chapter 5 organization and expression of ig genes oct 26 & 31, 2006
TRANSCRIPT
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Chapter 5 Organization and Expression of Ig Genes
Oct 26 & 31, 2006
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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
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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)
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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.
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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.
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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.
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Demonstration of DNA Deletion at an Ig Locus
Non-B cells:
sperm or liver cells
B-cells
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Demonstration of DNA Deletion at an Ig Locus
deleted sequence
比 大,因此在電泳時跑得比較慢
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Multigene organization of Ig genes
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-Chain Multigene Family
Mouse:
V region: 2 VV gene segments 4 JJgene segments (3 are functional) C region: 4 CCgene segments
Human: 30 VV, 4 JJ and 4 CC segments
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κ-Chain Multigene Family
Mouse:
V region: ~ 85 VV gene segments 5 JJgene segments (4 are functional) C region: 1 CCgene segment
Human: 40 VV, 5 JJ and 1 CC segments
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-Chain Multigene Family
Mouse:
V region: ~ 134 VV gene segments 13 DDHH gene segments 4 JJHHgene segments C region: 8 CCgene segments
Human: 51 VV, 27 DDHH, 6 JJ and 9 CC segments
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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.
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H-Chain DNA Undergoes V-D-J Rearrangements
(1st rearrangement)
(2nd rearrangement)
A mature , immunocompetentB cell expresses both IgM & IgD with identical antigenic specificity on its surface.
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L-Chain DNA Undergoes V-J Rearrangements
introns are removed
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Mechanism of V-region DNA Rearrangements
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Two unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment
One-turn RSS: located at 3’ to each V5’ to each Jand both sides of each DH gene segment
Two-turn RSS: located at 3’ to each V & VH and
5’ to each J & JH gene segment
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Recombination Signal Sequences (RSS)
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CACAGTG
23
nt
ACAAAAACC
GTGTCAC
12
nt
TGTTTTTGG
Vλ Jλ
//
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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.
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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.
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Process of Recombination of Ig Gene Segments
Double Strand Break Repair
Terminal deoxy-nucleotidyl Transferase
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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
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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
!!
!!
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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.
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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
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Generation of Ab Diversity
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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
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Junctional Flexibility Adds Diversity- 4 different joinings of V21- J1 in pre-B cell lines
(Flexible) (Precise)
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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.
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P-Addition Adds Diversity at Palindromic Sequences
{Palindromic sequences}
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N-Addition Adds Considerable Diversityby Addition of Nucleotides
add new (N) -nucleotides
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- 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
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P-nucleotide 及 N-nucleotide addition
有些什麼優缺點?
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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 MaturationAffinity Maturation
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Experimental Evidence for Somatic Mutation in V region of Ig Genes
Most of the mutations are clustered in the CDR1 and CDR2 hypervariable region.
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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
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Class Switching Among C-Region Genes
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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.
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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 C to C1 and then from C1 to C.
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Switch regions Class Switching from C to C1
Class Switching from C1 to C a circular excision product
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Expression of Ig Genes
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Co-expression of membrane forms of and H-chains by Alternative RNA Processing
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Expression of Membrane or Secreted Ig mRNAs
先暫時不考慮 C 的表現
(sIgM) (mIgM)
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Expression of Membrane or Secreted Ig mRNAs
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Expression of Membrane or Secreted IgM Molecules
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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.
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Synthesis, Assembly, and Secretion of Igs
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Membrane Form of Igs Are Anchored to the Membrane
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Regulation of Ig-Gene Transcription
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Overview of B-cell Development and Ig Expression