Genetic Screens

Hi in this video we're gonna be talking about genetic screens. So genetic screens is another really big technique that scientists use to evaluate the function of thousands of genes at once. So we've mainly been focusing on how to identify the function of one gene, which is good if you kind of already know about what genes to target. Right. So you knock out one gene that you think is involved in cell growth and you find out that it is Well that's good. But if you don't, if you have this whole genome sequence and you have no idea any of the genes in the then you're not going to be able to target one specific gene or one gene at a time. There's way too many genes to be able to do that. So genetic screens are important because you don't already have to have an idea of how the genome of the organism is laid out and where the genes are and what genes are probably doing what it just says, We have no idea. So let's test all the genes so or at least most of them. And so genetic screens super powerful. But how you do that is if you have to take the whole genome at once, then you have to be able to sort of start from that big huge amount of information and narrow down step by step to jeans with a particular function. So usually what you do with a genetic screen is you kind of think about what function you would want to look at. So you say, okay, I'm interested in cell growth. So I'm going to do a genetic screen and look for um look for organisms that have impairments and cell growth. So how you do this is you take the entire organism and you expose them to some kind of mutation. Now remember a mutation is going to cause mutations. And so what it does is it just causes random mutations all across the genome. Some of these organisms are going to die but most of them will have very weird effects, right? It could change the shape, it could say, into the size, it could change the growth, it could change metabolism, it could change color. Like I mean literally these mutations can cause any kind of phenotype. But remember you are interested in one phenotype and that phenotype with cell growth. So you're going to exposed cause all these mutations. And then you're going to search through the organisms and find the ones that have defects in cell growth or cell size, whichever, whatever your phenotype is. So if some are growing slower, you're gonna want those. If some are growing faster you're gonna want those and some are growing bigger, you're gonna want them. And then anything that looks weird, maybe shape or color or things like that. You say that's for somebody else to do. I'm interested in that phenotype. So you expose it to the region creates a lot of mutations, creates a lot of weird phenotype and then you pick all the ones with the phenotype that you're interested in? Once you have that group of mutant phenotype that you're interested in. You can do more tests on them and this is where it gets more specific and that identifies a specific gene that causes that phenotype. It can identify potentially more depending on what tests you do. And so um so I remember genetic screen. You you just organize the whole organism creates a lot of phenotype. You select for the phenotype you want. And then when you have a smaller selection of those little those phenotype that you're interested in, then other tests can go on to try to figure out, you know what where that gene is, what that gene is doing now, that's generally how genetic screens work. But there are some caveats and one of those things is that some of those mutations, like I said before cause the organism to die. And so if the organism is dead, you can't do any further tests on it. So genetic screens are only useful if the mutation is not lethal. And that is because you want to be able to do more tests on it. And if that organism dies, like you're not going to get any information about about what genes being mutated in that organism. So genetic screens are useful in sort of non essential genes, genes that cause a different phenotype. But it doesn't really matter. The cell can still survive without them. And um but you say well what if I wanted to study a gene that is so essential. It would kill the organism if it's mutated in this case use a special type of mutation called a conditional mutant. And this is again this is must be used to study lethal mutations. And it's called a conditional mutant because it's only a mutant under certain conditions. So you say how does that even work? Well it says that these mutations are only express So you don't get them expressed otherwise. But then at a mutant form is expressed under certain conditions. So an example of this is temperature. So a temperature sensitive conditional mutant will express its normal wild type phenotype at say 68°. But then if you raise the temperature to 72 it's all out it's going to express its mutant form. Then you'll see the phenotype. And that allows you to actually get a group of mutants with phenotype that would normally kill them. But you have enough time because you set the mutation you said this mutation is only gonna be expressed from the temperatures this side or this this degree and that allows you to study them and keep them alive long enough to study. So genetic screens are generally useful for non lethal mutations. But if you do want to study a lethal mutation you can through these conditional mutants. So again here's an example of a genetic screen we have these organisms here. We'll just say this is bacteria. But