Introduction To Biology8

        Let’s dive in today, and look at how geneticists use genetics. I’ve told you up till now, about some of the history of genetics, and how it gave rise to our understanding about genetic transmission of traits, about genetic mapping, linkage analysis, how all this help confirm the chromosome theory, and we move in a number of concepts about how scientific theories are developed: data is interpreted, intuitions are made. And how they’re actually proven, what sort of evidence, it takes time to achieve (not sure about this part) consensus around theories. That often takes sometimes years, many times decades, before full consensus is achieved around things. Today, I want turn a little bit to the experimental usage of genetics, in a more day-to-day fashion. And you recall this coat-of-arms that I put up here: Function, Gene, Protein, Biochemistry, Genetics. And I told you about how these were two different ways to study biological function. Today, I want to talk to you about how we use genetics to study biological functions. And in particular, I’m going to pick some examples of how we use genetics to study biological function, that have to do with the biological functions of biochemistry. So already, we’re beginning to look ahead to this connection between gene and protein, which molecular biology will establish for us. Suppose you want to do genetics. You got to study some organism. We talked already about Mendel’s choice of organism: the pea. We talked about some of its advantages and disadvantages. Advantages: You could get pure-breeding strains in the market. You could, when you’re done with the experiment, feed it to the other monks. There were a lot of things like that, that were advantageous about the pea, but it had problems of generation time. You can only get, certainly in Europe, a generation or so a year. In Northern Europe, maybe you could squeeze a second generation, and not so good. Fruit flies, very attractive system in many respects, because you could grow much larger numbers; the generation time is on the order of two weeks or so to go from a fertilized fly embryo, fly egg, developing into a fly, developing into a mature adult, able itself to have offspring, so very attractive. There are other systems that people study. Of course, one of the reasons they study the system is because it’s interesting, sorry, because it’s attractable. The other reason is because it’s interesting. So attractability is very important to a geneticist, right? The number of whale geneticist is few, for the most part. But, we also want to choose our system because what it will tell us about the system we want to study, like if you want to study distinctive things about the immune system, you might want to study them in mice. Or, you could even study them in people, though you can’t set up crosses in people, we’ll come to that on Monday. If you wanted to study things about basic aspects of development, you might want to study them in fruit flies. And if you did want to study basic biochemistry, the place to study basic biochemistry might best be done in single-celled organisms, which also have to carry out biochemical pathways like glycolysis and synthesis of amino acids, and things like that. They’re going to be, by far, the most attractable systems. And so, people are particularly fond of doing things like studying basic biochemistry, and many other aspects of basic molecular biology, to studying the organism yeast.

(3:59)    Yeast is a friend of human beings. Certainly, yeast is an intensely studied organism because of its practical benefit in the making of bread, in the making of beer; so, fermentation processes, rising and all that. But yeast also is a tremendously important organism for the geneticist. It is an extremely elegant for experimental system. Yeast is a fungus. It is a single-celled eukaryote. That is true nucleus, it’s got chromosomes that pair up. Its cells, to a first order approximation, are an awful lot like your cells in terms of having all the basic and important eukaryotic organelles in the nucleus, mitochondria, other things like that. So, yeast is a great motto for many purposes, and we’re not going to talk much about the cell biology of yeast, but I do want to talk about the husbandry of yeast. How it is that you grow yeast. So, the way a geneticist grows yeast is to take growth medium, that has lots of rich nutrients. You can take a broth of lots of amino acids and all sorts of stuff, and you know, a little bit of salt, and lots of water of course. And if you take a single yeast cell, and it’s got lots and lots of rich nutrients in this broth here, and you put this yeast cell into the broth, so we’ll do that. Here’s my flask, here’s my little rod which has a yeast cell, or a couple of yeast cells on the end of it. I put it in there, and I grow it at an appropriate temperature, let’s say 30 degrees for example would be a nice temperature, I could do that if I wanted to, then obviously, I grow it up and I get a culture of yeast in there. And I can tell, because nice clear broth is now cloudy with yeast that’s grown up in it. Now, I want to study these guys. So what I do, is I pour them out onto a Petri plate. The Petri plate has on it, a medium, a solid medium, an agar medium, that again has nutrients. And if I pour this out, and I pour out a lot of it, what will happen? Well, there’ll be yeast all over the place, and it’ll be very smutchy, there’ll be like yeast cells everywhere, and, you know, it’s not very organized. So what I want to do, is I want to take that and I want to dilute it. I want to take only a little bit of the broth, and put a little bit of the broth on my plate, maybe I’ll have diluted it first. And then, I want to spread it around with a little spreader, here’s a little glass spreader maybe or something. And, you know, push back and forth, so that really, they’re just individual single cells scattered around randomly. Scattered around. And so, this cell begins to grow and divide and divide and divide, and I get a colony, a little hill of cells, all of which descends from a single cell that was put into that position. And the reason I know that they all descend from a single cell, is because most of this plate does not have cells on it. Most of the plate is sparse; I’ve just got cell, cell, cell, cell, scattered about, and because of that I know these had to of been individual events. These things are called colonies.

