justin.ppt

• 1. The Organization and Expression of Ig Genes Justin Walter Immunology, Fall 2008

2. Introduction The vertebrate immune system is capable of responding to an essentially infinite array of foreign antigens Variable vs. constant regions Basis for variability organization and expression of Ig genes 3. Overview Historical perspective early theories Multigene organization of Ig genes Variable region gene rearrangements Basicsof mechanism key players: DNA signal sequences, specific enzymes Generation of Ab diversity sevenprimary routes 4. Historical perspective Ig sequence analysis revealed many dilemmas Extreme diversity of Ab specificity Variable regions vs constant regions Isotypes with similar Ag specificity but differing heavy-chain constant regions 5. Historical perspective Proponents of the one-gene-one-protein paradigm had trouble reconciling this model with the oddities of Igs. This led to an initial germ-line theory which suggested that a significant portion of the genome is dedicated solely to Ab coding. Argument: the immune system is THAT important 6. Historical perspective In contrast, somatic-variation theories emerged which suggested the opposite: Relatively small amount of Ig genes Specificity arises from mutation and/or recombination 7. Historical perspective Dreyer & Bennet (1965) Two-gene, one-protein model No precedent in any biological system 8. Historical perspective Tonegawa & Hozumi (1976) CompareIg DNA from embryonic (germline) and adult myeloma (somatic) cells Experimental data suggested that during differentiation, the V and C genes undergo rearrangement. 1987 Nobel prize 9. Tonegawa & Hozumi 10. Multigene Organization of Ig genes Each class of Ig components (kappa, lambda, heavy) encoded by separate multigene families on different chromosomes Each family contains several coding sequences, or gene segments 11. Multigene Organization of Ig genes 12. Multigene Organization of Ig genes & light chains: V (variable), J (joining), and C (constant) gene segments Heavy chains: V, D (diversity), J, and C gene segments A leader (L) sequence also precedes each V segment. Gene segments discovered by comparing DNA sequences with amino acid sequences of Igs Tonegawa, again. 13. Organization of Ig germ-line gene segments (mouse)Pre-rearrangement! 14. Variable-region rearrangement Multifaceted process, produces mature B cells which are committed to express specific Ab Specificity of Ab determined by the sequence of its rearranged variable genes. 15. Light-chain rearrangements V-J rearrangements Specificallowed rearrangements differ from species to species, but a big-picture view can suffice Rearrangement occurs in ordered steps but can be considered as random events which result in the random determination of Ab specificity 16. Kappa light-chain rearrangement & RNA processing Leader sequence targets nascent protein to ER and is subsequently cleaved 17. Heavy-chain rearrangements Requires two separate rearrangement events D-Jjoining V-DJ joining Differential polyadenylation & RNA splicing can result in mRNA with either Cu or C heavy chain genes B cells can express BOTH IgM and IgD with identical Ag specificity on its surface 18. Heavy-chain rearrangement The focus here is on IgM IgD B cells 19. Mechanism of Variable region DNA rearrangements Recombination signal sequences direct recombination 20. Mechanism of Variable region DNA rearrangements Recombination signal sequences direct recombinationV, JK, VH, JHVK, J, DH 21. Mechanism of Variable region DNA rearrangements A one-turn RSS can only join with a two- turn RSS Why might this be? 22. Mechanism of Variable region DNA rearrangements Gene segments are joined by a class of enzymes called recombinases Two recombination-activating genes encode proteins which act together to mediate V-(D)-J joining RAG-1 RAG-2 23. a. Sameb. oppositetranscriptional transcriptionalorientation orientation (most common) Addn. of P- nucleotides accomplished with repair enzymes 24. ? 25. Mechanism of Variable region DNA rearrangements Rearrangements may be productive or nonproductive. 26. Mechanism of Variable region DNA rearrangements Allelic exlusion Heavy-chain genes only expressed from one chromosome Light-chain genes only expressed from one chromosome Essential for specificity Expression of both alleles would result in a multispecific B cell 27. Mechanism of Variable region DNA rearrangements Allelic exlusion 28. Generation of Ab diversity Multiple germ-line gene segments Combinatorial V-(D)-J joining Junctional flexibility P-region nucleotide addition N-region nucleotide addition Somatic hypermutation Combinatorial association of light and heavy chains 29. Possibly as high as 1010! 30. Junctional flexibility 31. P/N-addition 32. Somatic hypermutation Nucleotide replacement, mediated by activation-induced cytidine deaminase (AID) Also plays a key role in class switching Frequency of 10-3 per bp per generation 100,000Xthe rate of spontaneous mutation! Approx 1 mutation every 2 cell divisions 33. Somatic hypermutation