it literally could be anything you want. A mutation comes in. It mutates all the D. N. A. So now all the D. N. A. It has different mutations in it. And you can see it causes these different phenotype. Some of these look like ovals. Some of them has this like weird X. In them which could be a color thing. Some of them are smaller. And if you imagine if you're working with bacteria you're working with thousands upon millions of individual cells and all of them could be different. So you select the one you're interested in and it looks like you're interested in cell size. So you select all the ones with mutations and cell size. And then you do more tests to identify the gene. So what kind of tests can you do to identify the gene and identify the genes function? There's a ton of them. I'm going to talk about two. The first is called a complementation test and both of these are sort of more genetic type tests and cell biology. And so if you take genetics or you have taken genetics you're gonna understand these very well. But the first is a complementation tests. And what the complementation test does is it takes to potential organisms say two of these small ones. And it says is it because the mutation in this cell and the mutation in this self are in the same gene. So we actually remember we started with and we have no idea what kind of where the mutations are. There's tons of them everywhere. But we have a lot of similar phenotype. But that doesn't mean that all the mutations are in the same gene that could be in multiple genes. So if you want to identify which gene is responsible and you want to identify the function, you can do complementation tests to figure out is this organism who looks small and this organism that looks small? Do both of them have a mutation in the same gene? So complementation test how you do them is you make two homicide is recessive organisms. Like I said, very genetic see here. So this would be the cross that you do. And you can see that if the phenotype is seen in the offspring. So if you still see the mutant phenotype in this case the small circle then the mutation is in the same gene. Because if you cross these and you still get this phenotype, then obviously the mutation is in the same gene. If the phenotype is not seen. So you see a wild type phenotype potentially, then the mutation is in different genes. And so that would look like if you did this cross, right. And what you would get, I know this is way more genetic city than you're probably used to. And that's fine. If you're if you haven't taken genetics, don't worry about it. But what you'll see is that in this case you're going to get a wild type phenotype because the mutations that called this circle and the mutations that cause this circle are in the R and the A jeans, they're not the same genes, they're in different genes. So the important part to realize here, you do a complementation test and you see if you still see a mutant phenotype mutant phenotype equal same gene, mutant phenotype equal same gene and wild type phenotype equals two different jeans. So, a complementation test is one type of test you can do to figure out whether the mutations are in the same gene. Second type of test is an episode basis analysis and what this does is it evaluates the order of protein pathways. So we've talked about this before, we will in terms of signaling pathways and signaling pathways. Remember, you start with something up here and it signals and it goes through these pathways. But remember we just mutated all sorts of things. So if we mutated this one that would block the pathway. If we mutated this one, that one would block the pathway. If we mutated this one that would block the pathway. But all of these would cause the same phenotype writing right on the edge here, Right. If we knocked out this one, this one this one or this one, they're all leading to the same thing, all of those mutations would have the same phenotype and through a genetic screen we don't know whether the mutation is here here here or here. An episode basis analysis is going to determine what the order is of these proteins. So it says that protein A potentially adds before protein B. Then a mutation and A. Will always stop B. But a mutation and B will only stop B. And A. Right? So if you have a mutation here it's going to block this entire pathway A. And B won't be produced. But if you have a mutation in B then A will still be produced whereas B. C. And D. Won't. So these are just two. Like I said, there's so many different um types of tests you can do to identify genes function. These are just a couple. But remember you have to combine them with genetic screens, you have to know you have to have some kind of phenotype. Have to have some kind of mutation. To be able to do these genetic screens. And the follow up tests are really really really really really important for identifying the genes function. So here's an example of an episode basis analysis. This is the same pathway that I did before. Except you can kind of imagine that there's C. And D. Here. So a protein A leads the protein B. And this is the normal pathway. If you have a mutation in A that's gonna block A. It's also gonna block B. But if you have a mutation and B. That's only going to block B. A will still be produced. And so these types of tests allow us to be able to take mutations that may all have the same phenotype, but determine is it a signaling pathway, and are they acting in a certain order? So with that, let's move on.