1. OF THE VARIABILITY OF ANTIGEN RECOGNIZING RECEPTORS
GENETIC BACKGROUND
OF THE VARIABILITY OF ANTIGEN RECOGNIZING RECEPTORS

2. There are an estimated only 20,000-25,000 human protein-coding genes.
Genetic background of antibody diversity
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.
BUT
There are an estimated only 20,000-25,000 human protein-coding genes.

3. Dogma of molecular biology Characteristics of immunoglobulin sequence
Gen
Protein
1 GENE = 1 PROTEIN
Dogma of molecular biology
Characteristics of immunoglobulin sequence
THEORIES
1 GENE
Somatic diversification theory
(high rate of somatic mutations in the V-region) V C
Before it was possible to examine the immunoglobulin genes directly, there were two main hypotheses for the origin of this diversity. The germline theory held that there is a separate gene for each different immunoglobulin chain and that the antibody repertoire is largely inherited. By contrast, somatic diversification theory proposed that the observed repertoire is generated from a limited number of inherited V-region sequences that undergo alteration within B cells during the individual's lifetime.
Germline theory (separate genes for each different Ab) V2 C2 Vn Cn V1 C1

4. Molecular genetics of immunogloublins
How can the bifunctional nature of antibodies be explained genetically?
In 1965, Dreyer & Bennett proposed that for a single isotype of antibody there may be:
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.
Dreyer and Bennett (1965) proposed a hypothesis that the V and C regions of immunoglobulin proteins are encoded by separate genes and that these two genes, namely, V and C genes, are brought together during differentiation of lymphocytes.
This was genetic heresy as it violated the then accepted notion that DNA was identical in every cell of an individual

5. The Dreyer - Bennett hypothesisC
A single C region gene is encoded in the germline and separated from the multiple V region genes V
A mechanism to rearrange V and C genes in the genome exists so that they can fuse to form a complete Immunoglobulin gene C V V
Aim: Find a way to show the existence of multiple V genes and rearrangement to the C gene

6. Approach DNA restriction enzymes to fragment DNA C V Germline DNA C V
Rearranged DNA
Tools:
cDNA probes to distinguish V from C regions
Germline (e.g. placenta) and rearranged B cell DNA (e.g. from a myeloma B cell)

7. Evidence for gene recombination
Cut myeloma B cell DNA with restriction enzymes V
Blot with a V region probe
Blot with a C region probe C
Size fractionate by gel electrophoresis
Germline DNA
Size fractionate by gel electrophoresis V C
Blot with a V region probe
Blot with a C region probe
V and C probes detect the same fragment
Some V regions missing
C fragment is larger cf germline DNA

8. Conclusion
There are many variable genes but only one constant gene V C
GERM LINE
V and C genes get close to each other in B-cells only C V
B-CELL
In nonlymphoid cells, the gene segments encoding the greater part of the V region of an immunoglobulin chain are some considerable distance away from the sequence encoding the C region. In mature B lymphocytes, however, the assembled V-region sequence lies much nearer the C region, as a consequence of gene rearrangement.
Rearrangement of gene segments into a single functional unit (gene)
PROTEIN
GENE

10. 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

11. 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: CL VL
~ 95 aa
~ 100 aa L CL VL
~ 95 aa
~ 100 aa JL
Extra amino acids provided by one of a small set of J or JOINING regions L CL VL
~ 208 aa L
Where do the extra 13 amino acids come from?

12. The germline organization of the human immunoglobulin
light-chain loci
The upper row shows the λ light-chain locus, which has about 30 functional Vλ gene segments and four pairs of functional Jλ gene segments and Cλ gene segments. The κ locus (lower row) is organized in a similar way, with about 35 functional Vκ gene segments accompanied by a cluster of five Jκ gene segments but with a single Cκ gene segment. In approximately half of the human population, the entire cluster of Vκ gene segments is duplicated (not shown, for simplicity).

