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Chapter 5 Organization and Expression of Ig Genes

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

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Chapter 5 Organization and Expression of Ig Genes

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  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. The vast diversity of antibody specificities • The presence in Ig heavy and light chains of a variable region at the amino-terminal end and a constant region at the carboxyl-terminal end • The existence of isotypes with the same antigenic specificity.

  8. 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.

  9. - 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.

  10. 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.

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

  12. 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. Southern blot

  14. Multigene organization of Ig genes

  15. l-Chain Multigene Family V region: 2 Vl gene segments 3 Jlgene segments(13 aa) C region: 3 Clgene segments – l1, l2, l3 subtypes (mouse) In humans: 30 Vl, 4 Jl and 4 Cl segments

  16. 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

  17. 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

  18. V-region gene rearrangements

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

  20. 51 26 6 1st rearrangement 2nd rearrangement A mature , immunocompetent B cell expresses both IgM & IgD with identical antigenic specificity on its surface.

  21. Mechanism of V-region DNA rearrangements

  22. Two unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment One-turn RSS: located at 3’ to each Vk, 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

  23. Recombination Signal Sequences (RSS)

  24. 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) (one-turn/two turn joining rule). • 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.

  25. Enzymatic Joining of Gene Segments Recombination-Activating Genes: RAG-1, RAG-2 - mediate V-(D)-J joining TdT: terminal deoxynucleotidyl transferase DSBR: double strand break repair enzymes

  26. TdT: Terminaldeoxynucleotidyltransferase DSBR: Double Strand Break Repair

  27. Inversional joining (signal joint): - two gene segments have opposite orientation • Deletional joining (coding joint): • two gene segments are in the same • transcriptional orientation

  28. deletion of the signal joint and intervening DNA as a circular excision product retention of both the coding joint and the signal joint (and inter- vening DNA) on the chromosome

  29. <15 nt

  30. Homework: If a mouse has a defect on RAG-1 or 2 , what will happen? If you knock out TdT or DSBR enzyme from a mouse, what will happen?

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

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

  33. Generation of Ab diversity

  34. 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

  35. Junctional Flexibility

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