1 00:00:05 --> 00:00:09 Let's dive in today and look at 2 00:00:09 --> 00:00:13 how geneticists use genetics. I've told you up until now about 3 00:00:13 --> 00:00:17 some of the history of genetics and how it gave rise to our 4 00:00:17 --> 00:00:21 understandings about genetic transmission in traits, about 5 00:00:21 --> 00:00:24 genetic mapping, linkage analysis, 6 00:00:24 --> 00:00:27 how all this helped confirm the Chromosomes Theory. 7 00:00:27 --> 00:00:31 And we wove in a number of concepts about how scientific theories are 8 00:00:31 --> 00:00:35 developed and data is interpreted and intuitions are made, 9 00:00:35 --> 00:00:39 and then how they're actually proven, what sort of evidence it takes to 10 00:00:39 --> 00:00:43 actually achieve conscientious around theories. 11 00:00:43 --> 00:00:47 And that often takes sometimes years, many times decades before 12 00:00:47 --> 00:00:51 full contentious is achieved around things. Today I want to turn a 13 00:00:51 --> 00:00:55 little bit to the experimental uses of genetics in a more day-to-day 14 00:00:55 --> 00:01:00 fashion. And you will recall this coat of arms that I put up here. 15 00:01:00 --> 00:01:05 Function. Gene. Protein. Biochemistry. 16 00:01:05 --> 00:01:10 Genetics. And I told you about how these were two different ways to 17 00:01:10 --> 00:01:15 study biological function. Today I want to talk a little bit 18 00:01:15 --> 00:01:20 about how we use genetics to study biological function. 19 00:01:20 --> 00:01:25 And, in particular, I'm going to pick some examples of how we use 20 00:01:25 --> 00:01:30 genetics to study biological function that have to do with the 21 00:01:30 --> 00:01:35 biological functions of biochemistry. 22 00:01:35 --> 00:01:40 So already we're beginning to look ahead to this connection between 23 00:01:40 --> 00:01:46 gene and protein, which molecular biology will 24 00:01:46 --> 00:01:51 establish for us. So, suppose you want to do genetics. 25 00:01:51 --> 00:01:57 You've got to study some organism. We talked already about Mendel's 26 00:01:57 --> 00:02:01 choice of organism, the pea. We talked about some of its 27 00:02:01 --> 00:02:05 advantages and disadvantages. Advantages you could get pure 28 00:02:05 --> 00:02:08 breeding strains in the market, you could, when you're done with the 29 00:02:08 --> 00:02:12 experiments, feed it to the other monks. There were a lot of things 30 00:02:12 --> 00:02:16 like that, that were advantageous about the pea, 31 00:02:16 --> 00:02:19 but it had problems of generation time. You would only get, 32 00:02:19 --> 00:02:23 certainly in Europe, a generation or so a year. In Northern Europe maybe 33 00:02:23 --> 00:02:27 you could squeeze a second generation in not so good. 34 00:02:27 --> 00:02:30 Fruit flies, a very attractive system in many respects because you 35 00:02:30 --> 00:02:33 could grow many, much larger numbers. 36 00:02:33 --> 00:02:36 The generation time is on the order of two weeks or so to go from a 37 00:02:36 --> 00:02:40 fertilized fly embryo, a fly egg developing into a fly, 38 00:02:40 --> 00:02:43 developing into a mature adult, able itself to have offspring. 39 00:02:43 --> 00:02:46 So, very attractive. There are other systems that people studied. 40 00:02:46 --> 00:02:50 And, of course, one of the reasons they study this system is because 41 00:02:50 --> 00:02:53 it's interesting, I'm sorry, because it's tractable. 42 00:02:53 --> 00:02:56 And the other reason is because it's interesting. 43 00:02:56 --> 00:03:00 So, tractability is very important to a geneticist, right? 44 00:03:00 --> 00:03:03 The number of whale geneticists is few, for the most part. 45 00:03:03 --> 00:03:07 But we also want to choose our system because of what it will tell 46 00:03:07 --> 00:03:11 us about the system we want to study. Like if you want to study 47 00:03:11 --> 00:03:15 distinctive things about the immune system, you might want to study them 48 00:03:15 --> 00:03:18 in mice, or if you could even study them in people, 49 00:03:18 --> 00:03:22 although you can't set up crosses in people. We'll come to that on 50 00:03:22 --> 00:03:26 Monday. If you wanted to study things about basic aspects of 51 00:03:26 --> 00:03:30 development, you might study them in fruit flies. 52 00:03:30 --> 00:03:34 And if you wanted to study basic biochemistry, the place to study 53 00:03:34 --> 00:03:38 basic biochemistry might best be done in single-celled organisms, 54 00:03:38 --> 00:03:42 which also have to carry out biochemical pathways like glycolysis 55 00:03:42 --> 00:03:46 and synthesis of amino acids and things like that. 56 00:03:46 --> 00:03:50 They're going to be, by far, the most tractable systems. 57 00:03:50 --> 00:03:54 And so, people are particularly fond for doing things like studying 58 00:03:54 --> 00:03:58 basic biochemistry and many other aspects of basic molecular biology 59 00:03:58 --> 00:04:02 to studying the organism yeast. Yeast is a friend of human beings. 60 00:04:02 --> 00:04:06 Certainly, yeast has been an intensely studied organism because 61 00:04:06 --> 00:04:11 of its practical benefits in the making of bread, 62 00:04:11 --> 00:04:16 in the making of beer. So, fermentation processes, 63 00:04:16 --> 00:04:20 dough rising and all that. But yeast also is a tremendously 64 00:04:20 --> 00:04:25 important organism for the geneticist. It is an extremely 65 00:04:25 --> 00:04:30 elegant experimental system. Yeast is a fungus. 66 00:04:30 --> 00:04:35 It is a single-celled eukaryote. That is true nucleus. It's got 67 00:04:35 --> 00:04:40 chromosomes that pair up. It's cells, through a first order 68 00:04:40 --> 00:04:45 approximation, that are an awful lot like your 69 00:04:45 --> 00:04:51 cells in terms of having all of the basic important eukaryotic 70 00:04:51 --> 00:04:56 organelles in the nucleus, mitochondria, other things like that. 71 00:04:56 --> 00:05:01 So, yeast is a great model for many 72 00:05:01 --> 00:05:06 purposes. And we're not going to talk much about the cell biology of 73 00:05:06 --> 00:05:10 yeast, but I do want to talk about the husbandry of yeast, 74 00:05:10 --> 00:05:15 how it is that you grow yeast. So, the way a geneticists grows 75 00:05:15 --> 00:05:20 yeast is take growth medium that has lots of rich nutrients. 76 00:05:20 --> 00:05:25 You could take a broth with lots of amino acids and all sorts of stuff, 77 00:05:25 --> 00:05:30 you know, a little bit of salt, lots of water of course. 