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The key experiment of Nobumichi Hozumi and Susumu Tonegawa

The key experiment of Nobumichi Hozumi and Susumu Tonegawa. Leader. pA. V κ. P. V κ. V κ. V κ. V κ. J. J. J. J. J. J. E. E. V κ - J κ. Prima ry RN A transcript. C κ. C κ. C κ. C κ. mRNA. AAAA. Trans lation. Protein. EXPRESSION OF THE KAPPA CHAIN.

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The key experiment of Nobumichi Hozumi and Susumu Tonegawa

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  1. The key experiment of Nobumichi Hozumi and Susumu Tonegawa

  2. Leader pA Vκ P Vκ Vκ Vκ Vκ J J J J J J E E Vκ-Jκ Primary RNAtranscript Cκ Cκ Cκ Cκ mRNA AAAA Translation Protein EXPRESSION OF THE KAPPA CHAIN Efficiency of somatic gene rearrangement?

  3. Ig light chain rearrangement: Rescue pathway 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 There is only a 1:3 chance of the join between the V and J region being in frame

  4. L VL JL CL Further diversity in the Ig heavy chain DH JH L VH 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 DHandVH to JHDH, Each light chain requires 1 recombination events VL to JL

  5. SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS During B-cell development VH1 VH1 VH1 D D D D D D D D JH JH VH2 JH JH VH2 VH3 120 VH 12 D 4 JH VH2 VH3 JH JH JH JH

  6. IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS Gene segments Light chain Heavy chain kappa lambda Variable (V) 132/40 105/30 123/65 Diversity (D) 0 0 27 Joining (J) 5 4 9 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

  7. VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES VH JH D V-Domains C-Domains VL JL VH-D-JH VL-JL B cells of one individual 1 2 3 4

  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. 10,530x 320 = 3,369600 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?12-23 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

  12. Severe combined immunodeficiency syndrome (SCID). 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. Vl Jl 7 23 12 7 9 9 Vk JH Jk 9 9 7 12 23 23 7 7 9 D 12 7 7 12 9 9 VH 9 7 23 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

  14. HEPTAMER - Always contiguous with coding sequence NONAMER - Separated fromthe heptamer by a 12 or 23 nucleotide spacer   JH JH 9 9 23 23 7 7 D D 12 12 7 7 7 7 12 12 9 9 9 9 VH VH 9 9 7 7 23 23 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

  15. 12-mer = one turn 23-mer = two turns Intervening DNA of any length 23 12 V 7 9 7 D J 9 Molecular explanation of the 12-23 rule

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

  17. V4 V5 V3 V6 V2 V7 9 V8 V9 9 23-mer 12-mer 7 7 D J V1 CONSEQUENCES OF RECOMBINATION Generation of P-nucleotides

  18. V4 V5 V3 V6 V2 V7 9 V8 V9 9 23-mer 12-mer 7 7 D J V1 Generation of N-nucleotides Terminal deoxynucleotidyl Transferase (TdT) Loop of intervening DNA is excised

  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?12-23 rule • How does the DNA break and rejoin? • Imprecisely, with the random removal and addition of nucleotidesto generate sequence diversity • Junctional diversity(P- and N- nucleotides, seeabove)

  20. 9 23 7 7 12 9 9 9 23 Coding joint Signal joint 12 V D J 7 7 V D J Junctional diversity Mini-circle of DNA is permanently lost from the genome 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 Junctional Diversity TTTTT TTTTT TTTTT 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. 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 GGGACAGGGGGC GlyGlnGly GGGACAGGGGGC 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?12-23 rule • How does the DNA break and rejoin? • Imprecisely, with the random removal and addition of nucleotidesto 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

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