(8:00)     Now, when yeast grows and divides like that, let’s take a moment and talk about its life cycle. We’ll introduce its life cycle here. Yeast: proper eukaryote, so it has a diploid stage. It grows as a diploid. And it can undergo mitosis, in which all of the chromosomes line up, as we talked about, they’ve already pre-replicated so they’ll be ready to divide up and give one to each daughter cell, and there you go. n for yeast is 16. Yeast happens to have 16 pairs of chromosomes, peas have 7, and humans have 23 pairs, every organism has its own. Yeast is 16. Now, what we do it is we undergo meiosis to make haploid cells, sperm or eggs, in the human population. Yeast also undergoes meiosis to make spores. It sporulates, and it produces spores, and it turns out these spores, of course, as you would expect, have n chromosomes. They undergo meiosis, just as we drew on the board. And these can come in two flavors. They happen to come, not in males and females, but in A and Alpha. There you go. A and Alpha cells can mate together, again, a diploid. They fertilize and can produce a diploid. They fuse to do that. And now you get back to your diploid from your haploid. So this looks just identical to the human genetic cycle here, but there is one difference. What’s the different? Sorry? Time? Yes, it’s true. Yeast can divide much more rapidly, yeast can have offspring extremely rapidly, over a course of a day or so, and humans take somewhat longer than that. They, for example, have to wait till they get out of college, to be able to reproduce, mostly. What else? There’s one other important thing. It turns out that yeast can also undergo mitosis as a haploid. In other words, the haploid cells of yeast, when it makes individual haploids, they can continue to go indefinitely. By contrast, your gametes, cannot.

(10:52)     You do not have an independent human stage in which you are haploid, or, you know, your gametes are haploid. Whereas yeast can hang out as a haploid for a very long time, until at the size it want sot mate. This is very convenient for geneticists. Geneticists like this, because this means we can grow the thing as a diploid, we can grow this thing as a haploid. When we want to mate them we can mate them together, but we can also study them alone. And you could imagine, this is going to be really good for studying recessive traits, right? So, that’s one of the reasons geneticists are fond of yeast. There are many reasons geneticists are fond of yeast. Just growing yeast smells very nice in the lab, for example. Try growing E. coli by comparison. So, it turns out that yeast is very happy if you grow it on a rich medium, but yeast can grow on minimal medium, with very few macromolecules. It needs a carbon source, which is some sugar it can ferment. It needs some simple source of nitrogen, it needs a source of phosphorus, and it needs some other trace salts and things like that, and it obviously needs some water. That’s it. Hey, think about what’s in a yeast cell. Like, it’s got phospholipid bilayers, but you’re not giving it any phospholipids. Why is it able to grow? It makes them. What about proteins? They’re made up of twenty amino acids. You’re not giving them any amino acids. Why? It makes them. Yeast is extraordinarily self-reliant. You, by contrast, is not as self-reliant. There are a number of amino acids which if I don’t give you, you can’t live, because you don’t actually have the ability to make those amino acids. But yeast is able to make the vast majority of things. Basically, you almost just need to give it the element. As for carbon sources and things like that, it’s very happy with a wide variety of fermentable sugars. You can give it glucose, you can give it sucrose, you can give it galactose, you can give it fructose, and it’ll deal. So yeast is very well set up metabolically. So it’s got all of these pathways, of the sort Bob has talked about (?), for being able to break down the things you give it, and being able to synthesis up the things it needs. Now, yeast of course, is not stupid, because if you give it amino acids, it’ll use it. If you give it all sorts of other things, it’ll use it. So yeast is able to use rich medium, that have lots of complex nutrients and macromolecules. So it has an ability, it has everything it needs to make these things, but it has an ability to regulate that. So the processes, the enzymatic pathways that produce complex macromolecules, amino acids, phospholipids etc, will be down regulated, shutoff, or at least decreased, if you provide it with these macromolecules.