13. Figure 2-15 part 1 of 2 J-joining CDR1 and CDR2 CDR3
Light-chain V-region genes are constructed from two segments. A variable (V) and a joining (J) gene segment in the genomic DNA are joined to form a complete light-chain V-region exon. Immunoglobulin chains are extra-cellular proteins and the V gene segment is preceded by an exon encoding a leader peptide (L), which directs the protein into the cell's secretory pathways and is then cleaved. The light-chain C region is encoded in a separate exon and is joined to the V-region exon by splicing of the light-chain RNA to remove the L-to-V and the J-to-C introns.
Variability is in V gene segments is in the sequences encoding the first and second hypervariable regions - the third hypervariable region is encoded by the junction of V and J.
CDR1 and CDR2
CDR3

14. Somatic rearrangement of kappa (κ) chain gene segmentsJκ Vκ
B-cell 2
35 Vκ
5 Jκ Vκ Jκ
Germline
During B-lymphocyte development Jk Jκ Vκ
B-cell 1
DNA

15. Expression of the kappa chain
Vκ-Jκ Vκ P pA Cκ E J
Primary RNA transcript Cκ E J Vκ
Leader
mRNA Cκ J Vκ
AAAA
Translation Cκ J Vκ
Protein

16. 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.

17. Further diversity in the Ig heavy chainVH JH DH L
Heavy chain: between 0 and 8 additional amino acids between JH and CH
The D or DIVERSITY region
Each heavy chain requires two recombination events:
DH to JH and VH to DHJH VL JL CL L
Each light chain requires one recombination event:
VL to JL

18. Heavy-chain V regions are constructed from three gene segments
Heavy-chain V regions are constructed from three gene segments. First the diversity (D) and J gene segments join, then the V gene segment joins to the combined DJ sequence, forming a complete heavy-chain V-region (VH) exon. For simplicity, only the first of the heavy-chain genes, Cμ, is shown here. Each immunoglobulin domain is encoded by a separate exon, and two additional membrane-coding exons (MC, colored light blue) specify the hydrophobic sequence that will anchor the heavy chain to the B-cell membrane.

19. The germline organization of the human immunoglobulin
heavy-chain loci *
The heavy-chain locus has about 40 functional VH gene segments, a cluster of about 23 D segments and 6 JH gene segments. For simplicity, a single CH gene (CH1–9) is shown in this diagram to represent the nine C genes. The diagram is not to scale: the total length of the heavy-chain locus is more than 2 megabases (2 million bases), whereas some of the D segments are only six bases long. L, leader sequence.
The organization of the heavy-chain locus, on chromosome 14, resembles that of the κ locus, with separate clusters of VH, DH, and JH gene segments and of CH genes.

20. SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS
40 VH
23 D
6 JH
VH1
VH2
VH3 D D D D JH JH JH JH D
VH1
VH2
VH3
During B-cell development JH
VH1
VH2 JH D

21. 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.

22. The numbers of functional gene segments available to
construct the variable and constant regions of human
immunoglobulin heavy chains and light chains

23. Variability of B-cell antigen receptors and antibodies
B cells of one individual VH D JH VL JL
V-Domains
C-Domains
VH-D-JH
VL-JL

24. Estimates of combinatorial diversity
Taking account of functional V D and J genes:
46 VH x 23 D x 6JH = 6,348 combinations
38 Vk x 5 Jk = 190 combinations
33 Vl 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 unstable
Certain V and J genes are also used more frequently than others.

