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The key experiment of Nobumichi Hozumi and Susumu Tonegawa EXPRESSION OF THE KAPPA CHAIN Vκ P Vκ J pA J E Cκ J E Cκ Vκ-Jκ Leader Vκ J Primary RNA transcript Vκ J Cκ AAAA mRNA Translation Vκ J Cκ Protein Efficiency of somatic gene rearrangement? 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 Non-productive rearrangement Light chain has a second chance to make a productive join using new V and J elements Spliced mRNA transcript Ck Further diversity in the Ig heavy chain L 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, L VL JL CL Each light chain requires 1 recombination events VL to JL SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS 120 VH VH1 VH2 12 D VH3 D D D 4 JH D JH JH JH JH During B-cell development VH1 VH2 VH3 VH1 D D JH JH VH2 D D JH JH 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 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 VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES B cells of one individual 2 3 1 4 V-Domains C-Domains VH D JH VL VH-D-JH JL VL-JL How does somatic gene rearrangement (recombination) work? 1. How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 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 = 3,369600 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 How does somatic gene rearrangement (recombination) work? 1. 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 NONAMER CACAGTG GTGACAC ACAAAAACC TGTTTTTGG CACAGTG GTGACAC GGTTTTTGT CCAAAAACA Somatic recombination to generate antibody diversity 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 Vk 7 23 12 7 23 9 12 9 7 12 9 9 9 VH 9 D 23 7 12 9 7 Jl 7 Jk 9 23 7 JH Recombination signal sequences (RSS) HEPTAMER - Always contiguous with coding sequence 9 VH 7 23 VH 7 12 9 23 7 D 7 9 12 9 9 12 NONAMER - Separated from the heptamer by a 12 or 23 9 nucleotide spacer 7 D 7 JH 23 7 12 9 9 23 7 JH 12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS Molecular explanation of the 12-23 rule 12-mer = one turn 23-mer = two turns 23 V7 Intervening DNA of any length 9 12 9 7D J Molecular explanation of the 12-23 rule V4 V1 V8 V9 V3 V2 V7 V6 V3 V4 V2 V5 9 9 23-mer • Heptamers and nonamers align back-to-back V6 Loop of intervening DNA is excised DJ V7 V8 V9 V1 7 12-mer 7 • The shape generated by the RSS’s acts as a target for recombinases V5 DJ • An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the 12-23 rule) CONSEQUENCES OF RECOMBINATION Generation of P-nucleotides V4 V5 V3 V6 V2 9 9 23-mer 7 V1 7 12-mer DJ V7 V8 V9 Generation of N-nucleotides V4 Terminal deoxynucleotidyl Transferase (TdT) V3 V2 9 9 23-mer 7 7 12-mer V1 V5 DJ V6 Loop of intervening DNA is excised V7 V8 V9 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 How does somatic gene rearrangement (recombination) work? 1. 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 3. 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) Junctional diversity Mini-circle of DNA is permanently lost from the genome 9 7 V 7 12 23 9 9 23 Coding joint 7 7 12 9 Signal joint DJ VDJ 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. Junctional Diversity V TCGACGTTATAT AGCTGCAATATA D J TTTTT Germline-encoded nucleotides TTTTT Palindromic (P) nucleotides - not in the germline (N) encoded nucleotides - not TTTTT Non-template 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 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 Frame 1 GGGACAGGGGGC GlyGlnGly Frame 2 GGGACAGGGGGC AspArgGly Frame 3 RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION 1. Somatic rearrangement of Ig gene segments occurs in a highly controlled manner 2. Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities 3. In each of us a huge B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains 4. This potential B-cell repertoire is able to recognize a wide array of antigens INDEPENDENT FROM ANTIGEN OCCURS IN THE BONE MARROW How does somatic gene rearrangement (recombination) work? 1. 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 3. 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 codominantly, how could B cells shut down the genes on one of their chromosomes? Allelic exclusion