In this section, we're going to look at some model manipulation to show you exactly how you break and join the DNA molecules in order to bring about gene rearrangement. Now, I didn't mention to you that the genes for the both the light and the heavy chain are very, very, very long chains. One of the first things that you have to do is actually unwrap them in the nucleus, and interestingly enough, when you unwrap them from the nucleus they actually make a little bit of RNA although it's never used. So, here I have a free DNA molecule and we have a double strands. So, here's the three prime, and down here in silver is the five prime, and of course, what I have on this end is a whole bunch of different variable leader regions. So, I'm going to just pick out one of the variable leader regions and you can see that at the downstream end of this, this variable leader region will have a recombination signal sequence. Understanding the actual way that the DNA moves around and breaks and rejoin during gene rearrangement is probably one of the trickier things I have to get across. So, I've done this animation and it may look a little bit crude but I'm hoping it does the job in terms of getting across the concepts. We're going to look again here at the signals that demarcate the places on the chromosome where we bring the different regions together. So, here we've got the one turn signal with its palindromic heptamer and its AT-rich nanomer and we have a corresponding 2-turn signal over here. So, in order to bring two regions together to join them, we need to juxtapose a 1-turn and a 2-turn signal. So, in this animation, I'm going to put the variable chain with the joining chain and you can see the variables upstream and the joining downstream, and I've left off the leader at the upstream end of the variable, and I've left up all of the other stuff at the downstream end of the joining which, of course, will continue on into the constant region. So, what we want to do is look at how we put these two things together. Now, in order to do that, I'm going to remind you that these things all illustrate a double strand of DNA, and so there's really one strand up here at the top running from five to three, and down here at the bottom there's another strand that's running from three to five. If I take out the middle part, you can see that I have a double-stranded DNA that has the recombination signal sequences lined up on both sides. Notice also that I'm leaving out a large chunk of DNA from the middle here. So, this one shows you the region where I'm just simply omitting riding at in what is probably a long string of Vs and maybe some J's in here as well, and again I'm emphasizing that the nucleotides are really part of both strands. One thing I can't do on this animation at least not easily is show you that this is a double helix and the these two strands are wrapped around each other but that's just would be confusing anyway. What we're going to do now is make a single stranded cut in the palindromic region here and on the corresponding side of the other DNA molecule there. Okay. So, the enzymes are going to come in and cut that and so we're going to get a cut here and a cut there. Once we've done that that means that the strand with the cuts, this sort of free to open up and move around. So, what I've done here is I've shown you this part of it moving around, and more to the point the one that is really going to matter is that the palindromic region here also can now move around and turn. Because what it's going to do is turn around and attack the other palindromic region of the other strand. So, here we're going to see that what happens is we have the palindromic region curving around and attacking the corresponding strand over here, and the same thing is happening over here. Now, when that happens, you get a cut here and here, and that's going to separate all of the intervening DNA. So, here you've got the sequences ready to get rejoined again. You have the double-stranded DNA molecule leading into this very unusual single-stranded loop over here. You have a corresponding setup with the single-stranded loop leading into the double-stranded DNA on the downstream end as well, and so I'm going to use these two loops to stick the V and the J together. The loops that are shown here with the single-stranded DNA were originally part of the palindromic sequences that's true for both sides. The one and two term sequences along with AT-rich sequences and any intervening Js and Vs have all been cut out and taken away, and that leaves these two loops held next to each other by the enzyme complex that is performing the ligations and rearrangement. So, let's take a closer look at these loops. So, here you can see the sugar phosphates are mostly in red and white, and the bases are the things that have the blue and the black. So, here you can see as you travel around, the sugar phosphate, the sugar's pretty in conspicuous but the phosphate is nice bright red and white as we go through here, and you can see they go up around down and so that one of the strands continues all the way on up and over and meet up the other one and forms this loop structure. Of the other thing you can see from this is that the basis are kind of flopping around to the outside. So, you've got that part going on, and if you'll recall, these are the bases that will come from the heptamer. Now, I'm going to line up that space-filling model to show you the way the molecules run in this loop area. So, here you can see the double-stranded DNA in both the model and the drawing, and that's going to extend indefinitely off upstream and indefinitely off downstream. Here is the loop. So, you can see the corresponding parts of the drawing and of the model. Enzymes are going to clip these loops but they're going to clip them in variable ways. So, we'll go back to doing just the drawing again, and so let's see we're going to have a clip here, and we're going to have a clip here. So, the enzymes that clip this loop will clip it somewhere in the loop but they can clip it in different places, and they don't have to clip it in corresponding ways on both loops. So, the enzymes clip this hairpin loop in variable places. What this will do is lead two open ends, one on the upstream region around where the variable is, and one on the downstream region where the joining is. So, when I do that, I will open them up and I will have two open ends. But notice the ends are not the same length on the regions that are attached to the variable. They are not the same, well actually, they're close to the same length on the ones in the j but they don't have to be, and more to the point they don't have to match or be complimentary and length to the open end of the gene you're joining it up to. So, let's open them up. We're going to flip this one up, we're going to flip that one up, we're going to take those and flip them down, and then we're going to ligate the open ends. Oh, but we have a problem. Because what we have on one side is a longer length of DNA than we have on the other side. At this point in the joining process, we're adding additional variability, they'll trim a few nucleotides off here but with the ligation process, you have to in many cases fill in to make up the difference in the length of one strand versus the length of the other. So, here we're going to fill this in and we're going to fill this in with nucleotides that match the palindromic region of the complimentary strand. So, that's why it's called P nucleotide addition. So, when we're done with this, we have the joint between the variable and the joining region, and that's going to be part of the third loop of the variable region of the light chain of the antibody. Now, it turns out that the heavy chain is going to be a good bit more complicated. So, what I have done is I've reproduced one of our classic heavy chain gene drawings up here at the top, and I'm showing you how it corresponds to the regions that we have in the gene after we have done the rearrangement with the P nucleotide addition. So, the heavy chain genes had even more variability. Because their third loop is going to be made up of the diversity region here, and the variable junctions that were put together when the diversity was joined first with the joining and then with the variable. So, let's take a closer look at that. Take that away, take that away and there is one more thing that can happen in this genes. You can see that we have two joints here and here, and we can add even more variability to them. I'm just going to show it on one of them. So, in other words, we can open this up and we can add in a number of nucleotides at random, and that's what's called n nucleotide addition. It only occurs in the heavy chain gene but it will occur in both of these joints, and interestingly all of this extra nucleotides that are thrown in will wind up in the third loop of the recognition region of the heavy chain gene. So, let's take a look at that. So, here is the heavy chain gene variable domain of the antibody, and you can see the corresponding regions for the D, the V, and the J, that is these are the regions of the protein that were coded for by those various regions of the gene.You can see that the joints will provide the information for this part of the loop and this part of the loop, and therefore all of this information goes into the third loop and not loops one and two. Quick look at the light chain. So, here is the light chain variable domain and in this case, loop3 will contain parts of the V, the J, and the joining region, and later on much later on, we will see that we can do some additional variation in the DNA that codes for any one of these three loops. So, all told, these processes lead to a great deal of random variation in the recognition region of the arms of the antibody. So, next we're going to look at the implications of all of this variation in constructing a whole bunch of different types of antibodies that gives us a chance to recognize just about anything that a pathogen can throw at us antigen wise.