(14:50)     That’s an interesting question of how it manages to regulate its biochemistry. Why does it care? Why doesn’t it, why not just have those pathways be on all the time? Yup. Sorry? Waste of energy, needs ATP, costs money. So in the beginning, they probably were on all the time. But then some yeast evolves a sort, some precursor of yeast evolves, that’s able to regulate it, that one is able to be more frugal with its energy, it outgrows it’s other ones, then…. Any place you can make a few ATPs here or there, you know, eventually the organism that does it will out complete the organism that doesn’t, and so, rather fine control of this, which is the topic we’ll come to in a couple of days: Gene Regulation, another kind of pathway regulation, is very important. Okay, so, we want to know how does it do it? What are the enzymes, what are the pathways, how does it actually make, oh I don’t know, arginine! How does it make arginine? Amino acid. How would you find out how yeast makes arginine? How can yeast synthesis arginine? You remember our picture, that biochemists wants to study a problem by grinding up the cell and purifying the components able to do something, so a biochemist might want to grind up the cell and purify an enzyme that can make arginine. From what, of course, is an interesting question. And the thing that made the thing that was used by the substrate, etc, etc. What would the geneticists do? How does a geneticist approach the problem of how does yeast make arginine? Find yeast that can’t make it. That’s what we do. So what we need is a mutant. A geneticist wants a yeast that can’t make it, a geneticist wants mutants. How do you find the mutant? You find the mutant by going on a mutant hunt. That is what geneticists refer to it as, and it’s is a very exciting thing. You go off, load up the guns, and go off into the bush on a mutant hunt. And so I want to talk about the strategy for a mutant hunt. How do we look for yeast that can’t make arginine? Sorry? A cannot. I’ve got a yeast that can make arginine, a normal wild type yeast that can grow at minimum medium without arginine supplied, and when I examined it, it’s got arginine in it. Yes? So who should I find? Proteins that contain arginine, and then, that doesn’t have the proteins that have arginine. Interesting. Now, I promise most proteins will have arginine, or the vast majority of them, and a yeast that lacked all those proteins that didn’t have arginine would not be much of a yeast. I think it would be pretty dead. So it’s a good thought if it was a more dispensable function, but that’s going to be tough. Or, maybe I can use the fact that it’s dead. So you know in a sense I could’ve find the yeast…hmmm… Yes, you had a thought on this, yes? Kill all yeasts that make arginine! Excellent. So, if I had a chemical agent that can kill yeast that can make arginine, I could only get the yeast that make… How do I that? Student: “The yeasts that make arginine has to use something to make the arginine, so focus on that and kill all the…”It’s a very interesting idea. Actually, you’re right. You could construct the chemical molecule in the arginine pathway, which, when it was broken down enzymatically, made some toxic product, and only those yeasts that couldn’t break it down would be able to grow, etc, etc. It’s a very clever idea.

