proteins. We have an amazing number of 185 00:15:05,000 --> 00:15:08,000 defenses against oxygen. But, on the other hand, as it 186 00:15:08,000 --> 00:15:11,000 accumulated in the atmosphere and organisms slowly over evolutionary 187 00:15:11,000 --> 00:15:15,000 time learned to deal with it, it then set us up for the 188 00:15:15,000 --> 00:15:18,000 possibility of respiration which, as you can see, is 18 times more 189 00:15:18,000 --> 00:15:21,000 efficient than in that ancient way of using glycolysis to make energy 190 00:15:21,000 --> 00:15:25,000 out of sugars. So that's more or less the story. 191 00:15:25,000 --> 00:15:29,000 This part is called photosystem II. 192 00:15:29,000 --> 00:15:34,000 This assembly of stuff is photosystem I. 193 00:15:34,000 --> 00:15:39,000 And I just wanted to show you this next slide because chlorophyll isn't 194 00:15:39,000 --> 00:15:44,000 just floating around like this. As you might guess, it's bound into 195 00:15:44,000 --> 00:15:49,000 proteins and things. And someone has figured out the 196 00:15:49,000 --> 00:15:54,000 structure of photosystem I. It consists of 12 proteins, 197 00:15:54,000 --> 00:16:00,000 96 chlorophylls and about 30 other molecules. 198 00:16:00,000 --> 00:16:04,000 And what it really does is it functions as an antenna. 199 00:16:04,000 --> 00:16:08,000 Some of the other molecules can absorb it at wavelengths that are 200 00:16:08,000 --> 00:16:12,000 different from chlorophyll. And all the energy gets funneled 201 00:16:12,000 --> 00:16:16,000 into the chlorophyll and into this process. And you'll probably 202 00:16:16,000 --> 00:16:20,000 recognize by now that proteins here we're seeing alpha helices and beta 203 00:16:20,000 --> 00:16:24,000 sheets in here as part of this structure. So the first organisms 204 00:16:24,000 --> 00:16:28,000 that learned how to do this were organisms we now know 205 00:16:28,000 --> 00:16:32,000 as cyanobacteria. They're a kind of bacteria that has 206 00:16:32,000 --> 00:16:36,000 two membranes like E. coli and like the other ones that 207 00:16:36,000 --> 00:16:41,000 we've talked about. You're familiar with these. 208 00:16:41,000 --> 00:16:45,000 There's the green scum you see on ponds. Here's a close-up. 209 00:16:45,000 --> 00:16:50,000 Sometimes they grow as filaments, the cells in a chain. You notice 210 00:16:50,000 --> 00:16:54,000 they're green. They're making chlorophyll. 211 00:16:54,000 --> 00:16:59,000 And what happened in plants was that apparently something probably 212 00:16:59,000 --> 00:17:03,000 related to the present-day cyanobacteria got trapped inside 213 00:17:03,000 --> 00:17:08,000 some early progenitor of what we now know as plants and green algae. 214 00:17:08,000 --> 00:17:15,000 And this trapped bacterium became a chloroplast. And it had all the 215 00:17:15,000 --> 00:17:23,000 machinery necessary to carry out this noncyclic photophosphorylation. 216 00:17:23,000 --> 00:17:31,000 The structure of these things, there's an outer membrane. 217 00:17:31,000 --> 00:17:35,000 Just similar to what I told you for the mitochondria. 218 00:17:35,000 --> 00:17:40,000 There's an inner membrane. And what's special about the 219 00:17:40,000 --> 00:17:45,000 mitochondrion then, there's another membrane inside 220 00:17:45,000 --> 00:17:54,000 that's known as the thylocoid. 221 00:17:54,000 --> 00:18:00,000 And that's where all the chlorophyll is. And the reason the out and the 222 00:18:00,000 --> 00:18:07,000 in is a little bit confusing in here is this part, which is probably the 223 00:18:07,000 --> 00:18:14,000 cytoplasm of the old bacterium, is pumped from what's known as the 224 00:18:14,000 --> 00:18:21,000 stroma of a chloroplast which is equivalent to the cytoplasm of the 225 00:18:21,000 --> 00:18:27,000 original bacteria into the lumen. So the chlorophyll that's in this 226 00:18:27,000 --> 00:18:32,000 membrane absorbs the light, pumps protons into the lumen 227 00:18:32,000 --> 00:18:37,000 building up a proton gradient, and then they flow back out in the 228 00:18:37,000 --> 00:18:42,000 other direction and make ATP. Here's a picture of a chloroplast 229 00:18:42,000 --> 00:18:47,000 once again. It looks an awful lot like the bacterium still that got 230 00:18:47,000 --> 00:18:52,000 captured. All this stuff on the inside, those are the thylocoid 231 00:18:52,000 --> 00:18:57,000 membranes that carry out this specialized stuff. So 232 00:18:57,000 --> 00:19:01,000 there you have it. That's how cells, 233 00:19:01,000 --> 00:19:05,000 the major ways that life has figured out how to make energy. 234 00:19:05,000 --> 00:19:08,000 When Penny Chisholm starts to talk to you she'll talk to you about how 235 00:19:08,000 --> 00:19:12,000 organisms adapt to various niches, things that live in the bottom of 236 00:19:12,000 --> 00:19:15,000 the ocean, things that live in various places. 