Welcome to lecture six. I hoped that actually welcome is a good word and that you're going to start feeling comfortable with this material because you will have seen some of it before. We're going to review a number of the illustrations that we did before, but this time we're going to go into a little more detail about the anatomical context and about the sequence of events that leads to the production of an effective antibody. I begin this lecture with a quote that's now over 10 years old from the Houston Chronicle, warning of the global health problems caused by HIV, by tuberculosis, and by malaria. I wish I could say things were better, but if anything, I think that these diseases are now a bigger threat than they ever were. In addition, we are now trying to deal with emerging tropical diseases and spreading craft tropical diseases like Dengue and West Nile virus. So, more than ever, it is becoming critically important that we develop appropriate vaccines. A vaccine is the best defense we have in a global health situation, we want to prevent these diseases because curing them is much more expensive. To develop a good vaccine, it helps to know how the immune system works. So, this lecture is going to go into a little more greater depth about how we produce appropriate antibodies with strategic FC stems, and an even better ability to recognize antigen. We're going to take them out of the bone marrow, we're going to activate them, we're going to put them into a follicle and improve the performances of the antibodies produced ultimately by the B cells. So, that's what we're doing here and now we're going to go and start from the beginning. Here are pictures of possible versions of heavy capal light and lambda light chains after they've been rearranged. Now, I mentioned to you that the human genome was administratively top heavy and so, when we look at these things, I want you to realize that I have left out a lot of the important parts of the gene that are involved in signaling how the gene should rearrange and how the gene should express itself by making and processing RNA. So, first, we're going to go look at the heavy chain gene, that is, this one up here and look at some more of the detail of the sequences that are important in controlling its function. Now, that means I have to add a lot of things and so to keep this thing still from, on your screen and still visible. I'm gonna just enlarge this part from here to here, this that I'm circling, and we're going to enlarge this and look at some more of the details of the sequences. All right. So, you can see there's a lot more different sequences in here than I showed you before. This cell, as you can see, has already rearranged it's heavy chain gene. So, the part that we will be using to make the antibody CDR, is up here in the variable domain coding region. This will be the heavy portion of it. Notice that we've selected a VL region here, and in doing so, we have activated the promoter sequence at the five prime end of the leader, and I'm showing that with a little lightning bolt. Let's see, you can see that I have put in here, I haven't put in, however many numerous VL regions might still be left, but I have left one VL region up in the upstream end so that you can see the inactivated promoter, and you can see that the downstream end of the V still has its complete recombination signal sequences. Again, the heptamer, the two-turn, the monomer that is the AT rich region. So, we're not going to use that one because the mRNA will attach where the lightning bolt is, and will transcribe through the next set of sequences. Here, if you look at it, you can see we have the diversity sequence in the middle, and the variable, and the leader on either side of it attached to it. You can see that in this case, we have still got the remains of the palindromic heptamer. In the middle of these, we also have the n-nucleotide addition sequences indicated in red. So, we have a solid sequence in here and we will have to attach these two together when we splice the leader onto the variable. So, when we get done making a message here, we're going to be doing a lot of splicing, and I've indicated all of those splicing sequences in this teal here. So, we're going to start making the message, we're going to get down through to the end of the J, and then we're going to keep transcribing into the constant region, but we will splice this J to the head where upstream end of whatever constant region we're doing. What this means, is of course, that the upstream splicing signals are going to be different from the downstream, but I've chosen to indicate them in one color. I could have gotten rid of all of the extra Js when I rearrange the gene, but I'd left one in here to just point out to you then an unused J may remain and may wind up in the primary transcript. This unused J will have its complete with combination signal sequences as well, the upstream version, and that's shown here. You can see again, we've got the AT rich nanomer, the two-term sequence, and the seven base haptemer are all still in here. Now, if you look at the diversity sequence, we really got rid of everything but the little sequence we're going to be using. That is, when we rearrange the gene, first we cut out all the downstream sequences when we put on the J, then we cut out all the upstream sequences when we put on the V, and when we did the re-arrangements, then, that got rid of the last of the recombination signal sequences, the AT, and two turn sequences as I put in the diversity. So that's the part that essentially is involved in rearranging so it can code for the variable domain. Now, as we traveled down, we can get into the