Transcript Defects in Ig-Gene Rearrangements

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