237 00:19:15,000 --> 00:19:19,000 They all have to make energy. Well, they all use some variation 238 00:19:19,000 --> 00:19:22,000 on these principles I've talked to you about. And she'll then show you 239 00:19:22,000 --> 00:19:26,000 how they're very cleaver at extracting energy out of all sorts 240 00:19:26,000 --> 00:19:30,000 of things by applying these principles in different ways. 241 00:19:30,000 --> 00:19:34,000 OK. So what we're going to start doing now is we're going to start 242 00:19:34,000 --> 00:19:39,000 talking about DNA. This is certainly a molecule that's 243 00:19:39,000 --> 00:19:43,000 fascinated me all my life. You should know from the first part 244 00:19:43,000 --> 00:19:48,000 that it's built up of units known as nucleotides that have a sugar. 245 00:19:48,000 --> 00:19:53,000 It's a ribose sugar that's missing one hydroxyl so it's a deoxyribose. 246 00:19:53,000 --> 00:19:57,000 The sugars are numbered 1, 2, 3, 4, 5. I showed you that. They'll be a 247 00:19:57,000 --> 00:20:02,000 phosphate. And then one of these nucleic acid 248 00:20:02,000 --> 00:20:06,000 bases, either a pyrimidine or a purine. And in DNA you find the 249 00:20:06,000 --> 00:20:11,000 pyrimidine bases are cytosine and thiamine. And in DNA the purine 250 00:20:11,000 --> 00:20:15,000 bases are adenine and guanine. And then these subunits are 251 00:20:15,000 --> 00:20:20,000 polymerized together. In essence, splitting out water to 252 00:20:20,000 --> 00:20:24,000 give you a polymer. And I didn't emphasize this too 253 00:20:24,000 --> 00:20:29,000 strong the first time I showed it to you. 254 00:20:29,000 --> 00:20:33,000 It's going to become a very big deal over the next few lectures as we 255 00:20:33,000 --> 00:20:37,000 begin to consider how nature had to figure out how to replicate DNA and 256 00:20:37,000 --> 00:20:42,000 all sorts of implications to go along with this, 257 00:20:42,000 --> 00:20:46,000 but there's a polarity to a strand of DNA. This is what's called the 5 258 00:20:46,000 --> 00:20:51,000 prime sugar. The primes indicate the numbers referring to the sugar, 259 00:20:51,000 --> 00:20:55,000 and the ones without primes are referring to numbers of atoms that 260 00:20:55,000 --> 00:21:00,000 make up part of the nucleic acid base. 261 00:21:00,000 --> 00:21:04,000 So if we're looking at a chain, this is a 5 prime carbon of the 262 00:21:04,000 --> 00:21:09,000 sugar, that's the 3 prime. And so what you can see, this bond 263 00:21:09,000 --> 00:21:14,000 which is really a phosphodiester bond, the phosphate group has formed 264 00:21:14,000 --> 00:21:19,000 an ester with this hydroxyl and with the hydroxyl that used to be here. 265 00:21:19,000 --> 00:21:24,000 So it's a phosphodiester bond and it's a 5 prime, 266 00:21:24,000 --> 00:21:29,000 3 prime bond. It joins the 5 prime carbon to the 3 prime 267 00:21:29,000 --> 00:21:33,000 carbon up here. So that means if you're looking at a 268 00:21:33,000 --> 00:21:37,000 chain of DNA, if you come down this way you're coming in the 5 to 3 269 00:21:37,000 --> 00:21:41,000 prime direction. If we come up the other way we're 270 00:21:41,000 --> 00:21:45,000 coming from the 3 prime end heading towards the 5 prime end. 271 00:21:45,000 --> 00:21:48,000 So you'll see me saying 5 prime, 3 prime. Now, as I told you, the 272 00:21:48,000 --> 00:21:52,000 principle force that holds the strands of the DNA together are 273 00:21:52,000 --> 00:21:56,000 hydrogen bonds, three of them between a G and a C 274 00:21:56,000 --> 00:22:00,000 and two of them between an A and a T. 275 00:22:00,000 --> 00:22:04,000 And then they are a pair of strands. And they're actually running in 276 00:22:04,000 --> 00:22:08,000 opposite polarities. This is something to contend with 277 00:22:08,000 --> 00:22:12,000 when we think about replication. 5 prime to 3 prime in one direction 278 00:22:12,000 --> 00:22:16,000 and 5 prime to 3 prime going in the opposite way here. 279 00:22:16,000 --> 00:22:21,000 And then, as you all know, it's called the double helix. 280 00:22:21,000 --> 00:22:25,000 So it's actually not flat like this in space. It's in a 3-dimensional 281 00:22:25,000 --> 00:22:29,000 twisted into a double helix and the base pairs are held together by 282 00:22:29,000 --> 00:22:33,000 hydrogen bonds between the bases on the opposite strands down the middle 283 00:22:33,000 --> 00:22:37,000 of the molecule. And I like this little movie I 284 00:22:37,000 --> 00:22:41,000 showed you because you can see it pretty well. The nitrogens are blue. 285 00:22:41,000 --> 00:22:44,000 It's easy to see the bases. And the hydrogen bonds are right in 286 00:22:44,000 --> 00:22:48,000 the middle. There's another force I didn't mention and it doesn't matter 287 00:22:48,000 --> 00:22:51,000 for this course, but when the bases sort of stack on 288 00:22:51,000 --> 00:22:55,000 top of each other there's actually a kind of extra stabilization that 289 00:22:55,000 --> 00:22:58,000 comes from that. It's a gorgeous molecule. 290 00:22:58,000 --> 00:23:02,000 You all know it encodes the genetic information. 291 00:23:02,000 --> 00:23:05,000 We're going to be talking about it a lot, but first thing, 292 00:23:05,000 --> 00:23:09,000 you know, I could just tell you it's the genetic information. 