regions that code for the heavy chain constant region. This drawing here shows you just three of the nine, because again, there's only so much space I've got. You can see if we look at the Mu and the Delta, these guys are preceded by a class switch signal, that's shown here in this stripy blue. If we class switch to the gamma three version, we will put this stripy blue with a stripy purple and cut out both the Mu and the Delta and we will never see them again. Now, notice we don't have a class which signal between the Mu and the Delta, so we have to either use both of them or neither of them. When we put together the message for the variable domain, we will put it together with what ever constant region is upstream and next to it. The mRNA will transcribe through, in this case, both the Mu and the Delta until it comes to this stop signal here. If, however, we've taken out the Mu and the Delta, the mRNA will be produced from the variable region on through the complete gamma three to the next stop signal which is down here. That stop signal of course is irrelevant if I still have my Mu and Delta because the message production will stop at the first stop signal. So, let's look a little bit more closely at these Mu and Delta regions and you can see that they're really quite complex. In addition to being preceded by the class switch signal, we have four sub-regions that code for immunoglobulin domains in the Mu. Recall, it had four immunoglobulin domains in the complete message. Each of these is flanked by splice signals because I'm going to put them together when I make the message. We can see we also have two more that are labeled MS, these are for the membrane spans. So, if we're going to make a soluble antibody, we will put together these four, we'll add the poly A tail here and then we'll finish processing the message and ultimately translate it. If we have a message that's a receptor, we'll put in the membrane spanning regions, we will splice out the first poly A tail region, we'll put in the second and then we will make that message. Of course on some occasions, especially in a mature B cell, we may splice the Delta part together. In that case, we put together the three immunoglobulin domain instructions, but one of which will have the hinge region and we will, in most cases, be making a membrane span. So, most of the time, we will also add in the membrane spans, and then ultimately the poly A tail. Those decisions, however, get made after the message transcript has been initially produced. Now, one of the things we're going to look at the part of this lecture is the process of class switching and if we class switch, we're going to bring together two of the class switch signals. So, if we class switch to the gamma three, we'll put together the gamma three's class switch signal with the upstream class switch signal for the Mu and the Delta, and then the modified gene will have the gamma three next to the J's and our message will be produced by transcribing through that whole gamma three sequence to the stop signal. Again, we will decide whether to splice a soluble version, don't use the membrane spans, or a membrane span version which will be used for memory cells using the membrane spans. So, if you look at all of these things you can see that there are a lot of places in this gene that will be involved in making decisions either during gene rearrangement or during class switching or later on deciding how to process the message to make its particular antibody. Though I change into our fortunately much more simple, here we have the Kappa and the Lambda and both of them have a variable domain coding region that just has the variable and joining and you can see here is the joint for the P nucleotides, the addition, and I don't have the red line for the N nucleotides. Notice when I put them together, again, I activated the promoter region in the leader. I have the same signals that point out that you do splicing, and I'm ultimately going to make a very similar finished message. One difference in the rearranged gene is that the cap may still have extra J's between the region that's going to be coded for the variable domain. Those will be transcribed in and then spliced out. Again, we have the constant region. In this case, the constant region does not have separate exons, you only have one immunoglobulin domain and only one exon, and that's true of the Lambda region as well. So, if you look at the Lambda region, you can see we have a very similar result when we're done processing the region that codes for the variable domain. The only differences we will never have extra J's between the J we're using and the constant that we're using because they come as payers. So, if you look at this one we find an unused J and constant in the downstream part and again, in this case, each of the constant regions is going to have to have its own transcription stops signal, and each again of the constant regions has only one domain. So, they have a single exon and there is no need to splice in that region of the message. So, for these messages, we're going to make a splicing region between the leader and the variable, and we're going to splice the end of the variable to the constant. In a Kappa, we might take out extra J's. In a Lambda, we might leave in extra J's, but when we're done we're going to make a message with a leader variable joining and constant region and put a poly A tail on it and of course cap it in the promoter region. So, again, this is a reminder that there is a lot of information in these genes that is necessary for their proper processing and expression and I didn't even talk about parts on translation. Oh, well, let's continue.