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GENETIC BACKGROUND OF THE VARIABILITY OF ANTIGEN
RECOGNIZING RECEPTORS
The total number of antibody specificities available to an individual is known as the antibody repertoire, and in humans is at least 1011, perhaps many more.
BUTThere are an estimated only 20,000-25,000 human protein-coding genes.
Genetic background of antibody diversity
Germline theory (separate genes for each different Ab)
V2 C2 Vn CnV1 C1
1 GENE
Somatic diversification theory
(high rate of somatic mutations in the V-region)
V C
Gen
Protein
1 GENE = 1 PROTEIN
Dogma of molecular biology
Characteristics of immunoglobulin sequence
THEORIES
Molecular genetics of immunogloublins
• A single C region gene encoded in the GERMLINE and separate from the V region genes
• Multiple choices of V region genes available• A mechanism to rearrange V and C genes in the genome so that they can
fuse to form a complete Immunoglobulin gene.
In 1965, Dreyer & Bennett proposed that for a single isotype of antibody there may be:
How can the bifunctional nature of antibodies be explained genetically?
This was genetic heresy as it violated the then accepted notion that DNA was identical in every cell of an individual
The Dreyer - Bennett hypothesis
VV
VV
V
V
VV
V
V
VV
V
A mechanism to rearrange V and C genes in the genome exists so that they can fuse to form a complete Immunoglobulin gene
CV
C
A single C region gene is encoded in the germline and separated from the multiple V
region genes
Aim: Find a way to show the existence of multiple V genes and rearrangement to the C gene
Tools:
• cDNA probes to distinguish V from C regions
C
VV
VV
V
V
VV
V
Germline DNA
• Germline (e.g. placenta) and rearranged B cell DNA (e.g. from a myeloma B cell)
• DNA restriction enzymes to fragment DNA
CV
V
VV
V
Rearranged DNA
Approach
CV
V
VV
V
CV
V
V
VV
Size fractionate by gel
electrophoresis
VV
V V
CV
Blot with a V region probe
Blot with a C region probe
Cut myeloma B cell DNA with restriction enzymes
V and C probes detect the same fragmentSome V regions missing
C fragment is larger cf germline DNA
VV
V V
CV
Evidence for gene recombination
V V V
Blot with a V region probe
Blot with a C region probe
C
V V
VV
V V
Size fractionate by gel
electrophoresis
Germline DNA
There are many variable genes but only one constant gene
V CV V V
GERM LINE
V and C genes get close to each other in B-cells only
CV V V
B-CELL
Conclusion
PROTEIN
GENE Rearrangement of gene segments into a single functional unit (gene)
The gene rearrangement concept
• Germline configuration
• Gene segments need to be reassembled for expression
• Sequentially arrayed
• Occurs in the B-cells precursors in the bone marrow (soma)
• A source of diversity BEFORE exposure to antigen
Ig gene sequencing complicated the model
Structures of germline VL genes were similar for Vk, and Vl,however there was an anomaly between germline and rearranged DNA:
Where do the extra 13 amino acids
come from?
CLVL
~ 95 aa ~ 100 aa
L CLVL
~ 95 aa ~ 100 aa
JL
Extra amino acids provided by one of a
small set of J or JOINING regions
L
CLVL
~ 208 aa
L
The germline organization of the human immunoglobulin light-chain loci
Figure 2-15 part 1 of 2J-joining
CDR1 and CDR2 CDR3
During B-lymphocyte development
Jk Jκ Jκ JκVκ Vκ VκB-cell 1
JκVκB-cell 2
35 Vκ 5 Jκ
Vκ Vκ Vκ Vκ Jκ Jκ Jκ JκGermline
Somatic rearrangement of kappa (κ) chain gene segments
DNA
pACκEJJ
Vκ-Jκ
VκVκ P
CκJVκ Protein
mRNACκJVκ AAAA
Translation
Expression of the kappa chain
Primary RNA transcript
CκEJJVκLeader
• In developing B cells, the immunoglobulin genes undergo
structural rearrangements that permit their expression.
• The V domains of immunoglobulin light chains are encoded
in two (V and J) different kinds of gene segments, that are
brought into juxtaposition by recombination.
Further diversity in the Ig heavy chain
VL JL CLL
CHVH JHDHL
Heavy chain: between 0 and 8 additional amino acids between JH and CH
The D or DIVERSITY region
Each light chain requires one recombination event:VL to JL
Each heavy chain requires two recombination events:DH to JH and VH to DHJH
Heavy-chain V regions are constructed from three gene segments
The germline organization of the human immunoglobulin heavy-chain loci
*
During B-cell development
VH2 JH JH
40 VH 6 JH
VH1 VH3 D JH JH
23 D
D DD
JH JH
JH JHD D
SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS
D DVH1 VH2 VH3
VH1 VH2
• The V domains of immunoglobulin heavy chains are encoded
in three (V, D and J) different kinds of gene segments, that are
brought into juxtaposition by recombination.
The numbers of functional gene segments available to construct the variable and constant regions of human
immunoglobulin heavy chains and light chains
VH D JH
VL JL
V-Domains
C-Domains
VH-D-JH VL-JL
Variability of B-cell antigen receptors and antibodies
B cells of one individual 1 2 3 4
Estimates of combinatorial diversityTaking account of functional V D and J genes:
46 VH x 23 D x 6JH = 6,348 combinations
38 V k x 5 Jk = 190 combinations33 V l x 5 Jl = 165 combinations
= 355 different light chains
If H and L chains pair randomly as H2L2 i.e. 6,348 x 355 = 2,253,540 possibilities
Due only to COMBINATORIAL diversity
In practice, some H + L combinations do not occur as they are unstableCertain V and J genes are also used more frequently than others.
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?
-Special - Recombitation Signal Sequences (RSS)
- Recognized by Recombination-Activating Genes coded proteins (RAGs)
12-23 rule
V, D, J flanking sequences
Vl 7 23 9
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
Vk 7 12 9 Jk7239
Jl7129
D7129 7 12 9
VH 7 23 9 JH7239
Recombination signal sequences (RSS)
12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS
VH 7 23 9
D7129 7 12 9
JH7239
HEPTAMER - Always contiguous with coding sequence
NONAMER - Separated fromthe heptamer by a 12 or 23
nucleotide spacer
VH 7 23 9
D7129 7 12 9
JH7239
23-mer = two turns 12-mer = one turn
Molecular explanation of the 12-23 rule
Intervening DNAof any length23
V 9712
D J79
23-mer
12-mer
Loop of intervening
DNA is excised
• Heptamers and nonamers align back-to-back
• The shape generated by the RSS’s acts as a target for recombinases
7
9
97
V1 V2 V3 V4
V8V7
V6 V5
V9 D J
V1 D J
V2
V3V4
V8
V7
V6
V5
V9
• An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the 12-23 rule)
Molecular explanation of the 12-23 rule
Gene segments encoding the variable region are joined by recombination at recombination signal sequences
23-mer
12-mer
V1 D J
V2
V3V4
V8
V7
V6
V5
V9
7
9
97
Consequences of recombinationGeneration of P-nucleotides
23-mer
12-mer
Loop of interveningDNA is excised
V1 D J
V2
V3V4
V8
V7
V6
V5
V9
7
9
97
Terminal deoxynucleotidyl Transferase (TdT)
Generation of N-nucleotides
Junctional diversity increases diversity by 6 orders of magnitude
Hipervariable and framework regions exist within the variable domains of Igs
HV3 in the light-chain is at the junction between rearranged V and J segments
In the heavy chain HV3 is formed by the D segment and the residues between the rearranged V and D segments and the D andJ segments .
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)