Transcript Document

Chapter 5
Organization and Expression of Ig Genes
l chain
k chain (n= ~85)
H chain (n= ~134)
Unique features of Ig genes (1)
- Vertebrates can respond to a limitless array of
foreign proteins.
- Every Ab molecule contains a unique a.a.sequence
in its V region, but only one of a limited number of
invariable sequences in its C region.
- Germ-line DNA contains multiple gene segments
encode portions of a single Ig H or L chain.
Unique features of Ig genes (2)
- The Ig gene segments carried in the germ cells
can’t be transcribed & translated into H & L chains
until they are rearranged into functional genes.
- During B-cell maturation in the bone marrow,
Ig gene segments are rearranged and generated
into more than 1010 combinations of V region.
- Each B cell has a unique combination and is
antigenically committed to a specific epitope.
Unique features of Ig genes (3)
- Mature B cells no longer contain identical
chromosomal DNA to germ-line DNA.
- After antigenic stimulation, further rearrangement
of C-region gene segments can generate changes
in isotypes without changing the specificity of Ig.
- Genomic rearrangement is an essential feature
of lymphocyte differentiation, and no other
vertebrate cell type has been shown to undergo
this process.
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Genetic model compatible with Ig structure
Multigene organization of Ig genes
V-region gene rearrangements
Mechanism of V-region DNA rearrangements
Generation of Ab diversity
Class switching among C-region genes
Expression of Ig genes
Regulation of Ig-gene transcription
Ab genes and Ab engineering
Genetic Model Compatible with Ig Structure
• The vast diversity of antibody specificities
• The presence in Ig heavy and light chains
of a variable region at the amino-terminal
end and a constant region at the carboxylterminal end
• The existence of isotypes with the same
antigenic specificity.
Germ-line theory:
The genome contributed by the germ cells,
egg and sperm, contains a large repertoire
of Ig genes.
Somatic-variation theory:
The genome contains a small number of Ig
genes, from which a large number of Ab
specificities are generated in the somatic cells
by mutation or recombination.
- How could stability be maintained in the
C region while some kind of diversifying
mechanism generated the V region?
- There must be mechanisms not only for
generating Ab diversity but also for
maintaining constancy.
- Neither the germ-line nor the somatic variation
theory could offer a reasonable explanation of
the central feature of Ig structure.
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 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.
Southern blot
Multigene organization of Ig genes
l-Chain Multigene Family
V region: 2 Vl gene segments
3 Jl gene segments(13 aa)
C region: 3 Cl gene segments – l1, l2, l3 subtypes (mouse)
In humans: 30 Vl, 4 Jl and 4 Cl segments
k-Chain Multigene Family
V region: 85 Vk gene segments
4 Jk gene segments
C region: 1 Ck gene segments (mouse)
In humans: 40 Vk, 5 Jk and 1 Ck segments
H-Chain Multigene Family
V region: 134 VH gene segments
13 DH gene segments
4 JH gene segments
C region:
8 CH gene segments (mouse)
In humans: 51 VH, 27 DH, 6 JH and 9 CH segments
V-region gene rearrangements
V Region gene rearrangements
- The H-chain V-region genes rearrange first,
then the L-chain V-region genes.
- The rearrangements are random events
51
26
6
1st rearrangement
2nd rearrangement
A mature , immunocompetent
B cell expresses both IgM &
IgD with identical antigenic
specificity on its surface.
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)
One turn/two-turn joining rule
- Signal sequences having a one-turn spacer (12 bp)
can join only with sequences having a two-turn
spacer (23 bp) (one-turn/two turn joining rule).
- This joining rule ensures that a VL segment joins
only to a JL segment and not to another VL segment.
- The rule likewise ensures that VH, DH, and JH
segments join in proper order and that segments
of the same type do not join each other.
Enzymatic Joining of Gene Segments
Recombination-Activating Genes: RAG-1, RAG-2
- mediate V-(D)-J joining
TdT: terminal deoxynucleotidyl transferase
DSBR: double strand break repair enzymes
TdT:
Terminal deoxynucleotidyl transferase
DSBR:
Double Strand Break Repair
Inversional joining (signal joint):
Deletional joining (coding joint):
-two gene segments are in the same - two gene segments have opposite
orientation
transcriptional orientation
deletion of the signal joint and
intervening DNA as a circular
excision product
retention of both the coding joint
and the signal joint (and intervening DNA) on the chromosome
<15 nt
Homework:
If a mouse has a defect on RAG-1 or 2 , what will happen?
If you knock out TdT or DSBR enzyme from a mouse, what
will happen?
Imprecise Joining
- productive and nonproductive
rearrangements
- productive rearrangement in
one allele is enough
- If rearrangement is not
produced, the B cell dies by
apoptosis.
Allelic Exclusion
A single B cell is only
specific for a single
epitope !!!
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
- 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
Somatic Hypermutation
- 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 100,000s-fold higher than the
spontaneous mutation rate, about 10-8/bp/peneration, 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
A Lymph Node
The Spleen
Peyer’s Patch
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
Class (isotype) switching
- Class-specific recombinase proteins 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 induces Cm to Cg1 or Ce (Chapter 11)
Expression of Ig Genes
Regulation of Ig-Gene Transcription