(18:50)     But I’d also have to know a lot about the pathway in advance. Supposed I didn’t know the pathway, suppose I know nothing about how arginine gets made. Yes? Excellent. So I take, so geneticists are simple-minded folk, and they like simple solutions: take medium, in which you’ve given the yeast arginine, grow it up, and then pour it out on a plate that doesn’t have arginine. So everybody got this idea? So, we’re going to take yeast, we’re going to grow it up in a medium which contains arginine, with arginine. So now yeast, those mutants that arose by chance, that are unable to make their own arginine, are still able to grow here, and then we dump it out, onto a plate, that has minimal medium, without arginine, no arginine, and those ones that can grow up are the ones that were not interested in, and the ones that don’t appear are the ones we’re interested in. But wait a second, that’s the problem, isn’t it? Cause they’re not there. How do we study them? They’re not…there. What can we do about them? Yes, you want to see if you can help us? Remove the ones that grow up. So get in there and scrape them off, now put some arginine on… We’re getting to the idea. Maybe we can set this up more elegantly though. Thoughts. How can we, how can we… Yes? Make a bet? Make a guess? I can make that guess, but how do I find them? He has a simple, simple, simple idea. Let me try a simple idea. How about, I grow up these yeasts, and instead of plating them on minimal medium, let’s be good to them. Let’s plate them on minimal medium… (is that me? Good. Interesting.)  Let’s plate them on minimal medium, plus arginine. Or actually, if we wanted to, we can even plate them on rich medium, we’ll be really good to them either way. So now, let’s let each one grow up, and here will be the arch: the ones that can grow and the ones that can’t grow with arginine. Now, let me take a plate that is minimal medium, and now, let me take a toothpick, put a little toothpick there, and carry it over, this colony to there. Let me take a toothpick and carry this guy to here, and a toothpick and carry this guy to here, and a toothpick, and a toothpick, and a toothpick. And all I have to do is keep transferring, one at a time, these colonies, and now, I can see that somewhere there was a colony that grew fine, when I gave it, say, rich medium, or minimal plus arginine, and a colony that didn’t grow when I put it on minimal medium. That would at least show, of course the issue is that I first have to find them by growing them on something that I’ve given them the arginine, and then I can see that they can’t grow. Alright. This is what geneticists basically do. What happens when I grew them on rich medium and I transfer them on minimal medium? Why might something not grow? Might be missing the ability to make tryptophane, might be missing the ability to make, you know, proline, might be missing the ability to make something else. So what I can do is, if I wanted to, make a very broad mutant hunt, I could just first grow on rich medium, and them plate on minimal medium, and any yeast that has lost the ability to make some essential nutrient, will be evident by its absence on the minimal medium plate. So we have a name for yeasts: Yeasts that are able to grow on minimal medium are called prototrophs. They are the wild type that can grow on minimal medium. They can make everything themselves. Yeasts that need help, that cannot grow by themselves, that need help, that need a supplement are called auxotrophs. “Auxo,” obviously meaning help. So it’s a mutant that has lost the ability to grow on minimal medium, and that it needs a supplement of some kind. So, if I wanted to, I could just first collect lots and lots and lots of auxotrophs, and then figure out what they need.

(24:27)     So, I might collect a large collection of auxotrophs, and then test to see if supplying arginine rescues them. I could also test tryptophane, I could also… So if I only, only, only care about finding arginine auxotrophs, I could just grow them on minimal plus arginine, and then test them on minimal. And then I would know in advance these guys all grew with arginine on minimal and didn’t grow without arginine, and I know it was arginine. Or, if I was in an expansive mood, I could test them on rich medium, collect everybody whose unable to grow on minimal, and then work out what the reason is. Is it arginine, is it proline, is it whatever. And it depends on how much work you’re interested in doing, and how complete the study is you want to do. Either way, we could end up with a collection of arginine auxotrophs: Organisms that are mutant for the ability to make their own arginine, and require it to be supplied to them in the medium. Alright. I might get, depending on how much work I want to do, dozens of independent colonies unable to grow without arginine. I might get hundreds if I’m willing to do enough work. I can get as many as I want. Our goal now is to study them, and find out why they’re unable to do that. I have a quick question: Those yeast cells we plated, were they haploid or diploid? We didn’t say, did we? So should they be haploid, or diploid? How many vote diploid? How many vote haploid? Many people voted haploid but aren’t willing to express their reasons why. Why haploid? Yeah. Ah. Right. Excellent, excellent. Although genes are not recessive, but okay. A little detail. Genotypes are recessive. Tell me a little more about what you’re thinking about? We’ll have it out later on this point. Yes. Yeah. So suppose we were looking in a haploid. I take your point, even if I’m nomenclature, I want to push back a bit there. So, supposedly it’s a diploid, and suppose we have now, two copies of this chromosome here in the diploid, and suppose there’s a gene over here that encodes an enzyme that we know is necessary to make arginine, or somebody knows it’s necessary to make arginine. Let’s imagine that that’s the case. In order to get a yeast, a haploid yeast, that is unable tot make arginine due to a mutation in this gene, you need to have some kind of a mutation in this copy. What about in the diploid yeast? In order to make this yeast unable to grow without arginine, do we need a mutation in both copies? Well, the answer is probably. The truth is actually a bit more complicated. But let’s suppose it was the case that even one copy of the functional gene was sufficient to carry out the enzymatic step, then the answer would be yeah, we need a mutation in both copies. What’s the chance in finding a yeast with mutations in both copies? It’s obviously much less than the chance of finding a yeast with the mutation in one copy. So, we’re much better to go searching in the haploid, where the phenotype will be revealed much more easily, by virtue of just a single mutation, rather to having to by chance, encounter on that had mutations in both copies.

