1. GENETIC BACKGROUND OF ANTIBODY DIVERSITY

2. STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES COMPLEMENT ACTIVATION
Heavy chain (H) VH VL CH
Light chain (L) CL
Antigen
Antigen binding
Variable domains
COMPLEMENT ACTIVATION
BINDING TO CELLS
DEGRADATION
TRANSPORT
Constans domains
Effector functions

3. AMINO ACID SEQUENCE OF IMMUNOGLOBULINS
Multiple myeloma (MM)
Plasma cell tumors – tumor cells reside in the bone marrow
Produce immunoglobulins of monoclonal origin, serum concentration mg/ml
Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification
AMINO ACID SEQUENCE OF IMMUNOGLOBULINS
Gel electrophoresis
Reduction
L H
50 kDa
Heavy chain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
25 kDa
Light chain
Variable
Constant

4. GENETIC BACKGROUND OF ANTIBODY DIVERSITYVL VH
S – S
S – S
Mechanism of the generation of variability?
Different rules for encoding the variable and constant regions?
Symmetric molecule  two identical VH and VL  both chromosomes encode for the same sequence?

5. V C V2 C Vn V1 DOGMA OF MOLECULAR BIOLOGY
Gen
Protein
1 GEN = 1 PROTEIN
DOGMA OF MOLECULAR BIOLOGY
CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE
THEORIES
1 GEN
High rate of somatic mutations in the V-region V C
Many GENES ( – ) V2 C Vn V1

6. 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.
This was genetic heresy as it violated the then accepted notion that DNA was identical in every cell of an individual

7. Proof of 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
Find a way to show the existence of multiple V genes and rearrangement to the C gene

8. Approach C V Germline DNA C V Rearranged DNA Tools:
A set of cDNA probes to specifically distinguish V regions from C regions
DNA restriction enzymes to fragment DNA
Examples of germline (e.g. placenta) and mature B cell DNA (e.g. a plasmacytoma/myeloma)

9. The experiment of Susumi Tonegawa 1976
V-CmRNS probe
CmRNS probe * *
B-cell V C
Embryonal cell

10. CONCLUSION
V and C genes get close to each other in B-cells only C V
B-CELL
There are many variable genes but only one constant gene V C
GERM LINE
GÉN SZEGMENSEK SZOMATIKUS ÁTRENDEZŐDÉSE EGY GÉNNÉ
Fehérje
Gén
GÉN SZEGMENSEK SZOMATIKUS ÁTRENDEZŐDÉSE EGY GÉNNÉ
Fehérje
Gén

11. Ig gene sequencing complicated the model
The structures of germline VL genes were similar for Vk, and Vl,
However there was an anomaly between germline and rearranged DNA: ? CL VL
~ 95aa
~ 100aa L CL VL
~ 95aa
~ 100aa JL
Some of the extra amino acids are provided by one of a small set of J or JOINING regions L CL VL
~ 208aa L
Where do the extra 13 amino acids come from?

12. SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTSJκ Vκ
B-cell 2
40 Vκ
5 Jκ Vκ Jκ
Germ line
During B-lymphocyte development Jk Jκ Vκ
B-cell 1
DNA

13. 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?

14. 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

15. 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 3 recombination events
JH to DH , VH to JHDH, and VHJHDH to CH VL JL CL L
Each light chain requires 2 recombination events
VL to JL and VLJL to CL

16. 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

17. 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

18. The key experiment of Nobumichi Hozumi and Susumu Tonegawa