78 00:05:30 --> 00:05:35 And if you take a single yeast cell and it's got lots and lots of rich 79 00:05:35 --> 00:05:40 nutrients in this broth here, you put your yeast cell into the 80 00:05:40 --> 00:05:45 broth, so I will do that. Here's my flask, here's my little 81 00:05:45 --> 00:05:50 rod which has a yeast cell or a couple of yeast cells on the end of 82 00:05:50 --> 00:05:55 it. I put it in there and I grow it at an appropriate temperature. 83 00:05:55 --> 00:06:00 Let's say 30 degrees, for example, would be a nice temperature. 84 00:06:00 --> 00:06:05 I could do that if I wanted to. Then a C obviously. I grow it up 85 00:06:05 --> 00:06:10 and I get a culture of yeast in there. And I can tell because this 86 00:06:10 --> 00:06:15 nice clear broth is now all cloudy with yeast that's grown up in it. 87 00:06:15 --> 00:06:20 Now I want to study these guys, so what I do is I pour them out onto a 88 00:06:20 --> 00:06:25 Petri plate. The Petri plate has on it a medium, a solid medium, 89 00:06:25 --> 00:06:30 an agar medium that again has nutrients. 90 00:06:30 --> 00:06:34 And if I pour this out, and I pour out a lot of it, 91 00:06:34 --> 00:06:39 what will happen? Well, there will be yeast all over the place and it 92 00:06:39 --> 00:06:43 will be very smootsie. There will be like yeast cells 93 00:06:43 --> 00:06:48 everywhere and it's not very organized. So, 94 00:06:48 --> 00:06:53 what I want to do is I want to take that and I want to dilute it. 95 00:06:53 --> 00:06:57 I want to take only a little bit of the broth and put a little bit of 96 00:06:57 --> 00:07:02 the broth on my plate. Maybe I'll have diluted it first. 97 00:07:02 --> 00:07:08 And then I want to spread it around with a little spreader, 98 00:07:08 --> 00:07:13 here's a little glass spreader maybe or something, and push it back and 99 00:07:13 --> 00:07:19 forth, so that really there are just individual single cells scattered 100 00:07:19 --> 00:07:24 randomly, scattered around. And so, then this cell begins to 101 00:07:24 --> 00:07:30 grow and divide and divide and divide and I get a colony. 102 00:07:30 --> 00:07:34 A little hill of cells all of which descend from a single cell that was 103 00:07:34 --> 00:07:39 put into that position. And the reason I know that they all 104 00:07:39 --> 00:07:43 descend from a single cell is because most of this plate does not 105 00:07:43 --> 00:07:48 have cells on it. Most of the plate is sparse. 106 00:07:48 --> 00:07:53 I've just got cells, cells, cells, cells scattered about. And because 107 00:07:53 --> 00:07:57 of that I know that these had of been individual events. 108 00:07:57 --> 00:08:03 These things are called colonies. Now, when yeast grows and divides 109 00:08:03 --> 00:08:10 like that, let's take a moment and talk about its life cycle. 110 00:08:10 --> 00:08:17 We'll introduce its life cycle here. Yeast proper eukaryote, 111 00:08:17 --> 00:08:24 so it has a diploid stage. It grows as a diploid. And it can 112 00:08:24 --> 00:08:31 undergo mitosis in which all of the chromosomes line up, as 113 00:08:31 --> 00:08:38 we talked about. They've already pre-replicated so 114 00:08:38 --> 00:08:44 that they'll be ready to divide up and give one to each daughter cell, 115 00:08:44 --> 00:08:50 and there you go. N for yeast is 16. Yeast happens to have 16 pairs of 116 00:08:50 --> 00:08:56 chromosomes. Peas had seven. Humans have 23 pairs. Every 117 00:08:56 --> 00:09:02 organism has its own yeast of 16. Now, what we do is we undergo 118 00:09:02 --> 00:09:07 meiosis to make haploid cells, sperm or eggs in the human 119 00:09:07 --> 00:09:12 population. Yeast also undergoes meiosis to make spores. 120 00:09:12 --> 00:09:17 It sporulates and it produces spores. And it turns out these 121 00:09:17 --> 00:09:22 spores, of course as you would expect, have N chromosomes. 122 00:09:22 --> 00:09:27 They undergo meiosis just as we drew it on the board. 123 00:09:27 --> 00:09:32 And these can come in two flavors. They happen to come not in male and 124 00:09:32 --> 00:09:37 females, but A and alpha, there you go. A and alpha cells can 125 00:09:37 --> 00:09:42 mate together to produce, again, a diploid. They fertilize 126 00:09:42 --> 00:09:47 and can produce a diploid. They fuse to do that. And you now 127 00:09:47 --> 00:09:52 get back to a diploid from your haploid. So, this looks just 128 00:09:52 --> 00:09:57 identical to the human genetic cycle here, but there is one difference. 129 00:09:57 --> 00:10:02 What's the difference? Sorry? Time. Yes, 130 00:10:02 --> 00:10:07 it's true. Yeast can divide much more rapidly. Yeast can have 131 00:10:07 --> 00:10:12 offspring extremely rapidly over a course of a day or so. 132 00:10:12 --> 00:10:17 And humans take somewhat longer than that. They, 133 00:10:17 --> 00:10:23 for example, have to wait until they get out of college to be able to 134 00:10:23 --> 00:10:28 reproduce mostly. What else? There's one other 135 00:10:28 --> 00:10:33 important thing. It turns out that yeast can also 136 00:10:33 --> 00:10:38 undergo mitosis as a haploid. In other words, 137 00:10:38 --> 00:10:43 the haploid cells of yeast, when it makes individual haploids, 138 00:10:43 --> 00:10:49 they can continue to grow indefinitely. By contrast, 139 00:10:49 --> 00:10:54 your gametes cannot. You do not have an independent human stage in 140 00:10:54 --> 00:11:00 which you are haploid, or your gametes are haploid. 141 00:11:00 --> 00:11:03 Whereas, yeast can hang out as a haploid for a very long time until 142 00:11:03 --> 00:11:06 it decides it wants to mate. This is very convenient for 143 00:11:06 --> 00:11:09 geneticists. Geneticists like this because it means we can grow the 144 00:11:09 --> 00:11:12 thing as a diploid, we can grow the thing as a haploid. 145 00:11:12 --> 00:11:15 When we want to mate them, we can mate them together, 146 00:11:15 --> 00:11:18 but we can also study them alone. And, you could imagine, this is 147 00:11:18 --> 00:11:21 going to be really good for studying recessive traits, 148 00:11:21 --> 00:11:24 right? So, that's one of the reasons why geneticists are fond of 149 00:11:24 --> 00:11:27 yeast. There are many reasons geneticists are fond of yeast. 150 00:11:27 --> 00:11:30 Just growing yeast, it smells very nice in the lab. 151 00:11:30 --> 00:11:40 For example, try growing E. coli by comparison. So, now, 152 00:11:40 --> 00:11:50 it turns out that yeast is very happy if you grow it on rich medium. 