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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 kappa Heavy chain lambda Variable (V) 40 30 65 Diversity (D) 0 0 27 Joining (J) 5 4 6 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 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 = 200combinations 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 = 3,369,600 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. There are other mechanisms that add diversity at the junctions between genes - JUNCTIONAL diversity GENERATES A POTENTIAL B-CELL REPERTOIRE Somatic recombination to generate antibody diversity Severe combined immunodeficiency (SCID) Omenn syndrome - RAG deficiency Lack of T-cells and B cells Early manifestation red rash on the face and shoulders, infections with opportunistic pathogens. (Candida albicans, Pneumocystis carnii pneumonia) Lack of 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 -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 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 9 VH 7 12 23 7 D 9 9 12 9 9 12 7 7 D NONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacer 23 7 12 9 7 JH 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 Intervening DNA of any length 23 V 7 9 12 9 7D J Molecular explanation of the 12-23 rule V4 V1 V8 V9 V2 V7 V3 V6 V3 V4 V2 V5 9 9 • Heptamers and nonamers align back-to-back V7 V8 V9 V1 7 12-mer 7 • The shape generated by the RSS’s acts as a target for recombinases V6 Loop of intervening DNA is excised D J 23-mer 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 V3 V5 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 V1 7 12-mer DJ V5 V6 Loop of intervening DNA is excised V7 V8 V9 Junctional Diversity V TCGACGTTATAT AGCTGCAATATA D J TTTTT Germline-encoded nucleotides TTTTT Palindromic (P) nucleotides - not in the germline (N) encoded nucleotides - not in the TTTTT Non-template 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 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)