293 00:23:09,000 --> 00:23:13,000 But one of the really big discoveries in biology was that DNA 294 00:23:13,000 --> 00:23:16,000 is the genetic information. And a point I'm trying to help you 295 00:23:16,000 --> 00:23:20,000 learn here, you know, I'm trying to teach you more than 296 00:23:20,000 --> 00:23:24,000 just facts. And I hope some of you at least will catch that. 297 00:23:24,000 --> 00:23:28,000 I'm trying to show you how biology is done. 298 00:23:28,000 --> 00:23:31,000 As an experimental scientist you don't sit down usually and figure it 299 00:23:31,000 --> 00:23:34,000 out. Instead you start doing experiments and you get all kinds of 300 00:23:34,000 --> 00:23:38,000 unexpected discoveries. And, in general, as people work in 301 00:23:38,000 --> 00:23:41,000 these unexpected discoveries ultimately they come to these grand 302 00:23:41,000 --> 00:23:45,000 new insights that, you know, sometimes would have been 303 00:23:45,000 --> 00:23:48,000 very hard to forget. So the real question that people 304 00:23:48,000 --> 00:23:52,000 wondered for a long time, and we'll talk more about the 305 00:23:52,000 --> 00:23:55,000 history of genetics, but people knew we clearly had 306 00:23:55,000 --> 00:23:59,000 inheritable traits. You could see it in your kids. 307 00:23:59,000 --> 00:24:03,000 People had been breeding plants and all sorts of things. 308 00:24:03,000 --> 00:24:07,000 Breeding domestic animals. They sort of understood the 309 00:24:07,000 --> 00:24:11,000 principle of inheritance. When I tell you about Mendel we'll 310 00:24:11,000 --> 00:24:15,000 begin to see how his thinking led to the idea that the inheritance wasn't 311 00:24:15,000 --> 00:24:19,000 just sort of like a liquid where everything mixed together. 312 00:24:19,000 --> 00:24:23,000 It came in units or particles which we know of as genes. 313 00:24:23,000 --> 00:24:27,000 And so the idea that genes had been accepted certainly by the beginning 314 00:24:27,000 --> 00:24:31,000 of this century anyway, but nobody knew what they were made 315 00:24:31,000 --> 00:24:37,000 of. They were made of -- The major properties that they had 316 00:24:37,000 --> 00:24:44,000 was they clearly encoded information in some way. 317 00:24:44,000 --> 00:24:50,000 They must replicate because one cell 318 00:24:50,000 --> 00:24:54,000 could give two and on and on and on. So if you were going to pass it 319 00:24:54,000 --> 00:24:58,000 down in an inherited way they have to be replicated. 320 00:24:58,000 --> 00:25:01,000 And the third thing was that people knew somehow they could mutate or 321 00:25:01,000 --> 00:25:05,000 the information content that they encoded could be changed. 322 00:25:05,000 --> 00:25:09,000 Again, you could see that, that you'd get something, an altered 323 00:25:09,000 --> 00:25:13,000 characteristic, and then it would be propagated down 324 00:25:13,000 --> 00:25:16,000 through that line. That was the principle of breeding 325 00:25:16,000 --> 00:25:20,000 that people had done for ages. And so they understood that. There 326 00:25:20,000 --> 00:25:24,000 was one other key thing they knew. They knew that these genes were in 327 00:25:24,000 --> 00:25:28,000 the nucleus. And I'll tell you the full story of how even that insight 328 00:25:28,000 --> 00:25:32,000 was arrived at. But I'll just show you for the 329 00:25:32,000 --> 00:25:36,000 moment this little movie. This shows some chromosomes that 330 00:25:36,000 --> 00:25:40,000 are all bunched up and are just pulled apart at the time of the cell 331 00:25:40,000 --> 00:25:44,000 division. Those chromosomes, as we now know, are made of DNA. 332 00:25:44,000 --> 00:25:48,000 But in essence what people had seen through the microscope was these 333 00:25:48,000 --> 00:25:52,000 chromosomes or colored things that they could stain. 334 00:25:52,000 --> 00:25:56,000 They could sort of see something had doubled. And just before the 335 00:25:56,000 --> 00:26:01,000 cell divided the two sets separated and each cell got a new set. 336 00:26:01,000 --> 00:26:05,000 So that's about what people knew. They had those properties. They're 337 00:26:05,000 --> 00:26:10,000 in the nucleus. They knew about as much as you do. 338 00:26:10,000 --> 00:26:15,000 They knew the major classes of biomolecules in a cell. 339 00:26:15,000 --> 00:26:20,000 So what do you think you would need to do to show that DNA is the 340 00:26:20,000 --> 00:26:25,000 genetic material, encodes the genes? 