Immunoglobulin Genes

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Chapter 5 Organization and Expression of Ig
Genes
l chain
k chain (n 85)
H chain (n 134)
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• 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.

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• Unique features of Ig genes (2)
• The Ig gene segments carried in the germ cells
• cant 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 108 combinations of V region.
• Each B cell has a unique combination and is
• antigenically committed to a specific epitope.

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• 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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• Chapter 5
• 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

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Genetic Model Compatible with Ig Structure
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Germ-line theory The genome contributed
by the germ cells, egg and sperm, contains a
large repertoire of Ig genes. Somatic-variati
on 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.
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- 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.
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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.
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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.
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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.
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Structure of Embryonic and Myeloma
k Chain DNA
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Multigene organization of Ig genes
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l-Chain Multigene Family
V region 2 Vl gene segments
3 Jl gene segments C region 3 Cl gene segments
l1, l2, l3 subtypes (mouse) In humans 30 Vl,
4 Jl and 4 Cl segments
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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
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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
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V-region gene rearrangements
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V Region gene rearrangements - The
H-chain V-region genes rearrange first, then
the L-chain V-region genes. - The
rearrangements are random events
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V-D-J Rearrangements in H-Chain DNA
(1st rearrangement)
(2nd rearrangement)
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V-J Rearrangements in L-Chain DNA
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Mechanism of V-region DNA rearrangements
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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

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Recombination Signal Sequences (RSS)
(palindromic)
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• 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).
• 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.

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Enzymatic Joining of Gene Segments Recombination-
Activating Genes RAG-1, RAG-2 -
mediate V-(D)-J joining
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• Deletional joining (coding joint)
• - two gene segments are in the
• same transcriptional orientation
• - deletion of the signal joint and
• intervening DNA as a circular
• excision product
• (b) Inversional joining (signal joint)
• - two gene segments have opposite
• orientation
• - retention of both the coding joint
• and the signal joint (and inter-
• vening DNA) on the chromosome
• TdT
• Terminal deoxynucleotidyl transferase
• DSBR
• Double Strand Break Repair

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Figure 4-7
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Figure 4-7 part 1 of 3
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Figure 4-7 part 2 of 3
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Figure 4-7 part 3 of 3
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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
and form a normal signal joint -
cannot properly join the coding sequences
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• Imprecise Joining
• productive and nonproductive
• rearrangements
• productive rearrangement in
• one allele is enough
• If rearrangement is not
• produced, the B cell dies by
• apoptosis.

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Allelic Exclusion
A single B cell is only specific for a single
epitope !!!
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Generation of Ab diversity
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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
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Junctional Flexibility
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Four different joinings of Vk21- Jk1
(Precise)
(Flexible)
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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.
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P-Addition
Palindromic sequences
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N-Addition
Palindromic sequences N-nucleotides
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• - 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

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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/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
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A secondary lymphoid follicle consisting of a
large germinal center
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A Lymph Node
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The Spleen
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Peyers Patch
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Experimental evidence for somatic mutation in V
region of Ig genes
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These data show

• The frequency of mutation increases in the course
  of the primary response (day 7 vs. day 14)
• Frequency of mutation is higher after secondary
  and tertiary immunizations than after primary
  immunization.
• The dissociation constant decreases during the
  transition from the primary to tertiary responses

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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 (8262 heavy X 320 light)
2,644,240
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Class Switching Among C-Region Genes
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Organization of H chain
Cm
V region
C region
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• 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)

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Switch regions

• Class switching needs 3 elements
• Switch regions
• Switch recombinase
• Cytokines

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Heavy-chain primary transcripts undergo
differential RNA processing
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sIgM
mIgM
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Expression of Membrane or Secreted IgM
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Simultaneous Expression of IgM and IgD (a)
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Simultaneous Expression of IgM and IgD (b)
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Synthesis, Assembly, and Secretion of Igs
Assembly of light and heavy chains occurs in
the cisternae of RER
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Regulation of Ig-Gene Transcription
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Three major classes of cis regulatory sequences
in DNA

• Promoter TATA box about 200 nucleotides upstream
  from initiation site. (B-cell promoters have an
  octomer sequence that is specific to B-cells)
• Enhancers Nucleotide sequences situated some
  distance upstream of downstream from a gene.
  Activatates transcription from the promoter
  sequence
• Silencers Nucleotide sequences that down
  regulate transcription.

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Regulation of Ig-Gene Transcription
Promoters
Enhancers
Silencers