Kuby Immunology 6/e
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Transcript Kuby Immunology 6/e
Chapter 7
Organization and Expression of Immunoglobulin Genes
Dr. Capers
Kindt • Goldsby • Osborne
Kuby IMMUNOLOGY
Sixth Edition
Chapter 5
Organization and Expression of
Immunoglobulin Genes
Copyright © 2007 by W. H. Freeman and Company
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 chain
During 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 – 1965
2 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 region
Proposed that there are only single copies of limited classes
Greater complexity was revealed later
Light 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
○D
○J
○C
VDJ segment codes for variable region
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 hypermutation
Gene conversion
CLASS-SWITCH recombination
IL-4 also involved
μ→δ→γ→ε→α
IgM→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 (antitumor 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