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Immunology 2008 Lecture 10 Genetic Basis of Antibody Diversity 16 October

Immunology 2008 Lecture 10 Genetic Basis of Antibody Diversity 16 October. Genetic Basis of Antibody Diversity. OUTLINE PROBLEM OF ANTIBODY GENE DIVERSITY GERMLINE vs. SOMATIC THEORIES THREE GENE FAMILIES ( H-chains, kappa, lambda ) GENE ORGANIZATION V/J/D REARRANGEMENT

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Immunology 2008 Lecture 10 Genetic Basis of Antibody Diversity 16 October

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  1. Immunology 2008 Lecture 10 Genetic Basis of Antibody Diversity 16 October

  2. Genetic Basis of Antibody Diversity OUTLINE PROBLEM OF ANTIBODY GENE DIVERSITY GERMLINE vs. SOMATIC THEORIES THREE GENE FAMILIES (H-chains, kappa, lambda) GENE ORGANIZATION V/J/D REARRANGEMENT CLASS SWITCHING SOMATIC MUTATION SOUTHERN BLOT ANALYSIS OF GENE ORGANIZATION

  3. How to Account for Antibody Diversity? ● An Ab combining site is made up of one VL and one VH ● The specificity of any combining site is determined by its amino acid sequence. ● There exist at least, say, 106 unique combining sites How many V-region genes must exist? If each V-region requires a unique gene, we might need only 103 L + 103 H-chains (combinatorial association of VLand VH)

  4. Germ-Line versus Somatic Theories Germ-line: One germ-line gene exists for every V-region i.e. Need thousands of genes Somatic: One germ-line gene diversifies during development. i.e. Need as few as 3 genes (VH, Vk, Vl) How to Account for Antibody Diversity? So...who’s right? Everybody is! 1) Many germ-line genes exist (Germ-Line diversity) 2) These genes are diversified somatically (Somatic diversity)

  5. Organization of Human Immunoglobulin Genes Three Ig Gene Families: H-Chains, k-Chains, l-Chains Each family has V-(D)-J-C, varying numbers of each gene and differing details of organization

  6. unique...

  7. Organization of Human Immunoglobulin Genes

  8. common to many other genes

  9. Organization of Human Immunoglobulin Genes Memory: Ab-secreting cells switch from IgM to IgG…

  10. unique to Ig

  11. Summary: Sources of Antibody Diversity 1) ~300 germline Vk-genes 2) 5 Jk-segments “Combinatorial Joining” of Vk &Jk 300 Vk x 5 Jk = 1,500 3) DNA rearrangement is imprecise 1,500 x 10 = 15,000 4) Somatic mutation of rearranged V-genes 15,000 x 10 = 150,000 different Vk- regions 5) Heavy and light chains associate randomly “Combinatorial Association” 150,000 X 150,000 = ~2x1010 combining sites.

  12. Peter Parham, 1990

  13. T-CELL ANTIGEN RECEPTOR the only other rearranging DNA...  Members ofIg Superfamily  a/b chains (homologous to Ig L & H-chains)  Similar gene structure/rearrangement (VVV-[DDD]-JJJ-C...) However: (1) TCR is only membrane-bound (2) Monovalent (3) No somatic mutation

  14. Genetic Events in B-Cell Differentiation Surface markers: IgM/IgD… DNA markers – Southern Blotting

  15.  Cut DNA with EcoRI  Separate fragments by agarose gel electrophoresis (size)  Blot & hybridize with C-kappa specific probe

  16. TUESDAY Immunoglobulin Biosynthesis, Chapter 9 ABO & Rh Blood Groups, Chapter 10 (PSET 2 Answer key) WEDNESDAY Clinical Correlate #1: Monoclonal Gammopathies (Dr. Sudhir Gupta) FRIDAY Inbreeding, Appendix 8 MHC & Transplantation, Chapter 11

  17. Fin

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