1. Chapter 5 Organization and Expression of Ig Genes Oct 26 & 31, 2006

2. 你需要學習的課題 : 1. 抗體基因是如何組成的？ 2. 抗體基因重組 (rearrangement) 的機制 3. 抗體的多樣性 (diversity) 是如何產生的？ 4. 細胞膜上的抗體如何轉變為分泌性抗體？ 5. 抗體的類別 (class) 如何變換？ - class switching

3. Central Feature of Ab Molecules: 1.Vast diversity of Ab specificities 2.A variable (V) region at the N-terminal end and a constant (C) region at the C- terminal end of Ab molecules 3.Different classes (or isotypes) of Ab (e.g., IgG and IgM) with identical V-region sequences (antigenic specificity)

4. The Two-gene model of Dryer and Bennett (1965) Two separate genes encode a single Ig H or L chain, one gene for the V region and the other for the C region.

5. The suggestion that two genes encoded a single polypeptide contradicted the existing one gene-one polypeptide principle and was without precedent ( 先例 ) in any known biological system.

6. Verification of the Dryer and Bennet Hypothesis (by Tonegawa and Hozumi, 1976) First direct evidence that separate genes encode the V and C regions of Ig and that the genes are rearranged in the course of B-cell differentiation. - Tonegawa was awarded the Nobel Prize for this work in 1987.

7. Demonstration of DNA Deletion at an Ig Locus Non-B cells: sperm or liver cells B-cells

8. Demonstration of DNA Deletion at an Ig Locus deleted sequence 比 大，因此在電泳 時跑得比較慢

9. Multigene organization of Ig genes

11. -Chain Multigene Family Mouse: V V region: 2 V gene segments J 4 J  gene segments (3 are functional) C C region: 4 C  gene segments V J C Human: 30 V, 4 J and 4 C segments

12. κ-Chain Multigene Family Mouse: V  V region: ~ 85 V  gene segments J  5 J  gene segments (4 are functional) C  C region: 1 C   gene segment V  J  C  Human: 40 V , 5 J  and 1 C  segments

13.  -Chain Multigene Family Mouse: V  V region: ~ 134 V  gene segments D H 13 D H gene segments J H 4 J H  gene segments C  C region: 8 C   gene segments V  D H J  C  Human: 51 V , 27 D H, 6 J  and 9 C  segments

14. V-Region Gene Rearrangements - The H-chain V-region genes rearrange first, then the L-chain V-region genes. - The rearrangements occur in an ordered sequence, but they are random events. - The arrangements of Ig and TCR genes are the only known site-specific DNA rearrangements in vertebrates.

15. H-Chain DNA Undergoes V-D-J Rearrangements (1st rearrangement) (2nd rearrangement) A mature, immunocompetent B cell expresses both IgM & IgD with identical antigenic specificity on its surface.

16. L-Chain DNA Undergoes V-J Rearrangements introns are removed

17. Mechanism of V-region DNA Rearrangements

18. Two unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment One-turn RSS: located at 3’ to each V  5’ to each J  and both sides of each D H gene segment Two-turn RSS: located at 3’ to each V & V H and 5’ to each J  & J H gene segment

19. Recombination Signal Sequences (RSS)

20. C A C A G T G 23 nt A C A A A A A C C G T G T C A C 12 nt T G T T T T T G G V λ J λ //

21. One turn/two-turn joining rule The rule ensures that V H, D H, and J H segments join in proper order and that segments of the same type do not join each other.

22. Gene Segments Are Joined by Recombinases - Recombination-Activating Genes: RAG-1, RAG-2 - The proteins encoded by RAG-1 and RAG-2 act synergistically and are required to mediate V-(D)-J joining. - Terminal deoxynucleotidyl transferase (TdT), another lymphoid-specific gene product, is also involved in V-(D)-J rearrangement.

23. Process of Recombination of Ig Gene Segments Double Strand Break Repair Terminal deoxy- nucleotidyl Transferase

24. Defects in Ig-Gene Rearrangements RAG-1 -/- or RAG-2 -/- mice: - lack RAG-1 or RAG-2 - cannot start the recombination process SCID (severe combined immunodeficiency) mice: - lack double strand break repair (DSBR) enzymes - can carry out synapsis, introduce d.s. breaks - cannot properly join the coding sequences

25. Imprecise Joining - productive and nonproductive rearrangements - productive rearrangement in one allele is enough - If rearrangement is not produced, the B cell dies by apoptosis. Ig-gene Rearrangements May Be Nonproductive !!