341 00:26:25,000 --> 00:26:30,000 Find somebody near you. I'll give you a minute or so. 342 00:26:30,000 --> 00:26:32,000 I'd like to hear what kind of ideas you come up with. 343 00:26:32,000 --> 00:26:35,000 Then I'll tell you how it happened. But I want to hear. Why don't you 344 00:26:35,000 --> 00:26:37,000 think about it and just see if you can come up with a couple ideas for 345 00:26:37,000 --> 00:27:13,000 me, what you'd need to figure out. 346 00:27:13,000 --> 00:27:17,000 Well, let's just see what kind of ideas anybody got. 347 00:27:17,000 --> 00:27:21,000 If you wanted to make me believe that DNA is doing that, 348 00:27:21,000 --> 00:27:25,000 or I think it's a protein for the moment, that's what I think is most 349 00:27:25,000 --> 00:27:30,000 likely, but what you do think? Anybody got an idea? 350 00:27:30,000 --> 00:27:36,000 Mess up the DNA and see if we can mess up the cell. 351 00:27:36,000 --> 00:27:42,000 How are we going to do that? I can break a cell open and I guess 352 00:27:42,000 --> 00:27:48,000 I can purify DNA and I can analyze it. And it's got four bases in it 353 00:27:48,000 --> 00:27:54,000 and it's got sugars and phosphates. At that point nobody could sequence 354 00:27:54,000 --> 00:28:00,000 DNA. We didn't even know the structure. 355 00:28:00,000 --> 00:28:04,000 Yeah? Take it out of one cell and put it into another. 356 00:28:04,000 --> 00:28:08,000 And what would you expect to happen then? 357 00:28:08,000 --> 00:28:17,000 OK. That's a really nice idea. 358 00:28:17,000 --> 00:28:21,000 Somehow if you took the DNA and moved it from one cell to another 359 00:28:21,000 --> 00:28:26,000 that the characteristic of this cell would be somehow carried 360 00:28:26,000 --> 00:28:31,000 over. OK. That's in fact the way it happened 361 00:28:31,000 --> 00:28:35,000 but not as simply as that, as I'll tell you, but that's exactly 362 00:28:35,000 --> 00:28:39,000 the essence of it. One little problem. 363 00:28:39,000 --> 00:28:43,000 Maybe we'll see if anybody has a thought on this. 364 00:28:43,000 --> 00:28:47,000 If I purify DNA, I mean nothing's ever really pure, 365 00:28:47,000 --> 00:28:51,000 right? You get it out and there's always little bits of stuff. 366 00:28:51,000 --> 00:28:55,000 And someone can always argue, well, yeah, it's 99% DNA, but it's 367 00:28:55,000 --> 00:29:00,000 the other bits you cannot get rid of. Yeah? 368 00:29:00,000 --> 00:29:03,000 If you used radioactive material, how is that going to help us? 369 00:29:03,000 --> 00:29:13,000 It does contain nitrogen. 370 00:29:13,000 --> 00:29:25,000 Well, it gets a little complicated. 371 00:29:25,000 --> 00:29:31,000 Certainly nucleic acids have like phosphate in them, but so does RNA. 372 00:29:31,000 --> 00:29:36,000 That's going to be hard. Maybe if I had a mixture of things 373 00:29:36,000 --> 00:29:41,000 and I wanted to prove whether something was let's say DNA, 374 00:29:41,000 --> 00:29:46,000 a protein or something, you need some really specific way of saying I 375 00:29:46,000 --> 00:29:51,000 did something to the DNA and not to the protein or something like that. 376 00:29:51,000 --> 00:29:56,000 Have you heard anything in this course that is really specific? 377 00:29:56,000 --> 00:30:02,000 Enzymes. Do you think that'd give you an idea of how you might do it? 378 00:30:02,000 --> 00:30:06,000 If I've got a tube and it's mostly DNA and maybe a bit of protein and 379 00:30:06,000 --> 00:30:10,000 something, and let's say his idea is working, that we can take the DNA 380 00:30:10,000 --> 00:30:14,000 from this cell and put it over into the second cell and see the 381 00:30:14,000 --> 00:30:19,000 characteristic change, if I wanted to do something to show 382 00:30:19,000 --> 00:30:23,000 that it was the DNA in the tube that was responsible, 383 00:30:23,000 --> 00:30:27,000 could you use an enzyme? And what kind of enzyme would you 384 00:30:27,000 --> 00:30:32,000 want? Well, what characteristics would you like it to have? 385 00:30:32,000 --> 00:30:38,000 Nature's probably made it for you already. Something that synthesizes 386 00:30:38,000 --> 00:30:44,000 DNA? Something that breaks down DNA? Say I treated this tube with some 387 00:30:44,000 --> 00:30:50,000 kind of enzyme and then I wanted to see the outcome, 388 00:30:50,000 --> 00:30:56,000 what would we want, an enzyme that did what? 389 00:30:56,000 --> 00:31:00,000 Anybody else got an idea? You're asserting that it's the DNA 390 00:31:00,000 --> 00:31:04,000 in my prep, I like your idea, but I need to prove it so I need to 391 00:31:04,000 --> 00:31:07,000 do something to show that it's actually the DNA and not the other 392 00:31:07,000 --> 00:31:11,000 stuff. So if I had an enzyme that did what to DNA? 393 00:31:11,000 --> 00:31:14,000 If I broke it down, yeah. We could treat it. 394 00:31:14,000 --> 00:31:18,000 And if your idea is right, we treat the stuff with something 395 00:31:18,000 --> 00:31:21,000 that specifically breaks down DNA it won't get transferred. 396 00:31:21,000 --> 00:31:25,000 Does that make sense? OK. I mean that's a way you could go at 397 00:31:25,000 --> 00:31:28,000 a proof of this. And, in fact, that's what happened. 398 00:31:28,000 --> 00:31:32,000 But I'm going to quickly tell you how it actually happened. 399 00:31:32,000 --> 00:31:36,000 And again, you know, as I say, I'm trying to tell you a 400 00:31:36,000 --> 00:31:40,000 few things that are besides here are the facts that you need to know on 401 00:31:40,000 --> 00:31:44,000 exam. There's a bigger picture here and this is how research goes, 402 00:31:44,000 --> 00:31:48,000 and particularly in an experimental science such as biology. 