153 00:11:50 --> 00:12:00 But yeast can grow on minimal media with very few macro molecules. 154 00:12:00 --> 00:12:08 It needs a carbon source which is some sugar that it can ferment. 155 00:12:08 --> 00:12:17 It needs a nitrogen. It needs some simple source of nitrogen. 156 00:12:17 --> 00:12:25 It needs some simple source of nitrogen. It needs a source of 157 00:12:25 --> 00:12:34 phosphorus. It needs some other trace salts and things like that. 158 00:12:34 --> 00:12:38 And obviously it needs some water. That's it. If you think about 159 00:12:38 --> 00:12:43 what's in a yeast cell, like it's got phospholipid bilayers. 160 00:12:43 --> 00:12:48 But you're not giving it any phospholipids. 161 00:12:48 --> 00:12:52 Why is it able to grow? It makes them. What about proteins? 162 00:12:52 --> 00:12:57 They're made up of 20 amino acids. You're not giving it any amino 163 00:12:57 --> 00:13:01 acids. Why? It makes them. Yeast is extraordinarily 164 00:13:01 --> 00:13:05 self-reliant. You, by contrast, are not as self-reliant. 165 00:13:05 --> 00:13:08 There are a number of amino acids which, if I don't give you, 166 00:13:08 --> 00:13:12 you can't live because you don't actually have the ability to make 167 00:13:12 --> 00:13:15 those amino acids. But yeast is able to make the vast 168 00:13:15 --> 00:13:19 majority of things. Basically, you almost just needed 169 00:13:19 --> 00:13:22 to give it the elements. As for carbon sources and things 170 00:13:22 --> 00:13:26 like that, it's very happy with a wide variety of fermentable sugars. 171 00:13:26 --> 00:13:30 You can give it glucose. You can give it sucrose. 172 00:13:30 --> 00:13:34 You can give it galactose. You can give it fructose and it 173 00:13:34 --> 00:13:38 will deal. So, yeast is very well set up 174 00:13:38 --> 00:13:42 metabolically. So, it's got all of these pathways 175 00:13:42 --> 00:13:46 of the sort Bob has talked about for being able to breakdown the things 176 00:13:46 --> 00:13:50 you give it and being able to synthesize up the things it needs. 177 00:13:50 --> 00:13:54 Now, yeast, of course, is not stupid. Because if you give it 178 00:13:54 --> 00:13:58 amino acids it will use it. If you give it all sorts of other 179 00:13:58 --> 00:14:04 things it will use it. So, yeast is able to use rich media 180 00:14:04 --> 00:14:14 that have lots of complex nutrients and macromolecules. 181 00:14:14 --> 00:14:23 So, it has an ability, it has everything it needs to make 182 00:14:23 --> 00:14:33 these things, but it has an ability to regulate that. 183 00:14:33 --> 00:14:37 So, the processes, the enzymatic pathways that produce 184 00:14:37 --> 00:14:41 complex macromolecules, amino acids, phospholipids, 185 00:14:41 --> 00:14:45 et cetera, will be down regulated, shut off, or at least decreased if 186 00:14:45 --> 00:14:49 you provide it with these macromolecules. 187 00:14:49 --> 00:14:53 That's an interesting question of how it manages to regulate its 188 00:14:53 --> 00:14:57 biochemistry. Why does it care? Why doesn't it, why not just have 189 00:14:57 --> 00:15:01 those pathways be on all the time? Sorry? Waste of energy. 190 00:15:01 --> 00:15:04 It needs ATP. It costs money. So, at the beginning probably they 191 00:15:04 --> 00:15:06 were on all the time, but some yeast evolves, 192 00:15:06 --> 00:15:09 or some precursor to yeast evolves that's able to regulate it. 193 00:15:09 --> 00:15:12 That one is able to be more frugal with its energy. 194 00:15:12 --> 00:15:14 It outgrows its other ones and then another, dah, dah, 195 00:15:14 --> 00:15:17 dah. Any place you can make a few ATPs here or there, 196 00:15:17 --> 00:15:20 eventually the organism that does it will out compete the organism that 197 00:15:20 --> 00:15:23 doesn't. And so, rather fine control of this, 198 00:15:23 --> 00:15:25 which is a topic we'll come to in a couple of days, 199 00:15:25 --> 00:15:28 gene regulation and other kinds of pathway regulation is 200 00:15:28 --> 00:15:33 very important. OK. So, we want to know how does it 201 00:15:33 --> 00:15:39 do it? What are the enzymes? What are the pathways? How does it 202 00:15:39 --> 00:15:46 actually make, oh, I don't know, 203 00:15:46 --> 00:15:53 arginine? How does it make arginine, amino acid? How would you find out 204 00:15:53 --> 00:16:00 how yeast makes arginine? How can yeast synthesize arginines? 205 00:16:00 --> 00:16:03 So, you remember our picture that the biochemist wants to study a 206 00:16:03 --> 00:16:07 problem by grinding up the cell and purifying a component able to do 207 00:16:07 --> 00:16:10 something. So, a biochemist might want to grind up 208 00:16:10 --> 00:16:14 the cell and purify an enzyme that can make arginine. 209 00:16:14 --> 00:16:17 Form what, of course, is an interesting question? 210 00:16:17 --> 00:16:21 And then the thing that made the thing that was used to substrate, 211 00:16:21 --> 00:16:24 et cetera, et cetera. What would a geneticist do? 212 00:16:24 --> 00:16:28 How does a geneticist approach the problem with how does 213 00:16:28 --> 00:16:33 yeast make arginine? Find a yeast that cannot make it, 214 00:16:33 --> 00:16:39 that's what we do. That is. So, what we need is a mutant. 215 00:16:39 --> 00:16:46 A geneticist wants a yeast that cannot make it. 216 00:16:46 --> 00:16:53 A geneticist wants mutants. How do you find the mutant? You 217 00:16:53 --> 00:17:00 find the mutant by going on a mutant hunt. 218 00:17:00 --> 00:17:04 That is what geneticists refer to it as. And it's a very exciting thing. 219 00:17:04 --> 00:17:08 You go off, load up the guns and go off into the bush on a mutant hunt. 220 00:17:08 --> 00:17:12 And so, I want to talk about the strategy for a mutant hunt. 221 00:17:12 --> 00:17:16 How do we look for a yeast that can't make arginine? 222 00:17:16 --> 00:17:20 Sorry? Cannot. I've got a yeast that can make arginine, 223 00:17:20 --> 00:17:24 because normal wild type yeast can grow on minimal media without 224 00:17:24 --> 00:17:28 arginine supplied. And, when I examine it, 225 00:17:28 --> 00:17:32 it's got arginine in it. Yes? So, who should I find? 226 00:17:32 --> 00:17:37 Proteins that contain arginine and then it doesn't have the proteins 227 00:17:37 --> 00:17:42 that doesn't have arginine. Interesting. Now, the problem is 228 00:17:42 --> 00:17:47 almost all proteins will have an arginine, or the vast majority of 229 00:17:47 --> 00:17:52 them. And a yeast that lacked all those proteins that didn't have 230 00:17:52 --> 00:17:57 arginine would not be much of a yeast. I think it would 231 00:17:57 --> 00:18:02 be pretty dead. So, it's a good thought if it was a 232 00:18:02 --> 00:18:06 more dispensable function. But that's going to be tough. 