25. How does somatic gene rearrangement
(recombination) work?
How is an infinite diversity of specificity generated from finite amounts of DNA?
Combinatorial diversity
How do V region find J regions and why don’t they join to C regions?
12-23 rule
-Special - Recombitation Signal Sequences (RSS)
- Recognized by Recombination-Activating Genes coded proteins (RAGs)

26. 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 23 9 Jl 7 12 9 Vk 7 12 9 Jk 7 23 9
There are two types of RSS. One consists of a nonamer (9 bp, shown in purple) and a heptamer (7 bp, shown in green) separated by a spacer of 12 bp (white). The other consists of the same 9- and 7-nucleotide sequences separated by a 23-bp spacer (white). D 7 12 9 VH 7 23 9 JH

27. Recombination signal sequences (RSS)
HEPTAMER - Always contiguous with coding sequence
NONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacer VH 7 23 9 D 12 JH   VH 7 23 9 D 12 JH
12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

28. Molecular explanation of the 12-23 rule
23-mer = two turns
12-mer = one turn
Intervening DNA
of any length 23 V 9 7 12 D J 7 9

29. Molecular explanation of the 12-23 ruleV1 D J V2 V3 V4 V8 V7 V6 V5 V9 V1 V2 V3 V4
Loop of intervening
DNA is excised V8 V7 V6 V5
Heptamers and nonamers align back-to-back
The shape generated by the RSS’s acts as a target for recombinases 7 9 V9 D J
23-mer
12-mer
An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the rule)

30. by recombination at recombination signal sequences
Gene segments encoding the variable region are joined
by recombination at recombination signal sequences
The recombination between a V (red) and a J (yellow) segment of a light-chain gene is shown here. A RAG complex (green) binds to one of the recombination signal sequences (RSSs) flanking the coding sequences to be joined. The RAG complex then recruits the other RSS. This process brings an RSS containing a 12-bp spacer together with one containing a 23-bp spacer. This is known as the 12/23 rule and ensures that gene segments are joined in the correct order. The DNA molecules are broken at the ends of the heptamer sequences (orange) and are then joined together with different topologies. The region of DNA that was originally between the V and J segments to be joined is excised as a small circle of DNA that has no function. The joint made in forming this circle is called the signal joint. Within the chromosomal DNA, the V and J segments are joined to form the coding joint. Formation of this joint involves the introduction of additional variability into the nucleotide sequence around the joint. Enzymes other than RAG involved in these processes are represented in blue. Nonamers are shown in purple, heptamers in orange, spacers in white.

31. Consequences of recombination
Generation of P-nucleotides V1 D J V2 V3 V4 V8 V7 V6 V5 V9 7 9
23-mer
12-mer

32. Junctional diversity increases diversity by
Generation of N-nucleotides V1 D J V2 V3 V4 V8 V7 V6 V5 V9
Terminal deoxynucleotidyl Transferase (TdT)
Loop of intervening
DNA is excised 7 9
23-mer
12-mer
The process is illustrated for a D to J rearrangement. The RSSs are brought together and the RAG complex cleaves (arrows) between the heptamer sequences and the gene segments (top panel). This leads to excision of the DNA that separates the D and J segments. The ends of the two DNA strands of the D and J segments are joined to form hairpins. Further cleavage (arrows) on one DNA strand of the D and J segments releases the hairpins and generates short single-stranded sequences at the ends of the D and J segments. The extra nucleotides are known as P nucleotides because they make a palindromic sequence in the final double-stranded DNA (as indicated on the diagram). Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the ends of the single strands. These nucleotides, which are not encoded in the germline, are known as N nucleotides. The single strands pair, and through the action of exonuclease, DNA polymerase, and DNA ligase the double-stranded DNA molecule is repaired to give the coding joint.
Junctional diversity increases diversity by
6 orders of magnitude

33. 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 and
J segments .
The domains of Ig heavy and light chains are shown here. The V regions of each polypeptide are encoded by different gene segments. The locations of intrachain and interchain disulfide bonds (S-S) are approximate. Areas in the dashed boxes are the hypervariable (complementarity-determining) regions. In the Ig μ chain, transmembrane (TM) and cytoplasmic (CYT) domains are encoded by separate exons.

34. How does somatic gene rearrangement
(recombination) work?
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? rule
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)