Unformatted text preview: Genetic basis of Ag receptor structure Not suprising, our main focus will be on the B cell receptor and at the end we'll talk about the T cell receptor and how it's similar to the the B cell receptor. I moved my office hours to 210C in BSRB. One of the things I want to emphasize are some of the differences on the receptors of the adapative immune response. They are generated somatically and expressed clonally. What do I mean by that? You inherit these receptors from Mom and Dad. These aren't inherited. You put together your own receptors. The repertoir of B cell receptors that you have might be very different from mom and dad. If you have different B cells or T cells triggered, they'll repsond and proliferate. They will have different specificity. The specificity of each clone will be different. That's why it's different from the macrophages that all express the same receptor. the antigen receptor that they express will have different specificity depending on the clone they derived from. because one of the characteristics .... the constant regions come in different isotypes. those different isotypes have different funcitonal properties. ... They exihist as botha membrane and secreted forms. how does a cell acheive that? One of the things that puzzled immunologists ios that H and L chans are separated into variable and constant regions. How can you have that? How is allelic exclusion achieved. Any single B cell lymphocyte will express only one of those cells. Like many discovereis in biology, the critical thing that enabled immunologists,.. is the development of restriction enzymes that will cut DNA at defined sequences. It should be noted that people who discovered RE that couldn't get a NIH grant because it was so novel. .... He took two different kinds of cells, one was a liver cell and the other cell was a malignant plasma cell. Myeloma is a plasma cell malignancy. He twook these two different kinds of cells. He took from the myeloma cell and isoalted the messenger for the light chain and took a two labeled probe that would encode that light chain. He then took the DNA and digested them with the endonucleases. He then took and hybridized these restriction fragments with his probe. What he saw came at a complete surprise. If he mixed them, the myeloma still only had one band and the DNA had two bands. It turns out the answer was that physical rearrangement of the DNA when you come committed, in this case, to making an L chain. When the cell becomes committed to making the immunoglobulin. It was much more associated with the .... to explain his results, if you cut with RE, you now generate two fragments. One region binds to variable region and one piece binds to the C region. But for the myeloma it's been rearranged that they both bind to the same one. We now know that the DNA is not so static. Also, the myeloma had lost one of its L chain and made it easier to interpret these results. When they looked at it more closely, it was more complicated than we had previously thought. Tere's a leader sequence in front of each of the variable regions. In order to be directed into thte phatway, it needs a leader sequence. That's important in having these proteins directing into the pathway. There were also multiple J regions. you sould realize that the J region for the antibody genes is not the J chain that is associated with polymeric Igs. Kanagawa wasn't an immunologist so he didn't know about J chain. T rearrangment that takes palce is a particular V regin gets associated with a J region. So there's now an intron remaining here. This intermediate .... This rearranged V kappa region has been rearranged in the promoter. it is transcribed, all of that is removed by splicing and the polyA tail is put on at the end. So now you have L associated V associated with J associated with C so that it is one reading frame. As the protein is secreted, the Leader sequnce is cleaved off. So the mature chain doesn't have a leader chian but it had to have that leader piece in order to be secreted. The events that happened is a particular L region is rearranged next to a J region. The initial primary transcript still contains introns. Splicing removes those introns, that mature message is translated into protein. The protein is processed such that the leader is removed. V and J make thvariable region. C is the constant region. We can see within the germline, you have multiple variable regions and multiple J regions. Any V region can be associated with any J region. Both of thsoe now are aspects of developing the immune response we make. If we look at te H chain, you'll see kthat there are multiple H chains. But the nearest one is IgM which you should know is ALWAYS the first made in an immune response. D region contributes to diversity. A particular D region now rearranges to a particular J region. Now, a That rearranging with the promoter region, thos introns are removed by splcing so t that... that is translated to make the nascent polypeptide... and the leader sequence is removed so that the mature H chain has a variable region comprised of VDJ and a constant region. It is just a schematic and not exactly right. One of the things that you should appreciate is that you have two alleles (one from mom and one from dad) and rearrangement can take place for both but only one results in the production of a functional protein. If you look at the condition of our two different alleles, we can show the diferent kinds of rearrangments can take place. We now cut with RE and we see two different bands. Of those rearrangements, we can only take one because of allelic exclusion because one cell can only make one type. It could be of nonsense or missense that it is not functional. There is a physical rearrangment of the DNA that takes place with the funcitonal variable region. You now have the same binding specificty. What you shouldnotice is associated with every isotype except delta is a switch region. these switch regions are highly repetitive DNA sequences. This is where now the rearrangment takes place. The DNA between these two switch regions. The consequence is that we now have a functional V region with a different constant region. You can see now that the switching... all these upstream genes are lost and can never be expressed. it is possible to express further the downstream constant regions. I made some new slides and thought I forgot to put them back in. We have this looping that is ... We have rearrangemtnat at Switch mu and switch gamma isotype. All of these upstream regions are deleted. It is possible to have some rearrangment ... It's going to make acircle that is now going to replicate. We have a new isotype making a new switching. You can see you can never go back upstream. You can never go from gamma back to mu because you've already deleted it. Isotype switching occurs with both the functional and nonn-functional allele. Once again, allelic exclusion, so only one functional H chain is expressed. It's possible to make, for a single B cell tao ake membrane IgM and mIgD of the same binding specificity. Those are the only isotypes made together. How that is achieved is that IgD and IgM are very near each other in the genome. It is also possible to make a longr transcript. From the message, the IgM is deleted and now you have the functional V region associated with the IgD region. Now, another thing is that remember tha tB cells are very clever. they can make membrane and secreted bound as a receptor. How? If you look at the gene. there are sequences called the M 1 M2 which encode the membrane bound version and if you want the secreted version, you have the S. To make membrane bound version, you process by removing the S region. We'll splice the IgD such that you remove so that the tail is the membrane bound version. what's important to realize that both the IgM and IgD of this cell have the same binding specificity. Because of this ability to have these long transcripts, one now can make IgM and IgD at the same time in the cell. Those genes could also be processed to make the secreted version. We have this transcript that takes place, they remove these exons, put a polya site here and now end up witha secreted version. You can now have the same iso type to make a secreted version and a membrane version. The secreted version is very hydorphilic. But in contrast, the sequcnes encoded by these membrane regions, are very hydrophobic. This is tre for all isotypes. Each constant region is coprised of multiple exons. How are V and J joined? You should pay attention to these s RSSsequcnes and they mean recombination signal sequences. Also the enzymes called RAG enzymes called recombination activating genes. We find that associated with each V region and each J region is an RSS. There's a sequence which is called a heptamer and a nonamter and between is t a two-turn RSS. One of ther lules is that only a 23 and a 12 can come together to make a rearrangement. They'r eobligatory for this to take place. If you have a RAG knockout mouse, you don't make functional B cell receptors. They are obligatory for this recombination to take place. You need to remembe the 12 23 rule but not which DNA segment. you'll find that v lamda can never recombine with a j kappa because they're both 23. What generates all the diversity? There's variablitity in joining. You have this l.. you have to fill in all this so now yo have a blunt end. It was not present in the germline. Now... One of the most amazingt things is called somatic hypermutationYou should remember this enzyme because t it is required cytidine deadmidase. ...
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This note was uploaded on 12/31/2009 for the course MIMG 185 taught by Professor Zack during the Fall '06 term at UCLA.
- Fall '06