233 00:18:06 --> 00:18:11 Or, maybe I can use the fact that it's dead. Now in a sense, 234 00:18:11 --> 00:18:16 can I find the yeast? Yes? You had a thought on this. 235 00:18:16 --> 00:18:20 Yes? Kill all yeast that make arginine, excellent. 236 00:18:20 --> 00:18:25 So, if I had a chemical agent that could kill yeast that can make 237 00:18:25 --> 00:18:30 arginine, I could only get the yeast that make it. How would I do that? 238 00:18:30 --> 00:18:34 That's a very interesting idea. You're right. You could construct 239 00:18:34 --> 00:18:38 the chemical molecule in the arginine pathway which when it was 240 00:18:38 --> 00:18:42 broken down enzymatically made some toxic product, 241 00:18:42 --> 00:18:46 and only those yeasts that couldn't break it down would be able to grow, 242 00:18:46 --> 00:18:50 et cetera, et cetera, and I could select. That's a very cleaver idea. 243 00:18:50 --> 00:18:54 But I'd also have to know an awful lot about the pathway in advance. 244 00:18:54 --> 00:18:58 So, suppose I didn't' know the pathway. Suppose I knew nothing 245 00:18:58 --> 00:19:03 about how arginine gets made. Yes? Excellent. So, 246 00:19:03 --> 00:19:09 I take, I mean geneticists are simpleminded folks and they like 247 00:19:09 --> 00:19:15 simple solutions. Take medium in which you've given 248 00:19:15 --> 00:19:21 the yeast arginine, grow it up, and then pour it out on 249 00:19:21 --> 00:19:27 a plate that doesn't have arginine. Everybody got this idea? So, we're 250 00:19:27 --> 00:19:32 going to take yeast. We're going to grow it up in medium 251 00:19:32 --> 00:19:37 which contains arginine with arginine. So, 252 00:19:37 --> 00:19:42 now yeasts, those mutants that arose by chance that are unable to make 253 00:19:42 --> 00:19:47 their own arginine are still able to grow here. And then we dump it out 254 00:19:47 --> 00:19:52 onto a plate that has minimal media without arginine, 255 00:19:52 --> 00:19:57 no arginine, and those ones that can grow up are the ones that we're not 256 00:19:57 --> 00:20:02 interested in. And the ones that don't appear are 257 00:20:02 --> 00:20:07 the ones we're interested in. But, wait a second, that's the 258 00:20:07 --> 00:20:12 problem, isn't it, because they're not here. 259 00:20:12 --> 00:20:17 How do we study them if they're not there? What can we do about that? 260 00:20:17 --> 00:20:22 Yes. You want to see if you can help us. Remove the ones that grew 261 00:20:22 --> 00:20:27 up. So, get in there, scrap them off, now put some 262 00:20:27 --> 00:20:34 arginine on. We're getting to the idea. 263 00:20:34 --> 00:20:42 Maybe we can set this up more elegantly, though. 264 00:20:42 --> 00:20:51 Thoughts? How can we, yes? Make a bet? Make a guess? 265 00:20:51 --> 00:21:00 I can make that guess, but how do I find them? 266 00:21:00 --> 00:21:06 Here's a simple, simple, simple idea. 267 00:21:06 --> 00:21:13 Let me try a simple idea. How about I grow up these yeast, 268 00:21:13 --> 00:21:19 and instead of plating them on minimal medium, 269 00:21:19 --> 00:21:26 let's be good to them. Let's plate them on minimal medium. 270 00:21:26 --> 00:21:32 Good. That's interesting. Let's plate them on minimal medium 271 00:21:32 --> 00:21:38 plus arginine. Or, actually, if we wanted to, 272 00:21:38 --> 00:21:43 we could even plate them on rich medium. We'll be really good to 273 00:21:43 --> 00:21:49 them. Either way. So, now, let's let each one grow up. 274 00:21:49 --> 00:21:55 And here will be the ones that can grow and the ones that can't 275 00:21:55 --> 00:22:00 grow with arginine. Now let me take a plate that is 276 00:22:00 --> 00:22:04 minimal medium. And now let me take a toothpick, 277 00:22:04 --> 00:22:08 put a little toothpick there and carry over this colony to there. 278 00:22:08 --> 00:22:13 Let me take a toothpick and carry this guy over to here and a 279 00:22:13 --> 00:22:17 toothpick and carry this guy to here, and a toothpick, 280 00:22:17 --> 00:22:21 and a toothpick, and a toothpick. And all I have to do is keep 281 00:22:21 --> 00:22:26 transferring, one at a time, these colonies. 282 00:22:26 --> 00:22:29 And now I can see that somewhere there was a colony that grew fine 283 00:22:29 --> 00:22:33 when I gave it, say, rich medium, 284 00:22:33 --> 00:22:36 or minimal plus arginine, and a colony that didn't grow when I 285 00:22:36 --> 00:22:40 put it on minimal medium. That would at least show, 286 00:22:40 --> 00:22:43 so, of course, the issue is I first have to find them by growing them on 287 00:22:43 --> 00:22:47 something where I've given the arginine and then I can see that 288 00:22:47 --> 00:22:51 they can't grow. All right. This is what 289 00:22:51 --> 00:22:54 geneticists basically do. What happens if I grew them on rich 290 00:22:54 --> 00:22:58 medium and I transferred them to minimal medium? Why might 291 00:22:58 --> 00:23:02 something not grow? It might be missing the ability to 292 00:23:02 --> 00:23:06 make tryptophan. It might be missing the ability to 293 00:23:06 --> 00:23:10 make proline. It might be missing the ability to make something else. 294 00:23:10 --> 00:23:14 So, what I can do is, if I wanted to, make a very broad mutant hunt. 295 00:23:14 --> 00:23:19 I could just first grow on rich medium and then plate on minimal 296 00:23:19 --> 00:23:23 medium and any yeast that has lost the ability to make some essential 297 00:23:23 --> 00:23:27 nutrient will be evident by its absence on the minimal medium plate. 298 00:23:27 --> 00:23:33 So, we have for yeasts. Yeasts that are able to grow on 299 00:23:33 --> 00:23:39 minimal media are called prototrophs. They are the wild type that can 300 00:23:39 --> 00:23:45 grow on minimal media. They can make everything themselves. 301 00:23:45 --> 00:23:51 Yeasts that need help, that cannot grow by themselves, 302 00:23:51 --> 00:23:57 that need help, that need a supplement are called auxotrophs. 