Hi. In this video, we're going to be talking about Functional Genomics. So, Functional Genomics, this is the type of genomics that studies the function, expression, and interactions of genes and proteins. Now, there are many different subdivisions of Functional Genomics that focus on different parts of gene expression. And so, there's going to be transcriptomics that studies the expression, the sequence and expression of transcripts. You have proteomics, which is going to be the expression of proteins, and you may ask, why are these different? Well, sometimes things get transcribed, but then they get suppressed before they're translated and can actually be very different between what transcripts are produced and what proteins are produced. And then, finally, there's interactomics, and this is going to be various interactions between DNA, RNA, and proteins. You know, how are proteins interacting with each other, how are proteins interacting with DNA, and how are proteins interacting with RNA? So these are the 3 main divisions that make up functional genomics.
Now, there are many different types of techniques that scientists who are studying functional genomics use. So the first, we're going to go through each one individually. The first that your book talks about are DNA microarrays. And microarrays are used to determine which genes are active in a particular cell under certain circumstances. So this is very specific. It can change from minute to minute in different environmental conditions. Right? Because gene expression changes are dynamic, they're not constant. So, it is different between cells, it is different between environmental conditions, it is different between age and development, and so DNA microarrays look at the active genes in a particular cell, at a particular time, in particular circumstances. So, if you want to know more, you have to do different cells at different times in different circumstances. This particular experiment is looking at cancer cells and normal cells under certain conditions. So, how this happens is you grow the cells in these plates, you take RNA, you take the mRNA. Right? This is the messenger RNA, so this is what's being expressed or trying to be expressed. You reverse transcribe it, that produces cDNA. You then label it differently, so we made the cancer cells red and the non-cancer cells green. We put this onto a microarray, which has different probes for pretty much every gene in the cell. And so, the probes that are binding, will then have these releases of color. So if the green binds, that means that gene is being expressed in normal cells, and if the red binds, that means that genes have been expressed in cancer cells. And you compare the relative levels of green and red, and you may find that in cancer cells, there's one gene that's just really expressed but not expressed very much in normal cells and vice versa. You may find a gene that's expressed very highly in normal cells and suppressed in cancer cells. And so DNA microarrays are looking at the expression of these genes, these RNA transcripts in different cells.
The second test is going to be a 2 hybrid test. This is performed in yeast, and this studies protein interactions. So how this is done is using a system called the gal system, but I didn't put it here because I feel like we've talked about the gal genes before, and I didn't want to necessarily confuse you. But, essentially, what this is is there's a promoter region here. This is usually the UAS region from the gal region. If you are familiar with it, you don't necessarily need to know it. And what happens is that in order for transcription to be activated, these two proteins need to come together because how this has been designed is that normally, this Gal system requires one protein to be activated. So what the scientists have done is they chopped this protein in half, and they have attached a bait protein onto it, onto one side, and a prey protein onto the other side. And so for activation, these two regions need to come together again, and the only way that they'll come together is if the bait and the prey are interacting. So what you see is in this case, the bait and the prey are 2 proteins that normally interact in the cell. So they come together, then these two regions will come together, and that will activate transcription. If the bait and the prey do not normally interact, then they won't come together and transcription will be halted. And you say, okay, well, how do you measure transcription? Well, you measure transcription through some type of gene called a reporter gene. Usually, this is something like GFP, which stands for green fluorescent protein, not super important, but know that when this protein is transcribed and produced, it produces a green color, and you can see it. And so if these two proteins come together and bind, transcription of GFP will be activated, and then you'll see green in the cell. If these two proteins do not bind together, this transcription will not be activated, and therefore, the cell will not be green. So that's how the 2 hybrid test works.
Another important test is ChIP or chromatin immunoprecipitation, which looks at protein-DNA interactions. So, how you do this is you have genomic DNA. There's protein binding onto it at various regions, whether to activate transcription, suppress it, you know, bind to enhancers, whatever they're doing. They're binding to genomic DNA. So what you do is you actually introduce chemicals that cross-link it, and what cross-link means, it means to just sort of bind it tightly together. So it's like a kind of super glue that really sticks it on there. So those proteins and DNA will be stuck to each other, like really, really difficult to remove. Then you cut the DNA into pieces, you take antibodies against these proteins you're interested in seeing are they binding DNA, and where are they binding. Then you can actually take the DNA, you introduce more chemicals. Right? And you separate the DNA from this protein that you've particularly acylated. And so then when you have the DNA, you know you can then sequence it and figure out what sequence this particular protein is binding to. Now, the important part here is specificity, right? Because the purpose of this is to look at which DNA sequence this protein is binding to. So the specificity од comes into the fact that the antibodies that you use are specific only for the one protein that you're investigating. Right? And so you're saying, does this POI protein bind to DNA, and where does it bind? Then you can sequence it and figure out, is it binding? Yes or no. Right? If you get no sequences, it's not binding to DNA. And then when you have the sequence, the normal sequencing reactions, you can say, okay, well, this POI protein is binding to this sequence, which is located on chromosome 3, you know, right here. And that allows you to figure out which proteins are binding to which DNA sequences and what that potentially does in relation to nearby genes.
And then, finally, the last one I'm going to talk about is reverse genetics, which is what is mentioned in your book, but there's actually kind of two sides of reverse genetics. There's forward genetics and reverse genetics, which are very tightly related, so I do want to introduce both of them. But reverse genetics works because you have a gene sequence and you know what's normal. So you disrupt it somehow. You introduce mutations, and then you see what happens when you have a mutation. Reverse genetics says you know the genotype, you mess it up, you cause a mutation, and you say, okay, I'm going to look at the phenotype. Forward genetics is the opposite of that, where you don't know the genotype, but you say you have a weird phenotype, say, that a fly only has 3 legs instead of 4. So you take all the 3-legged flies that you have, and you figure out what the genotype is. And so your book only mentions reverse genetics, but it is important to understand the reverse of this, the forward genetics part, so that you understand how those two play together. Reverse genetics goes from genotype to phenotype, whereas forward genetics goes from phenotype to genotype. So, those are some methods. With that, let's now move on.
