Download + - Heavy chain gene

Immunoglobulin Genetics
Problem…the immune system makes over
one billion different antibody proteins
• In 1950’s: central dogma stated DNA—to RNA—to
protein
• One gene for each protein
• Required millions of genes just for the immune system
• Does not seem possible, but most scientists thought it
might be
• Today we know the human genome is less than 30,000
genes
• So, what is really going on???
B lymphocyte development
B lymphocyte development (2)
Immunoglobulin Genetics
History
• Same C region could associate with many V regions
– IgG Ab with different specificities
• Same V region could associate with many C regions
– Same idiotype on IgG and IgM Ab
• V and C regions coded for by separate genes
Genes for immunoglobulin proteins are
found on different chromosomes
Light Chain Gene Families
Germ line gene organization
Lambda light chain genes; n=30
V1
L
P
L
V2
P
L
Vn
J
1
P
C
1
J
2
E
J
3
C
2
E
C
3
J
4
E
C
4
E
Kappa light chain genes; n=300
L
P
V1
L
P
V2
L
P
Vn
J 1
J 2
J 3
J 4
J 5
C
E
Light Chain Gene Families
Gene rearrangement and expression
L
V 1
P
L
V 2
P
V n
L
P
L
V 1
P
L
V 2 J4
J 5
P
L
V J
DNA
C
Primary transcript
RNA
L V J C
mRNA
Translation
DNA
C
E
E
RNA Processing
C
E
DNA Rearrangement
Transcription
J 1 J 2 J 3 J 4 J 5
RNA
L V J C
Protein
Transport to ER
V J C
Protein
V
C
Heavy Chain Gene Family
Germ line gene organization
Heavy chain genes; Vn=1000, Dn=15
L
P
V1
L
V2
P
L
D1 D2 D3
Vn
Dn
J1 J2 J3 J4 J5
P
C
C
E
C
C
C
C
3
1
2
4
C
C
C
1
2
CH1 H CH2 CH3 CH4
Introns separate exons coding for H chain domains
Heavy Chain Gene Family
Gene rearrangement and expression
L
P
V1
L
V2
P
D1 D2 D3
Vn
L
Dn
C
J1 J2 J3 J4 J5
C
E
P
DJ rearrangement
L
V1
L
V2
L
D1 D2 J4 J5
Vn
C
C
DNA
P
P
P
E
VDJ rearrangement
L
V1
L
P
V2 D2 J4
P
C
J5
C
DNA
E
Transcription
L
V2 D2 J4
C
J5
E
C
Primary transcript
RNA
Heavy Chain Gene Family
Gene rearrangement and expression
L V2 D2 J4 J5
C
L V2 D2 J4 J5
C
Primary transcript
E
C
C
E
RNA Processing
L V DJ C
L V DJ C
An
An
mRNA for C
mRNA for C
Translation
L V DJ C
L V DJ C
C heavy chain
C heavy
chain
Transport to ER
V DJ C
V
C
C heavy chain
V DJ C
V
C
C heavy
chain
Mechanism of DNA Rearrangements
• Recombination
signal sequences
(RSS)
– Nonmer
– Heptamer
– 1 or 2 turn signals
• Rag-1 and Rag-2
Nonamer
Nonamer
CACAGTG 23 bp
ACAAAAACC
GGTTTTTGT
12 bp
CACTGTG
23 bp
TGTTTTTGG
CCAAAAACA
12 bp
GTGACAC
Heptamer
GTGTCAC
Two turn
RSS
2
J
2
One turn
RSS
1
Kappa light chains
Lambda light chains
L V
Heptamer
L V
C
J
1
1
Heavy chains
L V
J
D
2
1 1
2
C
2
C
Order of Ig Gene Expression
Progenitor B cell
1st
Heavy chain gene
+
2nd
VDJ recombination
No
Productive rearrangement ?
-
Apoptosis
Yes
Transcribe/translate
 () Heavy chain
Maternal
 or
Pre B cell

Paternal
Order of Ig Gene Expression


or
Pre B cell
Light chain gene
+

VJ recombination
1st - 
2nd - 
3rd - 
4 th
No
Productive rearrangement ?
4th 
-
Apoptosis
Yes
Transcribe/translate
Light chain
+

Mature B cell
Order of Ig Gene Expression
Explains:
• Why an individual B cell only produces one
kind of Ig with one kind of heavy and one
kind of light chain
• Why an individual B cell makes only makes
antibodies of one specificity
• Why there is allelic exclusion
Origin of Antibody Diversity
History
• Definition – Diversity (repitoire) is the total
of all the Ab specificities that an organism is
capable of expressing
• Theories
– Germ line theory
– Somatic mutation theory
Origin of Antibody Diversity
Current concepts
• Multiple V genes
• V-J and V-D-J joining
• Junctional diversity
• N region insertions
– Amino acid
sequences not
encoded in the germ
line
V gene
D region
V gene
D region
CCCCCC
T GG
CCCCCC
T GG
C C C T GG
Pro
C C C C GG
Trp
Pro
V gene
D region
CCCCCC
T GG
CCCCCG
Pro
Pro
Arg
Generation of N region insertions
D
J
Break DNA
J
D
TdT adds nucleotides
-
D GTCAATG
J
Ligate
-
-
D GTCAATG J
N region insertion
+
Intron
Origin of Antibody Diversity
Current concepts
• Multiple V genes
• V-J and V-D-J joining
• Junctional diversity
• N region insertions
• Somatic mutation
• Combinatorial association
• Multispecificity
Generation of Diversity
B cell receptor
(Immunoglobulin)
Heavy
Light
V gene segments
1000
300
D gene segments
15
-
J gene segments
4
4
N region insertion
++
-
Junctional diversity
+++
+
Somatic mutation
+
VxDxJ
1000 x 15 x 4
Total
6 x 104
Combinatorial association
• Ag independent process
+
VxJ
300 x 4
1.2 x 103
7.2 x 107
• Clonal selection
ANTIBODY GENE REARRANGEMENTS
CLASS SWITCHING:
Selection of CH Genes
Mechanism Unclear, but Involves
Switch Region
Switch Recombinase
Isotype Specific Cytokine Signals
Copyright © 2007 by W. H. Freeman and Company
Class Switching among Constant-Region Genes