(28:45)     Now, the reason I’m a little bit cautious here, is cause, not withstanding the textbook, it’s not always the case that everything like this is a recessive trait. It’s possible that auxotrophy for arginine could be a dominant trait. So, how could that be? Well, auxotrophy could be a recessive trait, suppose there’s some enzymatic pathways, A goes to B goes to C goes to D, and this encodes an enzyme that carries out a particular biochemical step. Well, if the gene is broken, if the gene is missing, if the gene doesn’t make the protein, you guys all know that’s what happens, then, you don’t have the enzyme,  you can’t do the pathway. And, it is usually the case that having just one copy is sufficient, because having a little bit of the enzyme pathway may work slower, but it’ll still work just fine; you’ll eventually get arginine made. But it’s occasionally possible, I note, since you guys are sophisticated, that sometimes a gene can encode a protein which not only doesn’t work, but screws up the other working copies of the protein. Suppose the enzyme they did this was a tetramer. It had several subunits that had come together. A mutant copy of an enzyme, when it forms into a tetramer, might somehow, disrupt all the good other copies that are around, and that does happen sometimes. It can happen. That you can have an inability to make your own arginine be a dominantly inherited trait. So you actually have to test whether it’s recessive or dominant. Often, it will be recessive. So usually, most of these simple auxotrophies, are recessive traits. Traits. Just occasionally some are dominant. So now, suppose we get a whole collection of arg. (arginine) auxotrophs. And we’ll just give them a name, I don’t know, here’s my collection, we’ll call the first one, for the lack of anything terribly creative, arg 1, arg 2, arg 3, etc, each being an individual strain from growing up originally from a single colony that is unable to produce its own arginine. We now want to take this collection and characterize it. How many distinct genes does this affect. Are these mutants perhaps all in the same gene? Are they in a hundred different genes? How could we tell? Now, of course, if you’re a biochemist you already know the protein, you can see and…but if you know the answer, well, why are we asking already, right? A geneticist goes out and asks this question because here she wants to know all the possible ways you can disrupt a cell so it can’t make arginine. And we don’t know in advance what those ways are. So how are we going to be able to tell whether or not different mutation affect the same gene, the same function in yeast.

(31:57)     Geneticists do a variety of tests. The first test that a geneticist does, is to characterize a mutant by tests of recessivity, or dominance, whichever one you want to put it. We want to take each mutant, and test whether it is recessive or dominant as a phenotype, whether the phenotype, the auxotrophy for arginine, is recessive or dominant. So, here’s a mutant number one, a mutant cell, carrying this mutation, here, conceptually affects some gene, I’m going to label it arg 1. We don’t know where it is in the genome. There are other chromosomes here as well. Here’s my mutant cell. How am I going to find out whether or not the auxotrophy for arginine is recessive or dominant. Yup? With what? Cross it with a haploid that is a prototroph, or I can just say, cross it with  wild type, right? Perfect. So make a cross here, very good, with wild type. Plus there. How do I know it’s plus there? Cause it’s wild-type! Wild-type is the find, is the normal form, and so I said this is what we’re using as wild-type, it’s necessarily plus. Cause we’re measuring mutations relative to wild type. So what happens when we get here? We now, when we cross it, get a diploid, and arg 1, plus. Now, how do we know whether or not that phenotype was recessive or dominant? Sorry? What shows up when we try to grow it? So when we cross it, what kind of plate should we grow it on first? Should we grow it on minimal or rich? We better grow it on rich, because just in case it doesn’t, it can’t make it’s own arginine, we’d better first let it grow, and then test it. So let’s grow it on rich medium. We’ll cross these together, grow it on rich medium. So, grow on rich, test on minimal. Okay? And, we’ll be able to check out the phenotype, as to whether or not the phenotype is wild type or mutant. Alright. So, we could do that, and, we’ll test the first one and the second one and the third one and the fourth one, and for each of these, we’ll write down whether it is recessive or a dominant auxotrophy. Now, let me assume that all the ones we’re talking about are recessive phenotypes. Cause everything I’m about to say is very much harder if it turned out any of them were dominant. So we’re going to assume, let’s assume now, but it’s not always the case, we’ll assume that the collection, maybe arg 100, are all recessive auxotrophies. Their phenotype is recessive. Now, how do I tell if they’re in the same gene or not? So now I want to characterize my mutant by some other test that will tell me whether or not arg 1 and arg 2 are in the same gene. Suppose arg 1 and arg 2 are in different genes. Cross them. What will happen? Right. So, repeat that. If I cross together the two mutants, and they’re in different genes, each will be contributing a good copy, a functional copy, a wild type copy of one of the genes. So let’s walk this through. Interesting, interesting. So, suppose I take a situation where I’ve got arg 1, a mutation in a gene over here, in this chromosome. And on the other chromosome, I’ve got a wild type copy. My arg 1 mutant is mutated in a gene here, I’ve got this other gene here, which is normal, and I’m going to cross that now, by the strain that has a wild type copy here, for this first gene, but it has a mutation in the second gene.

