Marion 4

Receptors in the immune system have incredible structural diversity along with constant structure that allows for consistent function. Took until early to mid 1970s to prove genetic structure- experiment by Tonegawa. Did Southern hybridization. Used sequence-specific endonucleases (restriction enzymes cleave at sitesof 3-6 nucleotides in length) . He used electrophoresis to separate the fragments on basis of size. This produced a smear of DNA on gel. Transfer to nitrocellulose and incubate with radiolabelled DNA fragment. Where fragment is homologous to DNA on nitrocellulose, get a band on radiograph. See slide 1 in PP Figs. Tonegawa took DNA from germ line (mouse liver DNA) and made 2 gels with 2 probes: one V region probe for a particular myeloma protein (plasma cytomas secrete myeloma proteins). Detected a band of a particular size. Same for a constant region probe- looked as though V and C region genes were separate. Took Myeloma DNA for protein and did same thing. Now with V and C region probes, identified same piece of DNA. Proved one protein originated from 2 pieces of DNA. Probe came from digesting cDNA from mRNA for the protein derived from the known protein sequence.
In order to generate the gene that encoded the antibody, something had to happen to bring pieces of DNA together. Somatic recombination.
What does light/heavy chain locus look like? See slide 2.
In humans, there are a series of 40 individual kappa genes separated by hundreds of kb. Jkappa there are 5, then space, then Ck constant region. Front- there is a leader sequence (L) at 5’ for each V region gene. For lambda, there are 30 variable region genes.
Slide 3: In germ line DNA, genes that can encode antibodies are scattered. To generate a functional gene for light and heavy chains, they have to be brought together. For light chains, 1 somatic recombination brings 1V with 1J. Choice seems to be random. Mechanism controlling recombination is controlled by 2 proteins: Recombination Activating Gene 1 and 2 (RAG 1 and 2) are the 2 proteins.
Slide 4: For every V, at 3’end, there is a 7-mer separated by 12 or 23 base pairs. For Vkappa, this is 12. Then there is a nonamer. This is RSS- recombination signal sequence. RSS is recognized by RAG1 and 2. Recognition obeys rule called. 12/23 rule. Somatic recombination puts together only one 12 with one 23 (representing one turn and 2 turns of DNA helix). Rule ensures correct function of somatic recombination machinery to put V with J correctly.
Slide 11: (This is from the nri1152.pdf article) Rag 1 and 2 generate a double-stranded break and make a DNA hairpin. Generates palindrome. Resolves hairpin by cutting randomly . Exonuclease at 3’ end of J region chews away nucleotides ramdomly. Then V and J ligated together. Ku70/80 DNA ligase 4 and DNA protein kinase are all essential for this. DNA protein kinase was discovered in SCID mouse (Mel Bosma). Slide 13: RAG mediated recombination- RAG binds to RSS, makes a double-stranded break, generates hairpin, RAg protein resolves hairpin by DSB in DNA on 5’end of J in palindromic region, Vin 3’ region, ligates, makes VJ. Because process is random, and nucleotides at ends are removed randomly, most likely to generate out of frame, useless coding sequence.
Slide 12: What happens to DNA in between? Thrown away. RAGs join RSSs to create a signal joint. A piece of circular DNA forms and is lost.
Heavy chain:
Slide 14:3 gene segments involved in formation of heavy chain variable region gene. Rag complex pulls out D and J first to make DJ joint. P nucleotides (palindromic) Exonuclease removal of nucleotides is random again, but another enzyme is involved called terminal deoxynucleotidyl transferase (TdT). Adds nucleotides without a template to 3’ end of a piece of DNA. Functions during heavy chain only. TdT transfers random number of nucleotides. Prefers G> C>T>A. Region between D and J often rich in G and C. Rag genes then add V.
Light: one recombination event. V-J.
Heavy: D-J, then V-DJ.
In humans, if you look at RSS, spacing seen as on slide 3. V does not go to J because of spacers in heavy chain. 23 only joins with 12.
Why not VD? DNA must be made accessible. Early B cell development- cryptic promoter upstream of D and J turns on before V promoter.
Consequence (other than generating coding sequence for light and heavy chain Ig):
FR (framework region)low variability. CDR(complementarity determining region)- high variability.
Once generated, transcribed. Primary transcript(see drawing on paper).
Heavy chain more complex. Tm is transmembrane exon.
Slide 9 and 16: What determines whether you get mu (membrane or secreted) or delta (membrane) is RNA splicing. Splicing determines which poly A site is incorporated into mRNA.Same cell can produce IgM and IgD with same variable region. This is unique to lymphocytes.
Slide 7:There are multiple classes: G(1-4), A(1,2) M, E,D. We know about M and D, but what about the others?
B cell becomes activated, it will die, become an antibody secreting plasma cell, or become a memory cell (long-lived).
Memory antibody response switches from IgM to IgG. Ab affinity goes up all the time in a process called affinity maturation. B cell activated undergoes differentiation. Can switch isotype it makes, and antibody may become better at binding. How would you keep specificity, but inprove affinity? 2 processes.
1. isotype switching
2. somatic mutation and clonal selection. Slide 8: Every round division, 1 mutation randomly inserted into variable region. Most likely to generate nonsense or missense. But occasionally mutation inserts and is functional. If it is likely to work better- selected for cloning. CDRs tend to accumulate mutations thorugh clonal selection. Improved cells are more likely to be selected. Mutations in FR interfere with beta sheets.
Slide 17: isotype switching:
In germline, mu and delta close together, then gamma3, gamma1, gamma2b, gamma2a, epsilon, alpha. In front of each is a switch signal sequence. S is repetitive sequence. What determines which is switched? Transcription. In B cell switching, signals from T cells and cytokines determine recognition of switch sequence.
Slide 15: T cell receptor:
alpha like light chain-VJ. Beta like heavy chain-VDJ. Has TdT added N sequence. These are alpha beta cells.
There are others- gammadelta T cells are more like innate immunity.Gamma like light chain with its own locus with V and J genes. Delta sits in middle of alpha locus – lots of Ds and Js with its own constant region. Alpha rearrangement eliminates D. Delta uses same V regions as alpha, but develops along a different pathway.
Diversity is generated by V gene pairs, random joining, N-P sequences. For T cells you get 1014-18 potential different cells. Amazing.
He will modify and reprint notes- added some powerpoint figures to blackboard as well. These are referenced in my notes above. Images are from Janeway text. Note to All: the fifth edition of the Janeway, et al.Immunobiology text is in the Books section of Pubmed if you can’t get to the library. He uses the 6th ed, but a lot of the information is the same.