Download Kuby Immunology 6/e - Dr. Jennifer Capers

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