303 00:23:57 --> 00:24:03 Auxo obviously meaning help. So, it's a mutant that has lost the 304 00:24:03 --> 00:24:09 ability to grow on minimal medium and that it needs a supplement of 305 00:24:09 --> 00:24:15 some kind. So, if I wanted to, I could just first 306 00:24:15 --> 00:24:21 collect lots and lots and lots of auxotrophs and then figure out what 307 00:24:21 --> 00:24:27 they need. So, I might collect a large collection 308 00:24:27 --> 00:24:33 of auxotrophs. And then test to see if supplying 309 00:24:33 --> 00:24:40 arginine rescues them. I could also test tryptophan. 310 00:24:40 --> 00:24:48 So, if I only, only, only cared about finding arginine auxotrophs, 311 00:24:48 --> 00:24:55 I could just grow them on minimal plus arginine and then 312 00:24:55 --> 00:25:00 test them on minimal. And then I would know in advance, 313 00:25:00 --> 00:25:04 these guys all grew with arginine on minimal and didn't grow without 314 00:25:04 --> 00:25:08 arginine, and I'd know it was arginine. Or, 315 00:25:08 --> 00:25:11 if I was in an expansive mood, I could test them on rich medium, 316 00:25:11 --> 00:25:15 collect everybody who's unable to grow on minimal, 317 00:25:15 --> 00:25:19 and then work out what the reason is. Is it arginine? 318 00:25:19 --> 00:25:22 Is it proline? Is it whatever? And it depends how much work you're 319 00:25:22 --> 00:25:26 interested in doing and how complete the study is you want to do. 320 00:25:26 --> 00:25:30 Either way, we could end up with a collection of arginine auxotrophs. 321 00:25:30 --> 00:25:35 Organisms that are mutant for the ability to make their own arginine 322 00:25:35 --> 00:25:40 and require it to be supplied to them in the medium. 323 00:25:40 --> 00:25:45 All right. I might get, depending on how much work I'm 324 00:25:45 --> 00:25:50 willing to do, dozens of independent colonies 325 00:25:50 --> 00:25:55 unable to grow without arginine. I might get hundreds if I'm willing 326 00:25:55 --> 00:26:00 to do enough work. I can get as many as I want. 327 00:26:00 --> 00:26:08 Our goal now is to study them and find out why they're unable to do 328 00:26:08 --> 00:26:17 that. I have a quick question? Those yeast cells we plated, where 329 00:26:17 --> 00:26:25 they haploid or diploid? We didn't say, did we? So, 330 00:26:25 --> 00:26:33 should they be haploid or diploid? How many vote diploid? 331 00:26:33 --> 00:26:39 How many vote haploid? A lot of people vote haploid but 332 00:26:39 --> 00:26:45 aren't willing to express a reason why. Why haploid? 333 00:26:45 --> 00:26:51 Right. Excellent. Excellent, although genes are not 334 00:26:51 --> 00:26:57 recessive, but OK. A little detail. Phenotypes are 335 00:26:57 --> 00:27:02 recessive. Tell me a little more of what you're 336 00:27:02 --> 00:27:07 thinking about. We'll have it out later on this 337 00:27:07 --> 00:27:12 point, yes. So, suppose we were looking in a haploid. 338 00:27:12 --> 00:27:17 I take your point, even if on nomenclature I want to 339 00:27:17 --> 00:27:22 push back a bit. So, suppose it's a diploid and 340 00:27:22 --> 00:27:27 suppose we have now two copies of this chromosome here 341 00:27:27 --> 00:27:32 in the diploid. And suppose there's a gene over here 342 00:27:32 --> 00:27:36 that encodes an enzyme that we now is necessary to make arginine, 343 00:27:36 --> 00:27:41 or that somebody knows is necessary to make arginine. 344 00:27:41 --> 00:27:46 Let's image that that's the case. In order to get haploid yeast that 345 00:27:46 --> 00:27:50 is unable to make arginine due to a mutation in this gene, 346 00:27:50 --> 00:27:55 you need to have some kind of a mutation in this copy. 347 00:27:55 --> 00:28:00 What about in the diploid yeast? In order to make this yeast unable 348 00:28:00 --> 00:28:05 to grow without arginine, do we need a mutation in both copies? 349 00:28:05 --> 00:28:09 Well, the answer is probably. The truth is actually a bit more 350 00:28:09 --> 00:28:13 complicated, but let's suppose it was the case that even one copy of 351 00:28:13 --> 00:28:17 the functional gene was sufficient to carry out the enzymatic step, 352 00:28:17 --> 00:28:21 then the answer would be yeah, we'd need a mutation of both copies. 353 00:28:21 --> 00:28:25 What's the chance of finding a yeast that has a mutation in both 354 00:28:25 --> 00:28:29 copies? It's obviously much less than the chance of finding a yeast 355 00:28:29 --> 00:28:33 that had a mutation of one copy. So, we're much better to go 356 00:28:33 --> 00:28:37 searching in the haploid where the phenotype will be revealed much more 357 00:28:37 --> 00:28:41 easily by virtue of just the single mutation rather than having to, 358 00:28:41 --> 00:28:45 by chance, encounter one that had mutations in both copies. 359 00:28:45 --> 00:28:50 Now, the reason I'm a little bit cautious here is because 360 00:28:50 --> 00:28:54 notwithstanding the textbooks, it's not always the case that 361 00:28:54 --> 00:28:58 everything like this is a recessive trait. It's possible that 362 00:28:58 --> 00:29:03 auxotrophy for arginine could be a dominant trait. 363 00:29:03 --> 00:29:06 So, how could that be? Well, auxotrophy could be a 364 00:29:06 --> 00:29:09 recessive trait. Suppose there's some enzymatic 365 00:29:09 --> 00:29:12 pathway, A goes to B goes to C goes to D, and this encodes an enzyme 366 00:29:12 --> 00:29:16 that carries out a particular biochemical step. 367 00:29:16 --> 00:29:19 Well, if the gene is broken, if the gene is missing, if the gene 368 00:29:19 --> 00:29:22 doesn't make the protein, as you guys all know that that's 369 00:29:22 --> 00:29:25 what happens, then you don't have the enzyme, you can't do the pathway. 370 00:29:25 --> 00:29:29 And it is usually the case that having just one copy is sufficient. 371 00:29:29 --> 00:29:32 Because having a little bit of enzyme the pathway may work slower 372 00:29:32 --> 00:29:35 but it will still work just fine and you'll eventually get arginine made. 373 00:29:35 --> 00:29:39 But it's occasionally possible, I note since you guys are 374 00:29:39 --> 00:29:42 sophisticated, that sometimes a gene can encode a 375 00:29:42 --> 00:29:46 protein which not only doesn't work but screws up the other working 376 00:29:46 --> 00:29:49 copies of the protein. Suppose the enzyme that did this 377 00:29:49 --> 00:29:53 were a tetramer. It had several subunits that had 378 00:29:53 --> 00:29:57 come together. A mutant copy of an enzyme, 379 00:29:57 --> 00:30:03 when it forms into a tetramer, might somehow disrupt all the other 380 00:30:03 --> 00:30:09 good copies that are around. And that does happen sometimes. 