(37:19)  When I cross them together, I now get me a diploid cell here, which is arg 1 mutation there, plus there, plus copy here, and arg 2. Will having one copy, one working copy of this gene, be enough to make the enzyme? No? In other words, is the wild type phenotype dominant to this auxotrophy, or is this auxotrophy attributable to this gene recessive. Yes, why? Cause we assumed it. Why did we assume it. So I would be able to say this, right? Okay. If it wasn’t, we would be in trouble here. But by assuming that we’re working with a recessive phenotype, then we know that this will be enough to save the yeast. What about here? Enough to save the yeast. So it will grow without arginine. By contrast, suppose it was the case, that this cell here, arg 1, and suppose our other mutant, that we had isolated in our mutant hunt, was a mutation arg 2 in the same gene. Suppose these were the same gene. When I cross them together, I now have a cell that is arg 1, arg 2, rather, it’s genotype is arg 1 over arg 2, name of mutation. And can it grow? No growth without arginine. By contrast, the genotype here is arg 1 over plus, plus over arg 2. I could either write arg 2 over plus, but I just did that to indicate the chromosomes that they came from. Alright. This is called a test of complementation, because these two genes are able to compliment each other’s defect. If two mutations complement each other’s defect, then they are in different genes. Okay? So, (“why, that’s a noisy one”), so we’re able to make a complementation table. Suppose I take a bunch of yeasts. Wild type, wt, mutant number one, mutant number two, mutant number three, mutant number four. And suppose I cross them with each other, in all pair-wise combinations. I’ve assumed that all of these arginine auxotrophs have a recessive phenotype here. These are all my arg mutants. And I’m assuming this is recessive.

(41:13)     What happens when I cross them and I test to see whether they can grow without arginine. If I cross wild-type by wild-type, can it grow without arginine? Yeah. Normal phenotype. So pluses are going to mean prototrophic. Minus will going to mean auxotrophic for arginine. What happens when I cross wild-type with mutant number one? Grows, why? By assumption these are all recessive, I’m only testing recessive ones. Two, three, four. When I cross in this direction, wild-type by these guys, this is going to be a symmetric matrix, of course, right? Okay. Now, what happens when I cross mutant one by mutant one? I now have a diploid. Will it be able to grow without arginine? No, why not? Has no working copies of that gene. So I’m going to put a minus there. What about mutant two with mutant two? Minus. What about mutant three with mutant three? Minus. What about mutant four with mutant four? Minus. Now, what happens when I cross mutant one by mutant two? It depends. Might be plus or might be minus. If they’re in the same gene? Minus. Different genes? Could be plus. So, here, all this is compelled, but the kind of data… (oh, I’ll use a color. Isn’t not fun. Okay. They want me to use colors over there. Here we go.) Suppose the data were minus, minus, plus, plus, plus, plus, minus, minus, plus, plus, plus, plus, what would it be? What conclusion could we draw? Is mutant one and mutant three in the same gene? They complement each other. No. But is one in the same gene as two? Yes. In fact, this box, and this box here, define the genes beautifully. The groups that fail to complement, to find mutations in the same gene, these are called complementation groups, because they don’t compliment. Okay, it’s a little complicated but that’s alright. These are called complementation groups because all the members of the complementation group, mainly arg 1 and arg 2, fail to compliment each other. They could be called “failure to compliment groups,” but it would be too long. Okay, so, there you go. You can take hundreds of mutants, and organize them into complementation groups, and thereby know which ones go to the same gene. And now if I want to study the genes, I only have to study the distinct complementation groups. Last thing, which we’ll just have time to do, are what’s called tests of epistasis. We’ll probably earn just a moment or two over on this. Suppose a biochemist was collaborating with a geneticist and had study what here she thought was the pathway for making arginine. Some precursor alpha goes to precursor beta goes to precursor gamma goes to arginine.

