Chapter 5 Organization and Expression of Ig Genes

1. Chapter 5 Organization and Expression of Ig Genes l chain k chain (n= ~85) H chain (n= ~134)

2. Unique features of Ig genes (1) • Vertebrates can respond to a limitless array of • foreign proteins. • Every Ab molecule contains a unique a.a.sequence • in its V region, but only one of a limited number of • invariable sequences in its C region. • Germ-line DNA contains multiple gene segments • encode portions of a single Ig H or L chain.

3. Unique features of Ig genes (2) • The Ig gene segments carried in the germ cells • can’t be transcribed & translated into H & L chains • until they are rearranged into functional genes. • During B-cell maturation in the bone marrow, • Ig gene segments are rearranged and generated • into more than 1010 combinations of V region. • Each B cell has a unique combination and is • antigenically committed to a specific epitope.

4. Unique features of Ig genes (3) • Mature B cells no longer contain identical • chromosomal DNA to germ-line DNA. • After antigenic stimulation, further rearrangement • of C-region gene segments can generate changes • in isotypes without changing the specificity of Ig. • Genomic rearrangement is an essential feature • of lymphocyte differentiation, and no other • vertebrate cell type has been shown to undergo • this process.

5. 本章大綱: • Genetic model compatible with Ig structure • Multigene organization of Ig genes • V-region gene rearrangements • Mechanism of V-region DNA rearrangements • Generation of Ab diversity • Class switching among C-region genes • Expression of Ig genes • Regulation of Ig-gene transcription • Ab genes and Ab engineering

6. Genetic Model Compatible with Ig Structure

7. Germ-line theory: The genome contributed by the germ cells, egg and sperm, contains a large repertoire of Ig genes. Somatic-variation theory: The genome contains a small number of Ig genes, from which a large number of Ab specificities are generated in the somatic cells by mutation or recombination.

8. - How could stability be maintained in the C region while some kind of diversifying mechanism generated the V region? - There must be mechanisms not only for generating Ab diversity but also for maintaining constancy. - Neither the germ-line nor the somatic variation theory could offer a reasonable explanation of the central feature of Ig structure.

9. 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.

10. The suggestion that two genes encoded a single polypeptide contradicted the existingone gene-one polypeptideprinciple and was without precedent in any known biological system.

11. 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.

13. Structure of Embryonic and Myeloma k Chain DNA

14. Multigene organization of Ig genes

16. l-Chain Multigene Family V region: 2 Vl gene segments 3 Jlgene segments C region: 3 Clgene segments – l1, l2, l3 subtypes (mouse) In humans: 30 Vl, 4 Jl and 4 Cl segments

17. k-Chain Multigene Family V region: 85 Vk gene segments 4 Jkgene segments C region: 1 Ckgene segments (mouse) In humans: 40 Vk, 5 Jk and 1 Ck segments

18. H-Chain Multigene Family V region: 134 VH gene segments 13 DH gene segments 4 JHgene segments C region: 8 CHgene segments (mouse) In humans: 51 VH, 27 DH, 6 JH and 9 CH segments

19. V-region gene rearrangements

20. V Region gene rearrangements - The H-chain V-region genes rearrange first, then the L-chain V-region genes. - The rearrangements are random events

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

22. V-J Rearrangements in L-Chain DNA

23. Mechanism of V-region DNA rearrangements

24. Two unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment One-turn RSS: located at 3’ to eachVk, 5’ to each Jl, and both sides of each DH gene segment Two-turn RSS:located at 3’ to each Vl & VH and 5’ to each Jk & JHgene segment

25. Recombination Signal Sequences (RSS)

26. One turn/two-turn joining rule • - Signal sequences havinga one-turn spacer (12 bp) • can join only with sequences having a two-turn • spacer (23 bp). • This joining rule ensures that a VL segment joins • only to a JL segment and not to another VL segment. • The rule likewise ensures that VH, DH, and JH • segments join in proper order and that segments • of the same type do not join each other.

27. Enzymatic Joining of Gene Segments Recombination-Activating Genes: RAG-1, RAG-2 - mediate V-(D)-J joining

28. Deletional joining (coding joint): • - two gene segments are in the • same transcriptional orientation • - deletion of the signal joint and • intervening DNA as a circular • excision product • (b) Inversional joining (signal joint): • - two gene segments have opposite • orientation • - retention of both the coding joint • and the signal joint (and inter- • vening DNA) on the chromosome • TdT: • Terminaldeoxynucleotidyltransferase • DSBR: • Double Strand Break Repair

29. 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 and form a normal signal joint - cannot properly join the coding sequences

30. Imprecise Joining • productive and nonproductive • rearrangements • productive rearrangement in • one allele is enough • If rearrangement is not • produced, the B cell dies by • apoptosis.

31. Allelic Exclusion A single B cell is only specific for a single epitope !!!

32. Generation of Ab diversity

33. 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

38. Junctional Flexibility

39. Four different joinings of Vk21- Jk1 (Precise) (Flexible)

40. 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.

41. P-Addition {Palindromicsequences}

42. N-Addition {Palindromicsequences} N-nucleotides

43. - Up to 15 N-nucleotides can be added to both • the DH-JH and VH-DHJH joints. • Thus, a complete H-chain V region is encoded • by a VHNDHNJH unit. • N regions appears to consist of wholly random • sequences

44. Somatic Hypermutation -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 100,000s-fold higher than the spontaneous mutation rate, about 10-8/bp/peneration, in other genes. - B cells with higher-affinity Ig receptors will be preferentially selected for survival because of their greater ability to bind to the Ag. ----- Affinity Maturation

45. A secondary lymphoid follicle consisting of a large germinal center

46. A Lymph Node

47. The Spleen

48. Peyer’s Patch

49. Experimental evidence for somatic mutation in V region of Ig genes

50. 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