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Chapter 5 Organization and Expression of Ig Genes Oct 26 & 31, 2006 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 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 Cterminal end of Ab molecules 3. Different classes (or isotypes) of Ab (e.g., IgG and IgM) with identical V-region sequences (antigenic specificity) 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. 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. 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. Demonstration of DNA Deletion at an Ig Locus Non-B cells: sperm or liver cells B-cells Demonstration of DNA Deletion at an Ig Locus deleted sequence 比 大,因此在電泳 時跑得比較慢 Multigene organization of Ig genes l-Chain Multigene Family Mouse: V region: 2 Vl gene segments 4 Jl gene segments (3 are functional) C region: 4 Cl gene segments Human: 30 Vl, 4 Jl and 4 Cl segments κ-Chain Multigene Family Mouse: V region: ~ 85 Vk gene segments 5 Jk gene segments (4 are functional) C region: 1 Ck gene segment Human: 40 Vk, 5 Jk and 1 Ck segments H-Chain Multigene Family Mouse: V region: ~ 134 VH gene segments 13 DH gene segments 4 JH gene segments C region: 8 CH gene segments Human: 51 VH, 27 DH, 6 JH and 9 CH segments 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. 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. L-Chain DNA Undergoes V-J Rearrangements introns are removed Mechanism of V-region DNA Rearrangements 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 & JH gene segment Recombination Signal Sequences (RSS) Vλ Jλ C A C A G T G G T G T C A C 23 12 nt nt A C A A A A A C C T G T T T T T G G // One turn/two-turn joining rule The rule ensures that VH, DH, and JH segments join in proper order and that segments of the same type do not join each other. 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. Process of Recombination of Ig Gene Segments Double Strand Break Repair Terminal deoxynucleotidyl Transferase 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 Ig-gene Rearrangements May Be Nonproductive Imprecise Joining - productive and nonproductive rearrangements - productive rearrangement in one allele is enough !! !! - If rearrangement is not produced, the B cell dies by apoptosis. Only 1/3 attempts at VL – JL joining, and 1/3 subsequent attempts at VH – DHJH 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. Allelic Exclusion Ensures a Single Antigenic Specificity A single B cell is only specific for a single epitope !!! (1) * active alleles (2) 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. Generation of Ab Diversity 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 Junctional Flexibility Adds Diversity - 4 different joinings of Vk21- Jk1 in pre-B cell lines (Flexible) (Precise) 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. P-Addition Adds Diversity at Palindromic Sequences {Palindromic sequences} N-Addition Adds Considerable Diversity by Addition of Nucleotides add new (N) -nucleotides - 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 P-nucleotide 及 N-nucleotide addition 有些什麼優缺點? 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 ability to bind to the Ag. ----- Affinity Maturation Experimental Evidence for Somatic Mutation in V region of Ig Genes Most of the mutations are clustered in the CDR1 and CDR2 hypervariable region. 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 Class Switching Among C-Region Genes 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 VHDHJH unit can combine with any CH gene segment. This process is called class switching. Class (isotype) switching - Class-specific switch recombinases may bind to switch regions and facilitate DNA recombination. - Cytokines secreted by activated TH cells have been shown to induce B cells to class switch to a particular isotype. - IL-4, for example, induces class switching from Cm to Cg1 and then from Cg1 to Ce. Switch regions Class Switching from Cg1 to Ce Class Switching from Cm to Cg1 a circular excision product Expression of Ig Genes Co-expression of membrane forms of m and d H-chains by Alternative RNA Processing Expression of Membrane or Secreted Ig mRNAs 先暫時不考慮 Cd 的表現 (sIgM) (mIgM) Expression of Membrane or Secreted Ig mRNAs Expression of Membrane or Secreted IgM Molecules Therefore, processing of an Ig H-chain primary transcript can yield different mRNAs, which explains how a single B cell can produce secreted or membranebound forms of a particular Ig and simultaneously express IgM and IgD. Synthesis, Assembly, and Secretion of Igs Membrane Form of Igs Are Anchored to the Membrane Regulation of Ig-Gene Transcription Overview of B-cell Development and Ig Expression