381 00:30:09 --> 00:30:14 It can happen that you're going to have an inability to make your own 382 00:30:14 --> 00:30:20 arginine be a dominantly inherited trait. So, you actually have to 383 00:30:20 --> 00:30:26 test whether it's recessive or dominant. Often it will be 384 00:30:26 --> 00:30:32 recessive. So, usually most of these simple 385 00:30:32 --> 00:30:37 auxotrophs are recessive traits. Occasionally some are dominant. 386 00:30:37 --> 00:30:43 So, now, suppose we get a whole collection of Arg auxotrophs, 387 00:30:43 --> 00:30:49 and we'll just give them a name. I don't know. Here's my collection. 388 00:30:49 --> 00:30:54 We'll call the first one, for lack of anything terribly 389 00:30:54 --> 00:31:00 creative, Arg 1, Arg 2, Arg 3, et cetera, 390 00:31:00 --> 00:31:06 each being an individual strain from growing up originally for a single 391 00:31:06 --> 00:31:12 colony that is unable to produce its own arginine. 392 00:31:12 --> 00:31:17 We now want to take this collection and characterize it. 393 00:31:17 --> 00:31:22 How many distinct genes does this affect? Are these mutants perhaps 394 00:31:22 --> 00:31:28 all in the same gene? Are they in a hundred different 395 00:31:28 --> 00:31:32 genes? How could we tell? Now, of course, 396 00:31:32 --> 00:31:36 if you're a biochemist, you already know the protein you can 397 00:31:36 --> 00:31:39 see and dah, dah, dah. But, if you know the answer, 398 00:31:39 --> 00:31:42 well, why are asking then, right? A geneticist goes out to ask this 399 00:31:42 --> 00:31:46 question because he or she wants to know all the possible ways you can 400 00:31:46 --> 00:31:49 disrupt the cell so it cannot make arginine. And we don't know in 401 00:31:49 --> 00:31:53 advance what those ways are, so how are we going to be able to 402 00:31:53 --> 00:31:56 tell whether or not different mutations affect the same gene, 403 00:31:56 --> 00:32:00 the same function in yeast? It's an interesting question. 404 00:32:00 --> 00:32:10 Geneticists do a variety of tests. The first test that a geneticist 405 00:32:10 --> 00:32:20 does to characterize a mutant is by tests of recessivity or dominance, 406 00:32:20 --> 00:32:30 whichever way you want to put it. 407 00:32:30 --> 00:32:33 We want to take each mutant and test whether it is recessive or dominant 408 00:32:33 --> 00:32:37 as a phenotype, whether the phenotype, 409 00:32:37 --> 00:32:40 the auxotrophy for arginine is recessive or dominant. 410 00:32:40 --> 00:32:44 So, here's mutant number one, the mutant cell carrying this 411 00:32:44 --> 00:32:47 mutation here. Conceptually it affects some gene. 412 00:32:47 --> 00:32:51 I'm going to label it Arg 1. We don't know where it is in the genome. 413 00:32:51 --> 00:32:54 There are other chromosomes here as well. Here's my mutant cell. 414 00:32:54 --> 00:32:58 How am I going to find out whether or not the auxotrophy for arginine 415 00:32:58 --> 00:33:03 is recessive or dominant? Yup? With what? 416 00:33:03 --> 00:33:09 Cross it with a haploid that is a prototroph, or I could just say 417 00:33:09 --> 00:33:15 cross it with wild type, right? Perfect. So, make a cross 418 00:33:15 --> 00:33:21 here, very good, with wild type plus there. 419 00:33:21 --> 00:33:27 How do I know it's plus there? This is wild type. Wild type is 420 00:33:27 --> 00:33:32 defined as the normal form. And so, because I said this is what 421 00:33:32 --> 00:33:37 we're using as wild type, it's necessarily plus because we're 422 00:33:37 --> 00:33:42 measuring mutations relative to wild type. So, what happens when we get 423 00:33:42 --> 00:33:47 here? We now, when we cross we get a diploid, 424 00:33:47 --> 00:33:52 and Arg 1 plus. Now, how do we know whether or not that phenotype was 425 00:33:52 --> 00:33:57 recessive or dominant? Sorry? It's what shows up when we 426 00:33:57 --> 00:34:02 try to grow it. So, when we cross it, 427 00:34:02 --> 00:34:07 what kind of plate should we grow it on first? Should we grow it on 428 00:34:07 --> 00:34:12 minimal or rich? We better grow it on rich because 429 00:34:12 --> 00:34:17 just in case it doesn't, it can't make its own arginine, 430 00:34:17 --> 00:34:22 we better first let it grow and then test it. So, let's grow it on rich 431 00:34:22 --> 00:34:27 medium. We'll cross these together, grow it on rich medium. So, grow on 432 00:34:27 --> 00:34:32 rich, test on minimal. OK? And we'll be able to check out 433 00:34:32 --> 00:34:36 the phenotype as to whether or not the phenotype is wild type or mutant. 434 00:34:36 --> 00:34:41 All right. So, we could do that. 435 00:34:41 --> 00:34:45 And we'll test the first one and the second one and third one and the 436 00:34:45 --> 00:34:50 fourth one. And, for each of these, 437 00:34:50 --> 00:34:55 we'll write down whether it's recessive or a dominant auxotroph. 438 00:34:55 --> 00:34:59 Now, let me assume that all the ones we're talking about are 439 00:34:59 --> 00:35:04 recessive phenotypes. Because everything I'm about to say 440 00:35:04 --> 00:35:10 is very much harder if it turned out any of them were dominant. 441 00:35:10 --> 00:35:16 So, we're going to assume. Let's assume now, but it's not 442 00:35:16 --> 00:35:21 always the case, we'll assume that the collection, 443 00:35:21 --> 00:35:27 maybe Arg 100, are all recessive auxotrophies, the phenotype 444 00:35:27 --> 00:35:35 is recessive. Now, how do I tell if they're in the 445 00:35:35 --> 00:35:45 same gene or not? So, now I want to characterize my 446 00:35:45 --> 00:35:55 mutant by some other test that will tell me whether or not Arg 1 and Arg 447 00:35:55 --> 00:36:03 2 are in the same gene. Suppose Arg 1 and Arg 2 are in 448 00:36:03 --> 00:36:11 different genes. Cross them. What will happen? 449 00:36:11 --> 00:36:19 Right. So, to repeat that, if I cross together the two mutants and 450 00:36:19 --> 00:36:26 they're in different genes, each will have at least, the each 451 00:36:26 --> 00:36:34 will be contributing a good copy, a functional copy, a wild type copy 452 00:36:34 --> 00:36:40 of one of the genes. So, let's walk this through. 