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IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE
SEGMENTS
ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS
Chromosome 2
kappa light chain gene segments
Chromosome 22
lambda light chain gene segments
Chromosome 14
heavy chain gene segments
HOW MANY IMMUNOGLOBULIN GENE SEGMENTS
Gene segments
Light chain
kappa
Heavy chain
lambda
Variable (V)
40
30
65
Diversity (D)
0
0
27
Joining (J)
5
4
6
VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES
B cells of one individual
2
3
1
4
V-Domains
C-Domains
VH
D
JH
VL
VH-D-JH
JL
VL-JL
Estimates of combinatorial diversity
Taking account of functional V D and J genes:
65 VH x 27 DH x 6JH = 10,530 combinations
40 Vk x 5 Jk = 200combinations
30 Vl x 4 Jl = 120 combinations
= 320 different light chains
If H and L chains pair randomly as H2L2 i.e.
10,530x 320 = 3,369,600 possibilities
Due only to COMBINATORIAL diversity
In practice, some H + L combinations do not occur as they are unstable
Certain V and J genes are also used more frequently than others.
There are other mechanisms that add diversity at the junctions between genes
- JUNCTIONAL diversity
GENERATES A POTENTIAL B-CELL REPERTOIRE
Somatic recombination to generate
antibody diversity
Severe combined immunodeficiency (SCID)
Omenn syndrome - RAG deficiency
Lack of T-cells and B cells
Early manifestation
red rash on the face and shoulders,
infections with opportunistic pathogens. (Candida albicans, Pneumocystis carnii pneumonia)
Lack of palpable lymph nodes
How does somatic gene rearrangement
(recombination) work?
1.
How is an infinite diversity of specificity generated from finite amounts of DNA?
Combinatorial diversity
2.
How do V region find J regions and why don’t they join to C regions?
12-23 rule
-Special - Recobnitation Signal Sequences (RSS)
- Recognized by Recombination Activation Gene coded
proteins (RAGs)
PALINDROMIC SEQUENCES
HEPTAMER CACAGTG
CACAGTG
GTGACAC
GTGACAC
NONAMER ACAAAAACC
GGTTTTTGT
TGTTTTTGG CCAAAAACA
V, D, J flanking sequences
Sequencing upstream and downstream of V, D and J elements revealed
conserved sequences of 7, 23, 9 and 12 nucleotides in an arrangement that
depended upon the locus
Vl
7
Vk
7
23
12
7
23
9
12
9
7
12
9
9
9
VH
9
D
23
7
12
9
7
Jl
7
Jk
9
23
7
JH
Recombination signal sequences (RSS)
HEPTAMER - Always contiguous with
coding sequence
9
VH 7
23
9
VH
7
12
23
7
D
9
9
12
9
9
12
7
7
D
NONAMER - Separated from
the heptamer by a 12 or 23
nucleotide spacer
23
7
12
9
7
JH
9
23
7
JH
12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a
segment flanked by a 12mer RSS
Molecular explanation of the 12-23 rule
12-mer = one turn
23-mer = two turns
Intervening DNA
of any length
23
V 7
9
12
9
7D J
Molecular explanation of the 12-23 rule
V4
V1
V8
V9
V2
V7
V3
V6
V3
V4
V2
V5
9
9
• Heptamers and nonamers
align back-to-back
V7
V8
V9
V1
7
12-mer
7
• The shape generated by the
RSS’s acts as a target for
recombinases
V6
Loop of
intervening
DNA is
excised
D J
23-mer
V5
DJ
• An appropriate shape can not be formed if two 23-mer flanked elements attempted
to join (i.e. the 12-23 rule)
CONSEQUENCES OF RECOMBINATION
Generation of P-nucleotides
V4
V3
V5
V6
V2
9
9
23-mer
7
V1
7
12-mer
DJ
V7
V8
V9
Generation of N-nucleotides
V4
Terminal
deoxynucleotidyl
Transferase (TdT)
V3
V2
9
9
23-mer
7
V1
7
12-mer
DJ
V5
V6
Loop of
intervening
DNA is
excised
V7
V8
V9
Junctional Diversity
V
TCGACGTTATAT
AGCTGCAATATA D
J
TTTTT Germline-encoded nucleotides
TTTTT Palindromic (P) nucleotides - not in the germline
(N) encoded nucleotides - not in the
TTTTT Non-template
germline
Creates an essentially random sequence between the V region, D region and J region
in heavy chains and the V region and J region in light chains
How does somatic gene rearrangement
(recombination) work?
1.
How is an infinite diversity of specificity generated from finite amounts of
DNA?
Combinatorial diversity
2.
How do V region find J regions and why don’t they join to C regions?
12-23 rule
3.
How does the DNA break and rejoin?
Imprecisely, with the random removal and addition of nucleotides to
generate sequence diversity
Junctional diversity (P- and N- nucleotides, see above)