403 00:31:48,000 --> 00:31:52,000 The important early work on this came from a guy who was known as 404 00:31:52,000 --> 00:31:56,000 Frederick Griffith. He was in London. He was a 405 00:31:56,000 --> 00:32:00,000 physician. He was working in the 1920s. 406 00:32:00,000 --> 00:32:11,000 And he was studying pneumonia. That's an infection of the lungs -- 407 00:32:11,000 --> 00:32:20,000 -- by bacteria. 408 00:32:20,000 --> 00:32:25,000 There's more than one kind of bacterium that will cause pneumonia, 409 00:32:25,000 --> 00:32:30,000 but one of the really important ones clinically was streptococcus -- 410 00:32:30,000 --> 00:32:38,000 -- pneumonia. So it was a bacterium. 411 00:32:38,000 --> 00:32:43,000 It was given that name. We all have bacterium on us. 412 00:32:43,000 --> 00:32:49,000 I think I told you we have about ten to the twelfth on our skin, 413 00:32:49,000 --> 00:32:54,000 for example. And if streptococcus is on your skin it's not a problem, 414 00:32:54,000 --> 00:32:59,000 but if it gets into your lungs it's a problem. And so to live with all 415 00:32:59,000 --> 00:33:05,000 these bacteria with us our bodies have defenses. 416 00:33:05,000 --> 00:33:08,000 So we have this immune system, we'll talk about more, and a bunch 417 00:33:08,000 --> 00:33:11,000 of defender cells. Things that you know as white blood 418 00:33:11,000 --> 00:33:14,000 cells are defenders. Let's just see here. 419 00:33:14,000 --> 00:33:18,000 I'm going to show you this little movie. This is one of your white 420 00:33:18,000 --> 00:33:21,000 blood cells, a special kind of white blood cell. That little thing it's 421 00:33:21,000 --> 00:33:24,000 chasing is a bacterium. These round things are red blood 422 00:33:24,000 --> 00:33:28,000 cells. I mean doesn't it look like a dog going after a mouse, 423 00:33:28,000 --> 00:33:32,000 or a cat going after something? It's chasing it. 424 00:33:32,000 --> 00:33:36,000 It can tell it's there. This is remarkable. And it's a 425 00:33:36,000 --> 00:33:41,000 little pixilated, but this is real. It's going to 426 00:33:41,000 --> 00:33:45,000 catch it right about there. And it eats it. I mean we have 427 00:33:45,000 --> 00:33:49,000 these cells inside us. That's why you don't die even 428 00:33:49,000 --> 00:33:54,000 though we live in a world that's surrounded by bacteria. 429 00:33:54,000 --> 00:33:58,000 OK, so we'll go on. So getting pneumonia in those days was 430 00:33:58,000 --> 00:34:03,000 a really bad thing. You get infected, 431 00:34:03,000 --> 00:34:07,000 you get this in your lungs, and then you have four to six days this. So that's the bacterium. 432 00:34:07,000 --> 00:34:12,000 of high fever, and then the patient would reach Well, it turns out that streptococcus is a bacteria like 433 00:34:12,000 --> 00:34:17,000 what's termed as a ìcrisisî. And one of two things would happen. 434 00:34:17,000 --> 00:34:21,000 They'd either live or they'd die. And that was it. 435 00:34:21,000 --> 00:34:26,000 I mean this was no fun if somebody you knew had it because you didn't 436 00:34:26,000 --> 00:34:27,000 know the outcome. And the outcome wasn't necessarily 437 00:34:27,000 --> 00:34:23,000 very good. Now you call up the doctor and they pump you full of 438 00:34:23,000 --> 00:34:19,000 antibiotics, but antibiotics hadn't been discovered yet. 439 00:34:19,000 --> 00:34:15,000 So this was pretty serious business, and people were trying to understand 440 00:34:15,000 --> 00:34:11,000 what was happening. But what was going on during these 441 00:34:11,000 --> 00:34:07,000 four to six days that then led to one of these two outcomes? 442 00:35:07,000 --> 00:35:11,000 And it has around it something known as a capsule. And what that capsule 443 00:35:11,000 --> 00:35:15,000 is polysaccharide. Remember back to the second lecture 444 00:35:15,000 --> 00:35:19,000 when I was confusing you all by showing you how sugars could hook 445 00:35:19,000 --> 00:35:23,000 together in all a manner of different ways? 446 00:35:23,000 --> 00:35:27,000 Well, that's what polysaccharides are. You just hook a bunch 447 00:35:27,000 --> 00:35:31,000 of sugars together. And for this course you don't have 448 00:35:31,000 --> 00:35:34,000 to remember the linkages in particular. You just have to 449 00:35:34,000 --> 00:35:37,000 understand that there are different kinds of linkages, 450 00:35:37,000 --> 00:35:40,000 and every time you hook at it in a different way you get a different 451 00:35:40,000 --> 00:35:43,000 kind of polysaccharide out of it. But anyway, the bacterium make this 452 00:35:43,000 --> 00:35:46,000 capsule of polysaccharide. And it's full of hydroxyl groups 453 00:35:46,000 --> 00:35:49,000 from all those sugars so it attracts a lot of water around it. 454 00:35:49,000 --> 00:35:52,000 And what it does is it causes a problem for those defender cells 455 00:35:52,000 --> 00:35:55,000 that we just saw. Those would be, for example, 456 00:35:55,000 --> 00:35:59,000 a macrophagic kind of white blood cell. 457 00:35:59,000 --> 00:36:02,000 And it cannot eat something that's got the capsule. 