chapter 7 organization and expression of immunoglobulin genes dr. capers
TRANSCRIPT
Chapter 7Organization and Expression of Immunoglobulin
GenesDr. Capers
Kuby IMMUNOLOGYSixth Edition
Chapter 5Organization and Expression of
Immunoglobulin Genes
Copyright © 2007 by W. H. Freeman and Company
Kindt • Goldsby • Osborne
How does antibody diversity arise?
What causes the difference in amino acid sequences?
How can different heavy chain constant regions be associated with the same variable regions?
In germ-line DNA, multiple gene segments code portions of single immunoglobulin heavy or light chainDuring B cell maturation and
stimulation, gene segments are shuffled leaving coding sequence for only 1 functional heavy chain and light chain○ Chromosomal DNA in mature B cells is not
the same as germ-line DNA
Dreyer and Bennett – 19652 separate genes encode single
immunoglobulin heavy or light chain○ 1 for the variable region
Proposed there are hundreds or thousands of these
○ 1 for the constant regionProposed that there are only single copies of limited
classes
Greater complexity was revealed laterLight chains and heavy chains (separate
multi-gene families) are located on different chromosomes
DNA rearrangement: produces variable region
Later mRNA splicing: produces constant region
Kappa (κ) and lamda (λ) light chain segments:○ L – leader peptide, guides through ER○ V VJ segment codes for variable region
○ J○ C – constant region
Heavy chain○ L ○ V VDJ segment codes for variable region
○ D○ J○ C
Variable-region gene rearrangements
Variable-region gene rearrangements occur during B-cell maturation in bone marrow○ Heavy-chain variable region genes rearrange
first○ Then light-chain variable region○ In the end, B cell contains single functional
variable-region DNA sequence
○ Heavy chain rearrangement (“class switching”) happens after stimulation of B cell
Mechanism of Variable-Region DNA rearrangements Recombination signal sequences
(RSSs)○ Between V, D, and J segments○ Signal for recombination○ 2 kinds
- 12 base pairs (bp) – 1 turn of DNA- 23 bp – 2 turns of DNA- 12 can only join to 23 and vice versa
Mechanism of Variable-Region DNA rearrangements
Catalyzed by enzymes○ V(D)J recombinase
Proteins mediate V-(D)-J joining○ RAG-1 and RAG-2
Gene arrangements may be nonproductive○ Imprecise joining can occur so that reading frame is not complete○ Estimated that less than 1/9 of early pre-B cells progress to
maturity
Gene rearrangement video: http://www.youtube.com/watch?v=AxIMmNByqtM
Look at Figure 7-8 – VDJ recombination○ 1. Recognition of RSS by RAG1/RAG2 enzyme complex○ 2. One-strand cleavage at junction of coding and signal sequences○ 3. Formation of V and J hairpins and blunt signal end○ 4. ligation of blunt signal end to form signal joint
- 2 triangles on each end (RSS) are joined○ 5. Hairpin cleavage of V and J regions○ 6. P nucleotide addition (palindromic nucleotide addition – same if
read 5’ to 3’ on one strand or the other○ 7. Ligation of light V and J regions (joining)○ 8. Exonuclease trimming (in heavy chain)
- Trims edges of V region DNA joints
○ 9. N nucleotide addition (non-templated nucloetides)○ 10. Ligation and repair
Allelic Exclusion
Ensures that the rearranged heavy and light chain genes from only 1 chromosome are expressed
Generation of Antibody Diversity Multiple germ-line gene segments Combinatorial V-(D)-J joining Junctional flexibility P-region nucleotide addition N-region nucleotide addition Somatic hypermutation Combinatorial association of light and
heavy chains
○ This is mainly in mice and humans – other studied species differ in development of diversification
Ab diversity – Multiple gene-line segments AND combination of those segments
Ab diveristy – junctional flexibility Random joining of V-(D)-J segments
○ Imprecise joining can result in nonproductive rearrangements
○ However, imprecise joining can result in new functional rearrangements
Ab diversity – P-addition and N-addition
Ab diversity – somatic hypermutation Mutation occurs with much higher
frequency in these genes than in other genes
Normally happens in germinal centers in lymphoid tissue
Class Switching Isotype switching After antigenic stimulation of B cell VHDHJH until combines with CH gene
segment Activation-induced cytidine deaminase
(AID)Somatic hypermutationGene conversionCLASS-SWITCH recombination
IL-4 also involved
μ→δ→γ→ε→αIgM→IgD→IgG→IgE→IgA
Ig Gene Transcripts
Processing of immunoglobulin heavy chain primary transcript can yield several different mRNAs○ Explains how single B cell can have
secreted and membrane bound Ab
Regulation of Ig-Gene Transcription 2 major classes of cis regulatory sequences in DNA
regulate Promoters – promote RNA transcription in specific
direction Enhancers – help activate transcription
Gene rearrangement brings the promoter and enhancer closer together, accelerating transcription
Antibody Engineering Monoclonal Abs used for
many clinical reasons (anti- tumor Ab, for instance)
If developed in mice, might produce immune response when injected
○ Can be cleared in which they will not be efficient
○ Can create allergic response
Creating chimeric Abs or humanized Abs are beneficial
Rearrangement of TCR genes Similar to that of Ig
Rearrangement of α and γ chains○ V, J, and C segments
Rearrangement of β and δ chains○ V, D, J, and C segments
Generation of TCR diversity (a lot like Ig)○ Multiple germ-line gene segments○ Combinatorial V-(D)-J joining○ Junctional flexibility○ P-region nucleotide addition○ N-region nucleotide addition○ Combinatorial association of light and heavy
chains However, there is no somatic
mutation with TCR○ May be to ensure that after thymic
selection, the TCR doesn’t change to cause self-reactive T cell