453 00:36:40 --> 00:36:45 Interesting. Interesting. So, suppose I take a situation where 454 00:36:45 --> 00:36:50 I've got Arg 1, a mutation in a gene over here, 455 00:36:50 --> 00:36:55 on this chromosome, and on the other chromosome I've got a wild type copy. 456 00:36:55 --> 00:37:00 My Arg 1 mutant is mutated in a gene here. 457 00:37:00 --> 00:37:07 I've got this other gene here, which is normal. And I'm going to 458 00:37:07 --> 00:37:14 cross that now by the strain that has a wild type copy here for this 459 00:37:14 --> 00:37:21 first gene, but it has a mutation in the second gene. 460 00:37:21 --> 00:37:28 When I cross them together, I now get me a diploid cell here, 461 00:37:28 --> 00:37:35 which is Arg 1, a mutation there, plus there, plus copy 462 00:37:35 --> 00:37:41 here, and Arg 2. Will having one copy, 463 00:37:41 --> 00:37:47 one working copy of this gene be enough to make the enzyme? 464 00:37:47 --> 00:37:53 No? In other words, is the wild type phenotype dominant to this 465 00:37:53 --> 00:37:59 auxotrophy, or is the auxotrophy attributable to this 466 00:37:59 --> 00:38:04 gene recessive? Yes. Why? Because we assumed it. 467 00:38:04 --> 00:38:09 Why did we assume it? So I would be able to say this, 468 00:38:09 --> 00:38:14 right? OK. If it wasn't we'd be in trouble. But by assuming that we're 469 00:38:14 --> 00:38:19 working with a recessive phenotype, then we know that this will be 470 00:38:19 --> 00:38:24 enough to save the yeast. What about here? Enough to save 471 00:38:24 --> 00:38:30 the yeast so it will grow without arginine. 472 00:38:30 --> 00:38:38 By contrast, suppose it was the case that this cell here, 473 00:38:38 --> 00:38:47 Arg 1, and suppose our other mutant that we had isolated in our mutant 474 00:38:47 --> 00:38:56 hunt was a mutation Arg 2 in the same gene. Suppose these were the 475 00:38:56 --> 00:39:05 same gene. When I cross them together I now have a cell 476 00:39:05 --> 00:39:14 that is Arg 1, Arg 2. In other words, 477 00:39:14 --> 00:39:24 its genotype is Arg 1 over Arg 2, name of mutation. And can it grow? 478 00:39:24 --> 00:39:34 No growth without arginine. By contrast, the genotype here is Arg 1 479 00:39:34 --> 00:39:43 over plus, plus over Arg 2. I could even write Arg 2 over plus, 480 00:39:43 --> 00:39:51 but I just did that to indicate the chromosomes that they came from. 481 00:39:51 --> 00:39:59 All right. This is called a Test of Complementation because these two 482 00:39:59 --> 00:40:07 genes are able to compliment each other's defect. 483 00:40:07 --> 00:40:18 If two mutations compliment each other's defect then they are in 484 00:40:18 --> 00:40:30 different genes. OK? Boy, that's a noisy one. 485 00:40:30 --> 00:40:36 So, we're able to make a Complementation Table. 486 00:40:36 --> 00:40:43 Suppose I take a bunch of yeasts, wild type, WT, mutant number one, 487 00:40:43 --> 00:40:49 mutant number two, mutant number three, 488 00:40:49 --> 00:40:56 mutant number four. And suppose I cross them with each other in all 489 00:40:56 --> 00:41:02 pair-wise combinations. I've assumed that all of these 490 00:41:02 --> 00:41:07 arginine auxotrophs have a recessive phenotype here. 491 00:41:07 --> 00:41:12 These are all my Arg mutants, and I'm assuming that this is 492 00:41:12 --> 00:41:17 recessive. What happens when I cross them and I test to see whether 493 00:41:17 --> 00:41:22 they can grow without arginine? If I cross wild type by wild type, 494 00:41:22 --> 00:41:27 can it grow without arginine? Yeah. Normal phenotype. So, plus is 495 00:41:27 --> 00:41:32 going to mean prototrophic. Minus will mean auxotrophic for 496 00:41:32 --> 00:41:36 arginine. What happens when I cross wild type with mutant number one? 497 00:41:36 --> 00:41:41 It grows. Why? By assumption, these were all recessive. 498 00:41:41 --> 00:41:46 I'm only testing recessive ones. Two. Three. Four. When I cross 499 00:41:46 --> 00:41:50 in this direction, wild type by these guys. 500 00:41:50 --> 00:41:55 This is going to be a symmetric matrix, of course, 501 00:41:55 --> 00:42:00 right? OK. Now, what happens when I cross mutant one by mutant one? 502 00:42:00 --> 00:42:04 I now have a diploid. Will it be able to grow without 503 00:42:04 --> 00:42:08 arginine? No. Why not? It has no working copies 504 00:42:08 --> 00:42:12 of that gene, so I'm going to put a minus there. What about mutant two 505 00:42:12 --> 00:42:16 with mutant two? Minus. What about mutant three 506 00:42:16 --> 00:42:20 with mutant three? Minus. What about mutant four with 507 00:42:20 --> 00:42:24 mutant four? Minus. Now, what happens when I cross 508 00:42:24 --> 00:42:28 mutant one by mutant two? It depends. It might be plus or 509 00:42:28 --> 00:42:32 might be minus. If they're in the same gene, 510 00:42:32 --> 00:42:38 minus. Different genes, could be plus. So, here's some data. 511 00:42:38 --> 00:42:43 So, all this is compelled. But the kind of data, ooh, 512 00:42:43 --> 00:42:49 I'll use a color. Isn't that fun? They want me to use colors over 513 00:42:49 --> 00:42:54 there. Here we go. Suppose the data were minus, 514 00:42:54 --> 00:43:00 minus, plus, plus, plus, plus, minus, minus, plus, plus, plus, 515 00:43:00 --> 00:43:06 plus. What would it be? 516 00:43:06 --> 00:43:13 What conclusion could we draw? Is mutant one and mutant three in 517 00:43:13 --> 00:43:20 the same gene? They compliment each other? 518 00:43:20 --> 00:43:27 No. But is one in the same gene as two? Yes. In fact, 519 00:43:27 --> 00:43:34 this box and this box here define the genes beautifully. 520 00:43:34 --> 00:43:37 The groups that failed to compliment define mutations in the same gene. 521 00:43:37 --> 00:43:41 These are called Complementation Groups because they don't compliment, 522 00:43:41 --> 00:43:45 OK? It's a little complicated but that's all right. 523 00:43:45 --> 00:43:48 These are called Complementation Groups because all the members of 524 00:43:48 --> 00:43:52 the complementation group, namely Arg 1 and Arg 2, failed to 525 00:43:52 --> 00:43:56 compliment each other. They could be called failure to 526 00:43:56 --> 00:44:00 compliment groups, but it would be too long. 527 00:44:00 --> 00:44:04 OK? So, there you go. You can take hundreds of mutants 528 00:44:04 --> 00:44:09 and organize them into complementation groups and thereby 529 00:44:09 --> 00:44:13 know which ones go to the same gene. And now, if I want to study the 530 00:44:13 --> 00:44:18 genes, I only have to study the distinct complementation groups. 