Transcript vh event
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 SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS Vκ Jκ B-cell 2 Vκ Vκ 80 Vκ Vκ Vκ 5 Jκ Germ line Jκ Jκ Jκ Jκ During B-lymphocyte development Vκ B-cell 1 DNA Vκ Vκ Jk Jκ Jκ Jκ 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 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 , VH to JHDH, L VL JL CL Each light chain requires a single recombination event 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 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 recombination work? How is an almost 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 = 3,369,600 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 Reading D segment in 3 frames If recombination is truely random one expects a frameshift in the reading frame 2 out of 3 events. 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 How does somatic recombination work? How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity How do V region find J regions and why don’t they join to C regions? 12-23 rule 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) 5' V 3' J 7 9 12 9 23 RSS RSS - Recobnitation Signal Sequence 7 RSS RAG – Recombination Activation Genes hasítás cleavage RAG1 RAG2 5' V 3' OH P P J OH PALINDROMIC SEQUENCES 5' 3' 5' 3' V 5' 3' J 5' 3' V 5' 3' J 5' 3' V V J 5' 3' OH P HEPTAMER OH P NONAMER P HO P HO Ku protein J 3' 5' CACAGTG GTGACAC ACAAAAACC TGTTTTTGG CACAGTG GTGACAC GGTTTTTGT CCAAAAACA SOMATIC GENE REARRANGEMENT (RECOMBINATION) dsDNA nick, enzymatic deletion, religation Random recombination Antigen independent Defined order of recombination events Special recombinase enzymes RAG1/RAG2 Limited expression Special site (bone marrow, thymus) Narrow window of maturation How does somatic gene rearrangement (recombination) work? 1. How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 1. How do V region find J regions and why don’t they join to C regions? 12-23 rule 2. How does the DNA break and rejoin? Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity Junctional diversity 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. V4 V5 V3 V6 V2 9 9 23-mer 7 V1 7 12-mer DJ V7 V8 V9 V4 V5 V3 V2 9 9 23-mer 7 V1 7 12-mer DJ 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 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