1. ANTIBODY DIVERSITY

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

3. How can antibody spcificity and diversity be explained? Paul Ehrlich: side chain theory (1897) There are receptors for nutrients. There may be receptors (side chains) for foreign entities. BUT: the diversity is huge, hard to explain at that time What about pre-existing antibodies? Antibody is not produced by the host Information of the large antibody repertoire comes from the antigen itself. Direct: antibody comes from the antigen… BUT antibody level does not decline together with that of the antigen!! Instruction Theories: Antigen is produced by the host but the antigen teaches antibody to fold properly or it is synthesized on the antigen Problem: antibody is also produced by the descendents of the activated B cells 1950: Protein is created on nucleic acid template, but the paradox of coding for such diversity remained a mystery

4. Multiple myeloma (MM) Plasma cell tumors – tumor cells reside in the bone marrow Produce immunoglobulins of monoclonal origin, serum concentration 50-100mg/ml Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification AMINO ACID SEQUENCE OF IMMUNOGLOBULINS 50 kDa Heavy chain 25 kDa Light chain Gel electrophoresis V ariábilis C onstans 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Reduction L H

5. MOLECULAR GENETICS OF IMMUNOGLOUBLINS GERMLINE VARIATION OR SOMATIC MUTATION? 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, William Dreyer & Claude Bennett proposed that : How can the diversity 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

6. Many GENES (10 000 – 100 000) V2V2V2V2C VnVnVnVnC V1V1V1V1C 1 GEN High rate of somatic mutations in the V-region VC GenGenGenGen Protein 1 GEN = 1 PROTEIN DOGMA OF MOLECULAR BIOLOGY CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE THEORIES

7. Proof of the Dreyer - Bennett hypothesis V V V V V V V V V V V V 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 C A single C region gene is encoded in the germline and separated from the multiple V region genes Find a way to show the existence of multiple V genes and rearrangement to the C gene

8. Approach 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) C V V V V V V V V V Germline DNA C V V V V V Rearranged DNA

9. Liver cell B-cell 1.5. Kb B-cell V C 6.0 Kb V C 4.0 Kb DNA-extraction Digestion by restriction enzyme Gel electrophoresis Southern blot VC Kb 6,0 1,5 V-probe 4,0 CV C-probe Experiment of Susumu Tonegawa 1976 Basel C probe V 6,0 1,5 4,0

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

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