531 00:44:18 --> 00:44:23 Last thing, which we'll just have time to do, are what's called tests 532 00:44:23 --> 00:44:28 of epistasis. We'll probably run just a moment or two over on this. 533 00:44:28 --> 00:44:34 Suppose a biochemist were collaborating with a geneticist and 534 00:44:34 --> 00:44:40 had studied what he or she thought was the pathway for making arginine. 535 00:44:40 --> 00:44:47 Some precursor alpha goes to precursor beta, 536 00:44:47 --> 00:44:53 goes to precursor gamma, goes to arginine. And suppose 537 00:44:53 --> 00:45:00 specific genes were needed to encode specific proteins. 538 00:45:00 --> 00:45:05 I'll call them Arg A, Arg B, Arg C to catalyze each step 539 00:45:05 --> 00:45:10 of this biochemical reaction. The geneticist and the biochemist 540 00:45:10 --> 00:45:15 could collaborate with each other to study whether these mutants, 541 00:45:15 --> 00:45:20 these particular genes now that had been identified, 542 00:45:20 --> 00:45:26 affected each step of the pathway. And here's how they might do it. 543 00:45:26 --> 00:45:30 They might take wild type yeast, mutant, well, they wouldn't know in 544 00:45:30 --> 00:45:34 advance whether or not it was missing the ability to grow on each 545 00:45:34 --> 00:45:38 of, whether it was missing each of these enzymes, 546 00:45:38 --> 00:45:42 but let's think conceptually. Suppose we had a mutant that was, 547 00:45:42 --> 00:45:46 a strain that was wild type, Arg A minus, Arg B, minus, 548 00:45:46 --> 00:45:50 Arg C minus, unable to make this enzyme, this enzyme, 549 00:45:50 --> 00:45:54 this enzyme. And suppose we helped it along. Suppose we gave the 550 00:45:54 --> 00:45:58 mutant arginine. Suppose we supplement and grow it 551 00:45:58 --> 00:46:02 on media with arginine. Which ones will be able to grow with 552 00:46:02 --> 00:46:08 arginine? Can wild type grow if it's given arginine? 553 00:46:08 --> 00:46:13 What about Arg A minus? B minus? C minus? What if instead 554 00:46:13 --> 00:46:19 we offer it precursor gamma? Will wild type be able to grow if 555 00:46:19 --> 00:46:24 it's given precursor gamma? Sure. What about Arg A minus? 556 00:46:24 --> 00:46:30 No, because it still is stuck at this step. 557 00:46:30 --> 00:46:35 It cannot. What about Arg B minus? What about Arg C minus? Really? 558 00:46:35 --> 00:46:41 It hasn't got this enzyme. What's it going to do with gamma? 559 00:46:41 --> 00:46:47 It ain't got anything to do with gamma, no enzyme. 560 00:46:47 --> 00:46:53 Suppose I gave it beta. Wild type, can it grow? What about 561 00:46:53 --> 00:46:59 Arg A minus? No, because it can go from alpha to beta, 562 00:46:59 --> 00:47:04 but it can't go to gamma. It cannot grow. 563 00:47:04 --> 00:47:10 What about Arg B minus? I've given it beta, but it can't do 564 00:47:10 --> 00:47:16 anything with beta because it hasn't got this gene. 565 00:47:16 --> 00:47:21 What about Arg C minus? Wait a second. What did I just do? 566 00:47:21 --> 00:47:27 We're just backward. Sorry. If we gave it gamma, I just 567 00:47:27 --> 00:47:32 got lost here. If we gave it gamma it was able to 568 00:47:32 --> 00:47:36 grow, well, we are completely wrong, guys. It's able to grow here. 569 00:47:36 --> 00:47:40 Thank you. Let's go back on that. You should have caught me before. 570 00:47:40 --> 00:47:44 My mistake. If we have it gamma it's able to, if it's a mutant here 571 00:47:44 --> 00:47:48 it can grow because it bypasses this problem. And having gamma is enough. 572 00:47:48 --> 00:47:52 If I gave it beta, sorry, if I gave it gamma and its 573 00:47:52 --> 00:47:57 mutation was here it can grow. Sorry. 574 00:47:57 --> 00:48:02 Now, if I gave it here beta, and its mutation was here, it can 575 00:48:02 --> 00:48:07 still grow, right? But if its mutation is here it 576 00:48:07 --> 00:48:12 can't and if its mutation is here it can't. That's better. 577 00:48:12 --> 00:48:17 I was getting worried there for a while myself. Suppose I gave it 578 00:48:17 --> 00:48:22 alpha. Wild type can grow. If I give this guy alpha, will that 579 00:48:22 --> 00:48:27 help if he's mutant in A? No. Can it help if he's mutant in 580 00:48:27 --> 00:48:32 B? No. Can it help if he's mutant in C? 581 00:48:32 --> 00:48:37 No. Sorry. There we go. I usually start at the other end of 582 00:48:37 --> 00:48:42 this picture. So, what you can see is these mutants 583 00:48:42 --> 00:48:47 have different phenotypes with respect to being able to supplement 584 00:48:47 --> 00:48:52 them with different chemicals. Now, let me ask in our last two 585 00:48:52 --> 00:48:57 minutes, I'll run two minutes over here. Suppose I gave you a mutant 586 00:48:57 --> 00:49:03 that was a double homozygote. Suppose it was Arg B minus, 587 00:49:03 --> 00:49:09 Arg B minus, sorry, Arg B minus and Arg C minus. Suppose it was a 588 00:49:09 --> 00:49:16 double mutant, it lacked both this and this. 589 00:49:16 --> 00:49:23 Which line of my table would it resemble? Would it look like the 590 00:49:23 --> 00:49:30 first line, the second line or the third line of my table? 591 00:49:30 --> 00:49:36 Second line. Why's that? If I'm lacking B, I'm already in 592 00:49:36 --> 00:49:42 trouble here. And also lacking C doesn't matter. 593 00:49:42 --> 00:49:49 So, I will look, just like a mutant who lacks B. 594 00:49:49 --> 00:49:55 So, in other words, I'm able, if I know something about the 595 00:49:55 --> 00:50:02 biochemistry of a pathway and I can break my arginine mutants up into 596 00:50:02 --> 00:50:08 different kinds of phenotypes here by their response to different steps 597 00:50:08 --> 00:50:15 in a pathway, I can then look at combinations of mutants. 598 00:50:15 --> 00:50:20 And I can say if I have a double mutant missing both B and C, 599 00:50:20 --> 00:50:25 does it look like B or does it look C when I put them together? 600 00:50:25 --> 00:50:31 And it turns out that if it looks like B then B was further upstream 601 00:50:31 --> 00:50:36 in the pathway. So, it turns out that geneticists 602 00:50:36 --> 00:50:40 and biochemists can collaborate based on the phenotype of the 603 00:50:40 --> 00:50:45 organism sometimes to infer aspects of the biochemical pathway. 604 00:50:45 --> 00:50:49 These are the kinds of things a geneticist does to be able to 605 00:50:49 --> 00:50:54 characterize mutants on a mutant hunt. Next time what I want to do 606 00:50:54 --> 00:50:58 is talk about characterizing mutants in a very different kind of organism, 607 00:50:58 --> 51:03 namely the human being.