458 00:36:02,000 --> 00:36:06,000 Now, here's a picture of one of these capsules on one of these kinds 459 00:36:06,000 --> 00:36:10,000 of bacteria. You can sort of see it out here. It's polysaccharide. 460 00:36:10,000 --> 00:36:14,000 That's the main part of the bacterium. And here's another 461 00:36:14,000 --> 00:36:18,000 pixilated thing of one of these white blood cells eating a bacterium 462 00:36:18,000 --> 00:36:22,000 that doesn't have a capsule. But watch what happens if the 463 00:36:22,000 --> 00:36:26,000 bacterium has a capsule. It cannot get a hold of it. 464 00:36:26,000 --> 00:36:30,000 It just cannot quite grab hold of it. So what happened during those 465 00:36:30,000 --> 00:36:34,000 days, though, was this capsule which is a foreign entity to your body 466 00:36:34,000 --> 00:36:38,000 gets recognized in your immune system. 467 00:36:38,000 --> 00:36:42,000 And your immune system made antibodies that could recognize that. 468 00:36:42,000 --> 00:36:47,000 We'll talk about what these are, but all you need to know for the 469 00:36:47,000 --> 00:36:52,000 moment is that they're proteins and they can be tuned to recognize some 470 00:36:52,000 --> 00:36:57,000 chemical entity with a very, very high degree of specificity. 471 00:36:57,000 --> 00:37:02,000 So what the body was doing during this thing was trying to make 472 00:37:02,000 --> 00:37:07,000 antibodies that would help it recognize this capsule. 473 00:37:07,000 --> 00:37:10,000 And then it decorates the capsule with these things. 474 00:37:10,000 --> 00:37:14,000 And once it puts antibodies stuck all over the surface now it can get 475 00:37:14,000 --> 00:37:18,000 a hold of it. And, again, a fact you don't have to know. 476 00:37:18,000 --> 00:37:22,000 This whole process is called opsonization. The reason they use 477 00:37:22,000 --> 00:37:26,000 the word opsin because opsin is the Greek word for seasoning. 478 00:37:26,000 --> 00:37:30,000 And it was as if these white blood cells liked to have their bacteria 479 00:37:30,000 --> 00:37:34,000 seasoned correctly before they can eat them. 480 00:37:34,000 --> 00:37:37,000 And what's really going on is that they're decorating them with 481 00:37:37,000 --> 00:37:40,000 antibodies. So what was going on after a person got sick, 482 00:37:40,000 --> 00:37:43,000 it was a race between their immune system trying to make antibodies 483 00:37:43,000 --> 00:37:47,000 which would let their immune system suppress the infection and the 484 00:37:47,000 --> 00:37:50,000 bacterium which is replicating unchecked for the first few days. 485 00:37:50,000 --> 00:37:53,000 And that's why it was such a scary business, because you didn't know 486 00:37:53,000 --> 00:37:57,000 what the outcome was and things could tip it one way 487 00:37:57,000 --> 00:38:03,000 or the other. Well, this did suggest a kind of 488 00:38:03,000 --> 00:38:13,000 therapy. The kind of therapy would be to isolate a capsule 489 00:38:13,000 --> 00:38:25,000 to inject a horse. 490 00:38:25,000 --> 00:38:28,000 Get the antibodies from the horse. Why a horse? A horse is huge, 491 00:38:28,000 --> 00:38:32,000 right? It makes a lot of antibodies. 492 00:38:32,000 --> 00:38:39,000 A lot better than injecting a mouse if you want to get antibodies. 493 00:38:39,000 --> 00:38:46,000 So get antibodies and then inject the patient. It's a good idea in 494 00:38:46,000 --> 00:38:53,000 principle. So you're sort of short-circuiting this whole process. 495 00:38:53,000 --> 00:39:00,000 The problem was there were more than 20 kinds of capsules. 496 00:39:00,000 --> 00:39:07,000 And so what people had to do was they had to isolate 497 00:39:07,000 --> 00:39:17,000 the bacterium -- 498 00:39:17,000 --> 00:39:30,000 -- from the patient, determine the type of capsule. 499 00:39:30,000 --> 00:39:35,000 Let's say it's sort of from capsule 1 up to capsule type 20, 500 00:39:35,000 --> 00:39:40,000 which one it was, and then inject the correct antibody. 501 00:39:40,000 --> 00:39:46,000 So this was nerve-racking because it took a while for the bacteria to 502 00:39:46,000 --> 00:39:51,000 grow so it was a pretty tight time window. And if you saw the patient 503 00:39:51,000 --> 00:39:57,000 right away that's good, but if they were partway down the 504 00:39:57,000 --> 00:40:02,000 infection not so good. So the one other thing to do this, 505 00:40:02,000 --> 00:40:06,000 they didn't bother all the way to isolate the capsule. 506 00:40:06,000 --> 00:40:10,000 What they would usually do is use heat-killed bacteria and then you'd 507 00:40:10,000 --> 00:40:14,000 have the capsule and everything. The bacterium is dead, it cannot do 508 00:40:14,000 --> 00:40:19,000 anything, and they'd inject the horse with that. 509 00:40:19,000 --> 00:40:23,000 And that would get you the antibodies with the capsule. 