26. Only 1/3 attempts at V L – J L joining, and 1/3 subsequent attempts at V H – D H J H joining, are productive. As a result, < 1/9 (11%) of the early-stage pre-B cells in the bone marrow progress to maturity and leave the bone marrow as mature immunocompetent B cells.

27. Allelic Exclusion Ensures a Single Antigenic Specificity A single B cell is only specific for a single epitope !!! Once a productive rearrangement is attained, its encoded protein is expressed and the presence of this protein acts as a signal to prevent further gene rearrangement. (1)(2) * active alleles

28. Generation of Ab Diversity

29. Antibody Diversity Seven means of generation of Ab diversity: 1. Multiple germ-line V, D, and J gene segments 2. Combinatorial V-(D)-J joining 3. Junctional flexibility 4. P-region nucleotide addition (P-addition) 5. N-region nucleotide addition (N-addition) 6. Somatic hypermutation 7. Combinatorial association of light and heavy chains

31. Junctional Flexibility Adds Diversity - 4 different joinings of V  21- J  1 in pre-B cell lines (Flexible)(Precise)

32. Since CDR3 makes a major contribution to Ag binding by the Ab molecule, amino acid changes generated by junctional flexibility can make a major contribution to Ab diversity.

33. P-Addition Adds Diversity at Palindromic Sequences {Palindromic sequences}

34. N-Addition Adds Considerable Diversity by Addition of Nucleotides add new (N) -nucleotides

35. - Up to 15 N-nucleotides can be added to both the D H - J H and V H - D H J H joints. - Thus, a complete H - chain V region is encoded by a V H ND H NJ H unit. - N regions appears to consist of wholly random sequences

36. P-nucleotide 及 N-nucleotide addition 有些什麼優缺點？

37. Somatic Hypermutation Adds Diversity in Already-rearranged Gene Segment - Somatic hypermutation occurs only within germinal centers, structures that form in secondary lymphoid organs within a week or so of immunization with an Ag that activates a T-cell-dependent B-cell response. - Somatic hypermutation occurs at a frequency approaching 10 -3 /bp/generation. This rate is at least 100,000-fold higher than the spontaneous mutation rate, about 10 -8 /bp /generation, in other genes. - B cells with higher-affinity Ig receptors will be preferentially selected for survival because of their greater Affinity Maturation ability to bind to the Ag. ----- Affinity Maturation

38. Experimental Evidence for Somatic Mutation in V region of Ig Genes Most of the mutations are clustered in the CDR1 and CDR2 hypervariable region.

39. Antibody Diversity Seven means of generation of Ab diversity: 1. Multiple germ-line V, D, and J gene segments 2. Combinatorial V-(D)-J joining 3. Junctional flexibility 4. P-region nucleotide addition (P-addition) 5. N-region nucleotide addition (N-addition) 6. Somatic hypermutation – after Ag stimulation 7. Combinatorial association of light and heavy chains

40. Class Switching Among C-Region Genes

41. Organization of H chain V region C region After antigenic stimulation of a B cell, the H-chain DNA can undergo a further rearrangement in which the V H D H J H unit can combine with any C H gene segment. This process is called class switching.

42. Class (isotype) switching - Class-specific switch recombinases may bind to switch regions and facilitate DNA recombination. - Cytokines secreted by activated T H cells have been shown to induce B cells to class switch to a particular isotype. - IL-4, for example, induces class switching from C  to C  1 and then from C  1 to C .

43. Switch regions Class Switching from C  to C  1 Class Switching from C  1 to C  a circular excision product

44. Expression of Ig Genes

45. Co-expression of membrane forms of  and  H-chains by Alternative RNA Processing

46. Expression of Membrane or Secreted Ig mRNAs 先暫時不考慮 C  的表現 (sIgM) (mIgM)

47. Expression of Membrane or Secreted Ig mRNAs

49. Expression of Membrane or Secreted IgM Molecules

50. Therefore, processing of an Ig H-chain primary transcript can yield different mRNAs, which explains how a single B cell can produce secreted or membrane- bound forms of a particular Ig and simultaneously express IgM and IgD.

51. Synthesis, Assembly, and Secretion of Igs

52. Membrane Form of Igs Are Anchored to the Membrane

53. Regulation of Ig-Gene Transcription

54. Overview of B-cell Development and Ig Expression