(44:56)     And suppose specific genes were needed to encode specific proteins, I’ll call them arg A, arg B, arg C, to catalyze each step of this biochemical reaction. The geneticist “amby by (no idea what this is supposed to be)” chemist could collaborate with each other, to study whether these mutants, these particular genes now, that have been identified, affected each step of the pathway. And here’s how they might do it. They might take wild-type yeast, mutant that they wouldn’t know in advance whether it’s missing each of these enzymes, but let’s think conceptually. Suppose we had mutant that had strains of arg A-, arg B-, arg C-, unable to make this enzyme, this enzyme, this enzyme. And suppose we helped it along. Suppose we gave the mutant, oops, arginine. Suppose we supplement, and grow it on medium with arginine. Which one will be able to grow with arginine? Can wild-type grow if it’s given arginine? What about arg A-? B-? C-? What if instead, we offer it precursor gamma? Will wild-type be able to grow if it’s given precursor gamma? Sure. What about arg A-? No, because it’s still stuck at this step. It can’t. What about arg B-? What about arg C-? Really? It hasn’t got this enzyme. What’s it got to do with gamma? Ain’t got anything to do with gamma. No enzyme. Suppose I gave it beta. Wild-type, can it grow? What about arg A-? No, because it can go from alpha to beta but it can’t go to gamma. Can’t grow. What about arg B-? I’ve given it beta, but it can’t do anything with beta, because it hasn’t got this gene. What about arg C-? Wait a second, what did I just do? If we gave it gamma, I just got lost here, if we gave it gamma, it was able to grow, we’re completely wrong guys. It’s able to grow here. Thank you. Let’s go back on that. You should have caught me before my mistake. We gave it gamma, if it’s a mutant here, it can grow, cause it bypasses this problem. And having gamma is enough. If I gave it beta, sorry, if I gave it gamma, and its mutation was here, it can grow. Sorry. Now, if I gave it here, beta, and its mutation was here, it can still grow, right? But if its mutation is here it can’t, and if it’s mutation is here it can’t. Whoo, that’s better. I was getting worried there for one myself. Suppose I gave it alpha. Wild-type can grow. If I gave this guy alpha, would that help if he’s mutant in A? No. Can it help if it’s mutant in B? No. Can it help if he’s mutant in C? No. Sorry, there we go. I usually start at the other end of this picture. So what you can see is these mutants have different phenotypes with respect to being able to supplement them with different chemicals. Now, let me ask, in our last two minutes, I’ll run two minutes over here. Suppose I gave you a mutant that was a double homozygote. Suppsoe it was arg B- and arg C-. Suppose it was a double mutant. It lacked both this and this. Which line of my table would it resemble? Would it look like the first line, the second line, or the third line of my table? The second line? Why’s that? If I’m lacking B, I’m already in trouble here. And also, lacking C doesn’t matter. So I will look just like a mutant that lacks B. So in other words, I’m able, if I know something about the biochemistry of the pathway, and I can break my arginine mutants up into different kinds of phenotypes here, by their response to different steps in a pathway, I can then look at combinations of mutants, and I can say if I had a double mutant, missing both B and C, does it look like B or does it look like C when I put them together. And it turns out, if it looks like B, then B was further upstream in the pathway. So it turns out that geneticists and biochemists can collaborate based on the phenotype of the organisms sometimes, to infer aspects of the biochemical pathway. These are the kinds of things a geneticist does to be able to characterize mutants on a mutant hunt. Next time, what I want to do it talk about characterizing mutants in a very different kind of organism. Namely, the human being.