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Organization and Expression of Immunoglobulin Genes

Organization and Expression of Immunoglobulin Genes. 미생물학교실 권 형 주. B-Cell Development. Generation of antibody diversity. Environment : 10 7 – 10 9 개 이상의 antigenic shapes (non-self)  Genome 에 존재하는 antibody 이상의 antibody 생산이 요구됨. How can all these diversity be generated?

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Organization and Expression of Immunoglobulin Genes

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  1. Organization and Expression of Immunoglobulin Genes 미생물학교실 권 형 주

  2. B-Cell Development

  3. Generation of antibody diversity Environment : 107 – 109개 이상의 antigenic shapes (non-self)  Genome에 존재하는 antibody 이상의 antibody 생산이 요구됨 How can all these diversity be generated? - Germ-line theories - Somatic-variation theories • Ehrlich side-chain theory • : antigen-induced selection • Instructive hypothesis • : flexible antibody molecule •  acted on by antigen to form • a complementary binding site • - Dreyer & Bennett : existence of a constant region • and a variable region •  encoded by at least two different genes (??) •  Tonegawa : somatic recombination •  somatic mutation

  4. Tonegawa’s bombshell-Immunoglobulin gene rearrange Detection of Ig gene rearrangement by Southern blot hybridization - Embryonic cell (nonlymphoid cell)의 Ig DNA - Committed cells of B lymphocyte lineage (adult myeloma cells) -- VJ rearrangement (pre-B cell) : somatic recombination

  5. Multigene Organization of Ig Genes Mouse

  6. Variable-Region Gene Rearrangements Light-chain DNA undergoes V-J rearrangements

  7. Heavy-chain DNA undergoes V-D-J rearrangements

  8. Mechanism of Variable-Region DNA Rearrangements - Recombination signal sequences (RSSs) direct recombination

  9. - Gene segments are joined by recombinases V(D)J recombinase : recombination-activating genes RAG-1 and RAG-2 Circular DNA isolated from thymocytes in which the DNA encoding the chains of the TCR undergoes rearrangement in a process like that involving the Ig genes

  10. Ig-gene rearrangements may be productive or nonproductive • Allelic exclusion ensures a single antigenic specificity • Allelic exclusion: A single specificity is maintained because only one of the two parental • alleles of Ig is expressed by every B cell clone from its earliest maturation stage

  11. Generation of antibody diversity • Multiple germ-line gene segments • Combinatorial V-J and V-D-J joining generates diversity

  12. Junctional flexibility adds diversity • P-addition adds diversity • at palindromic sequences • N-addition adds considerable diversity • by addition of nucleotides

  13. Somatic hypermutation adds diversity in already-rearranged gene segments Somatic hypermutation : Immunoglobulin heavy and light chain genes undergo structural modifications after antigen stimulation • Antigenic stimulation 후에 발생되는 structural alteration • light chain 과 heavy chain 의 variable region 에서 • single base change • Germinal center- cells which have produced • a higher-affinity antibody are selected for survival • - Dependent on T cells and germinal center • - Athymic mice lack T cells and germinal center • - no affinity maturation • Affinity maturation

  14. Immunoglobulin gene diversification differs among species

  15. Class switching among constant-region genes • Switching regions : 2 – 3 kb upstream from each CH segment (except Cd) Proposedmechanism for class switching induced by IL-4 in rearranged Ig heavy chain genes

  16. AID (activation-induced cytidine deaminase) mediates both somatic hypermutation and class switching • AID : RNA editing enzyme • : C  U, repair G-C  A-T Experimental demonstration of the role of the enzyme AID in class switching and hypermutation

  17. Expression of Ig Genes • Heavy-chain primary transcripts • undergo differential RNA processing • : Alternative RNA processing

  18. Simultaneous expression of IgM and IgD

  19. Synthesis, assembly, and secretion of Igs

  20. Regulation of Ig-Gene transcription • Promoter : AT-rich sequence (TATA box) • oct-2 (found only in B cells) • - Enhancer : in an orientation-independent manner • Ig-gene expression is inhibited in T cells : Ig-gene rearrangement (H, L chain –only in B cells : TCR-gene rearrangement- T cells  why????, k-chain 3’ enhancer(3’kE) mutation  Ig-genePU.1 binding site

  21. Antibody genes and antibody engineering Human antibody from mice bearing a human artificial chromosome (HAC) that includes entire human heavy- and light-chain loci.

  22. scFv : single-chain fragment variable • Phage display technology

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