Transcript Document

The key experiment of Nobumichi Hozumi and Susumu Tonegawa
EXPRESSION OF THE KAPPA CHAIN
Vκ
P
Vκ J
pA
J
E
Cκ
J
E
Cκ
Vκ-Jκ
Leader
Vκ J
Primary RNA transcript
Vκ J
Cκ
AAAA
mRNA
Translation
Vκ J
Cκ
Protein
Efficiency of somatic gene rearrangement?
Ig light chain rearrangement: Rescue pathway
There is only a 1:3 chance of the join between the V and J
region being in frame
Vk
Jk
Non-productive rearrangement
Light chain has a second chance to make
a productive join using new V and J elements
Spliced mRNA transcript
Ck
Further diversity in the Ig heavy chain
L VH DH JH
CH
The heavy chain was found to have further amino acids (0 – 8)
between the JH és CH genes
D (DIVERSITY) region
Each heavy chain requires 2 recombination events
JH to DH and VH to JHDH,
L VL
JL
CL
Each light chain requires 1 recombination events
VL to JL
SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE
SEGMENTS
120 VH
VH1
VH2
12 D
VH3
D
D
D
4 JH
D
JH JH JH JH
During B-cell development
VH1
VH2
VH3
VH1
D
D JH JH
VH2
D
D JH JH
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
Heavy chain
kappa
lambda
Variable (V)
132/40
105/30
123/65
Diversity (D)
0
0
27
Joining (J)
5
4
9
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
How does somatic gene rearrangement
(recombination) work?
1.
How is an infinite diversity of specificity generated from finite amounts of
DNA?
Combinatorial diversity
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 = 200 combinations
30 Vl x 4 Jl = 120 combinations
= 320 different light chains
If H and L chains pair randomly as H2L2 i.e.
10,530 x 320 = 3,369600 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.
GENERATES A POTENTIAL B-CELL REPERTOIRE
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
NONAMER
CACAGTG
GTGACAC
ACAAAAACC
TGTTTTTGG
CACAGTG
GTGACAC
GGTTTTTGT
CCAAAAACA
Somatic recombination to generate
antibody diversity
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

VH
7
12
9
23
7
D 7
9
12
9
9
12
NONAMER - Separated from
the heptamer by a 12 or 23
9
nucleotide spacer
7
D
7 JH
23
7
12
9
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
23
V7
Intervening DNA
of any length
9
12
9
7D J
Molecular explanation of the 12-23 rule
V4
V1
V8
V9
V3
V2
V7
V6
V3
V4
V2
V5
9
9
23-mer
• Heptamers and nonamers
align back-to-back
V6
Loop of
intervening
DNA is
excised
DJ
V7
V8
V9
V1
7
12-mer
7
• The shape generated by the
RSS’s acts as a target for
recombinases
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
V5
V3
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
7
12-mer
V1
V5
DJ
V6
Loop of
intervening
DNA is
excised
V7
V8
V9
Severe combined immunodeficiency
syndrome (SCID).
Omen syndrome RAG deficiency
Early onset
loose bowel movements
Red scaly rashes all over the body
Opportunistic infections (Candida albicans, Pneumocystis carnii pneumonia)
No 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
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)
Junctional diversity
Mini-circle of DNA is
permanently lost from the
genome
9
7
V
7
12
23
9
9
23
Coding joint
7
7
12
9
Signal joint
DJ
VDJ
Imprecise and random events that occur when the DNA breaks and
rejoins allows new nucleotides to be inserted or lost from the
sequence at and around the coding joint.
Junctional Diversity
V
TCGACGTTATAT
AGCTGCAATATA D
J
TTTTT Germline-encoded nucleotides
TTTTT Palindromic (P) nucleotides - not in the germline
(N) encoded nucleotides - not
TTTTT Non-template
in the 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
Reading D segment in 3 frames
Analysis of D region from different antibodies show
that the same D region can be translated in all three frames to make
different protein sequences and hence antibody specificities
GGGACAGGGGGC
GlyThrGlyGly
Frame 1
GGGACAGGGGGC
GlyGlnGly
Frame 2
GGGACAGGGGGC
AspArgGly
Frame 3
RESULT OF SOMATIC GENE REARRANGEMENT AND
ALLELIC EXCLUSION
1. Somatic rearrangement of Ig gene segments occurs in a highly
controlled manner
2. Single B-cells become committed to the synthesis of one unique
H-chain and one unique L-chain variable domain, which
determine their specificities
3. In each of us a huge B-cell repertoire is generated consisting of
B-cell clones with different H- and L-chain variable domains
4. This potential B-cell repertoire is able to recognize a wide array
of antigens
INDEPENDENT FROM ANTIGEN
OCCURS IN THE BONE MARROW
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
How B cells express one light chain species and one heavy chain species even
though every B cell possesses a maternal and paternal locus of both genes.
Since all other genes known at the time appeared to be expressed codominantly, how could B cells shut down the genes on one of their
chromosomes?
Allelic exclusion