510 00:40:23,000 --> 00:40:27,000 So what Griffith was doing was he was fiddling around 511 00:40:27,000 --> 00:40:32,000 with this system. And there was one other discovery 512 00:40:32,000 --> 00:40:36,000 that he made. Perhaps it wouldn't surprise you that since the bacteria 513 00:40:36,000 --> 00:40:40,000 surrounded by a molecule absorbs water that the capsules would look 514 00:40:40,000 --> 00:40:45,000 sort of glistening. They absorbed a lot of water. 515 00:40:45,000 --> 00:40:49,000 You can see how they look here. So what they discovered was if they 516 00:40:49,000 --> 00:40:54,000 have a capsule you get what are called smooth colonies. 517 00:40:54,000 --> 00:40:58,000 And the word colony in this thing just refers to it started out as one 518 00:40:58,000 --> 00:41:03,000 bacterium and it kept dividing and dividing and dividing. 519 00:41:03,000 --> 00:41:07,000 And maybe there are ten to the eighth or ten to the ninth bacteria 520 00:41:07,000 --> 00:41:11,000 in that little colony. But you can see it. They've all 521 00:41:11,000 --> 00:41:15,000 got capsules on the outside so it attracts a lot of water and it looks 522 00:41:15,000 --> 00:41:19,000 wet. And those are what you saw. But what they found is if you 523 00:41:19,000 --> 00:41:23,000 waited or grew the cultures up that you would see some things that 524 00:41:23,000 --> 00:41:27,000 looked dry or they called them rough. And these turned out to be bacteria 525 00:41:27,000 --> 00:41:33,000 that lacked a capsule. And so if you might start with a 526 00:41:33,000 --> 00:41:40,000 smooth strain S here and then isolate from it a rough strain it 527 00:41:40,000 --> 00:41:48,000 might designate it in that kind of way. So this is the sort of thing 528 00:41:48,000 --> 00:41:55,000 that Griffith was fooling around with. So he started with doing this 529 00:41:55,000 --> 00:42:02,000 kind of experiment. He took a smooth strain making a 530 00:42:02,000 --> 00:42:08,000 capsule type two, OK? So he was injecting a mouse 531 00:42:08,000 --> 00:42:14,000 with this. And what happened was the mouse was dead. 532 00:42:14,000 --> 00:42:20,000 This was a virulent form of the bacterium. So if he took the rough 533 00:42:20,000 --> 00:42:26,000 mutant, injected the mouse, the mouse is alive and you saw why. 534 00:42:26,000 --> 00:42:32,000 If it doesn't have a capsule, 535 00:42:32,000 --> 00:42:36,000 the mouse has defender cells and white blood cells could eat it. 536 00:42:36,000 --> 00:42:41,000 Then he had heat-killed S3. So this was a strain of streptococcus 537 00:42:41,000 --> 00:42:46,000 that had a different capsule, a type 3 capsule, but it was 538 00:42:46,000 --> 00:42:50,000 heat-killed. Why was he working with heat-killed stuff? 539 00:42:50,000 --> 00:42:55,000 Because that's what you injected the horses with to get it. 540 00:42:55,000 --> 00:43:00,000 So what do you think would happen here? 541 00:43:00,000 --> 00:43:06,000 Since the bacteria are dead, probably not a big surprise the 542 00:43:06,000 --> 00:43:13,000 mouse is alive. Now, I don't know whether he did 543 00:43:13,000 --> 00:43:19,000 this on purpose or he did it as a control, but what he did was he 544 00:43:19,000 --> 00:43:26,000 injected at the same time then R2 plus heat-killed S3. 545 00:43:26,000 --> 00:43:32,000 So he's got two things that don't do anything, he injects a mouse, 546 00:43:32,000 --> 00:43:38,000 and uh-oh, the mouse dies. That is a weird result. 547 00:43:38,000 --> 00:43:42,000 That is actually also, though, the first really key step to 548 00:43:42,000 --> 00:43:47,000 understanding that DNA is a genetic material. It doesn't look like it 549 00:43:47,000 --> 00:43:52,000 at this point probably, but it was. This is how we learned 550 00:43:52,000 --> 00:43:56,000 this really enormous fact from these experiments. He wasn't trying 551 00:43:56,000 --> 00:44:00,000 to figure it out. He was trying to work out something 552 00:44:00,000 --> 00:44:04,000 else, as you can see, but it was a bizarre finding. 553 00:44:04,000 --> 00:44:08,000 So what would you think? I've put in something that used to have a R2 554 00:44:08,000 --> 00:44:12,000 capsule. So did it get rejuvenated somehow by this heat-killed thing or, 555 00:44:12,000 --> 00:44:16,000 as you'd suggested, did some characteristic get 556 00:44:16,000 --> 00:44:20,000 transferred from here or whatever? So he isolated the bacteria out of 557 00:44:20,000 --> 00:44:24,000 this, and what he found now was he had a live bacterium 558 00:44:24,000 --> 00:44:29,000 that was making S3. So something had been transferred 559 00:44:29,000 --> 00:44:35,000 from this set of dead bacteria into bacteria that were alive, 560 00:44:35,000 --> 00:44:41,000 and the characteristic had been passed from the dead bacterium to 561 00:44:41,000 --> 00:44:47,000 the new bacterium, the other bacterium. 