1. The key experiment of Nobumichi Hozumi and Susumu Tonegawa

2. 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
Efficiency of somatic gene rearrangement?

3. 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 Ck
Non-productive rearrangement
Light chain has a second chance to make
a productive join using new V and J elements
Spliced mRNA transcript

4. Further diversity in the Ig heavy chainL 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, VL JL CL L
Each light chain requires 1 recombination events
VL to JL

5. SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS
120 VH
12 D
4 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

6. 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
Variable (V) / / /65
Diversity (D)
Joining (J)
Gene segments Light chain Heavy chain
kappa lambda

7. VH-D-JH VL-JL 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

8. How does somatic gene rearrangement
(recombination) work?
How is an infinite diversity of specificity generated from finite amounts of DNA?
Combinatorial diversity

9. 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, 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

10. 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
-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

11. Somatic recombination to generate
antibody diversity
Immunoglobulin genes are composed of separated segments of DNA that become joined together by a process called somatic recombination to make a functional gene. In heavy chain genes there are three gene segments, the variable or V segment, a diversity or D gene segment, and the joining or J segment. Light chain genes, such as those shown here, have only two gene segments - the V and the J segments. Gene segments that can be recombined have specific sequence motifs adjacent to them, called recombination signal sequence, or RSS motifs. A protein complex containing the products of the recombination activator genes, RAG1 and RAG2, binds specifically to the RSS motifs, in this example flanking a V gene segment and a J gene segment. The individual gene segments, to whose flanking RSS motifs the RAG protein complexes bind, are selected at random from a number of copies present at each gene locus. The Rag protein complexes bring together the gene segments to be recombined, and cleave the DNA exactly at the junction of the gene segment and it's adjoining RSS motif. The cleavage creates a hairpin of DNA at the ends of the gene segments and double stranded breaks at the ends of the RSS motifs. Additional proteins, DNA-dependent protein kinase, Ku, Artemis and a dimer of DNA ligase and XRCC4, are incorporated into a large complex with the RAG proteins. These RSS ends are joined, forming what is called the "signal joint", to create a closed circle of DNA that plays no further role in the recombination process. The DNA hairpins at the ends of the gene segments are then cleaved. An additional enzyme, terminal deoxynucleotidyl transferase or TdT, is recruited and adds additional nucleotides to the ends of the DNA strands The other enzymes in the complex ligate together the two ends of the gene segments, completing the recombination process.

12. Severe combined immunodeficiency syndrome (SCID).
SCID is characterized by a lack of functional T cells and B cells and the inability to make an adaptive immune response. Infants with SCID typically show infections with opportunistic pathogens. Panel a shows chronic Candida albicans infection in the mouth of an infant with SCID. SCID can be caused by various genetic defects, one of which is complete loss of RAG function. Panel b shows an infant with Omenn syndrome, a similar immunodeficiency which is due to a genetic defect that results in 80% loss of RAG activity. The bright red rash on the face and shoulders, which is due to chronic inflammation, is a characteristic of this condition. Unless an immune system can be reconstituted by bone marrow transplantation from a healthy donor, babies with SCID or Omenn syndrome die in infancy. Panel a courtesy of Fred Rosen; panel b courtesy of Luigi Notarangelo.
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

13. 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 D 7 12 9 VH 7 23 9 JH

14. 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

15. 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

16. 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
23-mer
12-mer V9 D J
An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the rule)

17. V1 D J 9 7 CONSEQUENCES OF RECOMBINATION Generation of P-nucleotides
23-mer
12-mer

18. V1 D J 9 7 Generation of N-nucleotides V4 V5
Terminal deoxynucleotidyl Transferase (TdT)
Loop of intervening
DNA is excised 7 9
23-mer
12-mer

19. 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, see above)

20. Mini-circle of DNA is permanently lost from the genome
Junctional diversity 7 12 9 23 7 12 9 23
Mini-circle of DNA is permanently lost from the genome
Signal joint
Coding joint V D J V D J
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.

21. V D J TCGACGTTATAT AGCTGCAATATA TTTTT Junctional Diversity
Germline-encoded nucleotides
Palindromic (P) nucleotides - not in the germline
Non-template (N) encoded nucleotides - not 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

22. GGGACAGGGGGC GlyThrGlyGly GlyGlnGly AspArgGly
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
GlyGlnGly
AspArgGly
Frame 1
Frame 2
Frame 3

23. RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION
Somatic rearrangement of Ig gene segments in a highly controlled manner
Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities
In each of us huge B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains
This potential B-cell repertoire is able to recognize a wide array of various antigens
INDEPENDENT ON ANTIGEN
OCCURS IN THE BONE MARROW

24. 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
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 co-dominantly, how could B cells shut down the genes on one of their chromosomes?
Allelic exclusion