562 00:44:47,000 --> 00:44:53,000 So this is about what Griffith did, but this problem was picked up by a 563 00:44:53,000 --> 00:44:59,000 scientist at Rockefeller, Oswald Avery who worked as part of a 564 00:44:59,000 --> 00:45:04,000 team. And he took this finding and started 565 00:45:04,000 --> 00:45:10,000 to work on it and tried to figure out, because he saw in this result a 566 00:45:10,000 --> 00:45:16,000 way of finding out what was the genetic material because somehow 567 00:45:16,000 --> 00:45:21,000 what was in that heat-killed S3 was the stuff that would transfer 568 00:45:21,000 --> 00:45:27,000 genetic information into another bacterium. So he made one 569 00:45:27,000 --> 00:45:31,000 really big discovery. And that was you didn't need the 570 00:45:31,000 --> 00:45:35,000 mouse at all. All that was happening was the mouse, 571 00:45:35,000 --> 00:45:38,000 by dying, was in essence selecting for smooth bacteria. 572 00:45:38,000 --> 00:45:42,000 So he could simplify things by just taking a rough bacteria, 573 00:45:42,000 --> 00:45:45,000 taking the heat-killed extract, putting it in, and now he'd just 574 00:45:45,000 --> 00:45:49,000 look for smooth colonies. Didn't need any mice at all. 575 00:45:49,000 --> 00:45:52,000 So he was able to see the characteristic of the capsule being 576 00:45:52,000 --> 00:45:56,000 transferred from some kind of heat-killed mess of things into a 577 00:45:56,000 --> 00:46:00,000 rough bacterium and changing it into a live bacterium. 578 00:46:00,000 --> 00:46:04,000 So he started fractionating, and he did exactly the kind of this was called transformation. 579 00:46:04,000 --> 00:46:08,000 approach that you suggested. And he purified and he purified and matters in the thing was taking DNA and putting it in. 580 00:46:08,000 --> 00:46:12,000 he purified using as his assay this ability to pass on this smooth 581 00:46:12,000 --> 00:46:16,000 characteristic. And what he ended up with was 582 00:46:16,000 --> 00:46:20,000 virtually pure DNA but, as I said, you know, always never 583 00:46:20,000 --> 00:46:24,000 quite pure. And somebody can always argue, well, you've got a little bit 584 00:46:24,000 --> 00:46:28,000 of something else in there. So he did a really key experiment 585 00:46:28,000 --> 00:46:33,000 and he treated with DNAs, your experiment. 586 00:46:33,000 --> 00:46:00,000 And it lost the transforming activity. So this process of doing plasma and you stick it into E. coli so you can grow it up, that 587 00:45:50,000 --> 00:46:04,000 Initially it described that phenomenon. Now that we know what 588 00:46:18,000 --> 00:46:33,000 So if you do a UROP somewhere here, you clone a piece of DNA into a 589 00:46:47,000 --> 00:47:01,000 process of taking the naked DNA and putting it inside the bacteria, 590 00:47:01,000 --> 00:47:11,000 you'll call it transformation. Now, in fact, this result wasn't 591 00:47:11,000 --> 00:47:16,000 accepted right away. This was published in 1944. 592 00:47:16,000 --> 00:47:20,000 And the general realization that DNA was the genetic material really 593 00:47:20,000 --> 00:47:25,000 didn't come until the ë50s. Yet this result proved it, if you 594 00:47:25,000 --> 00:47:30,000 will, but part of the problem was the world wasn't yet ready to accept 595 00:47:30,000 --> 00:47:34,000 that DNA was a genetic material. And maybe you can see the problem. 596 00:47:34,000 --> 00:47:37,000 It looked like a monotonous molecule. It only had four things 597 00:47:37,000 --> 00:47:40,000 that were different in it. And if you isolated they were all 598 00:47:40,000 --> 00:47:43,000 often kind of there and about the same amount. People thought it was 599 00:47:43,000 --> 00:47:46,000 just an analyst GATC. It didn't sound like anything had 600 00:47:46,000 --> 00:47:49,000 encoded information. Proteins looked really attractive. 601 00:47:49,000 --> 00:47:52,000 Twenty different amino acids that all had different characteristics, 602 00:47:52,000 --> 00:47:55,000 so that was a great place for storing information. 603 00:47:55,000 --> 00:47:58,000 So the world wasn't quite ready to accept it, even though the 604 00:47:58,000 --> 00:48:02,000 experimental evidence was there. And so the result came later. 605 00:48:02,000 --> 00:48:07,000 Now, the last thing I just want to show you, because there's a kind of 606 00:48:07,000 --> 00:48:12,000 direct link from that Avery experiment to you guys because a 607 00:48:12,000 --> 00:48:17,000 year or two ago it was the 50th anniversary of the discovery of DNA. 608 00:48:17,000 --> 00:48:22,000 And Avery worked with a team of two other people called MacLeod and 609 00:48:22,000 --> 00:48:27,000 McCarty. This was at the 50th anniversary, the meeting down at 610 00:48:27,000 --> 00:48:32,000 Cold Spring Harbor celebrating it. McCarty was the only member of the 611 00:48:32,000 --> 00:48:36,000 team alive. There he was. I asked him to autograph my program. 612 00:48:36,000 --> 00:48:41,000 There was his signature. He just died a little while ago, 613 00:48:41,000 --> 00:48:45,000 and so there's no living connection to that anymore, 614 00:48:45,000 --> 00:48:50,000 but I have a picture to show you guys that takes you back from that 615 00:48:50,000 --> 00:48:54,000 experiment to his signature right there. OK? So I'll tell you some 616 00:48:54,000 --> 00:48:57,000 more stuff next lecture. Have a good weekend.