IgTCR基因重排原理及多样性

1、 Ig gene rearrangement in antibody producing cells - Southern blot analysis non lymphoid cell : V,C in the distant fragments mature B cell : V,C in the same fragments somatic recombination Figure 4.1 - V gene rearrangement Figure 4.2 V region of light chain : V gene segment (VL protein) (VL gene) J

2、gene segment (JL gene) V region of heavy chain : V gene segment (VH protein) (VH gene) D gene segment (DH gene) J gene segment (JH gene)- The numbers of functional gene segments for the V region of human heavy and light chains pseudogenes : not functional protein Figure 4.3- The germline organizatio

3、n of the Ig genes gene family Figure 4.4 Rearrangement of V,D,J gene segments - DNA rearrangements are guided by conserved noncoding DNA sequence Figure 4.5 (recombination signal sequence, RSS) heptamer : contiguous with the coding sequence space : 12 or 23 nucleotides long nonamer : second conserve

4、d sequence - coding sequence-heptamer-space-nonamer (12/23) - 12/23 rule - exception : D-D joining (fusion) in human 5% - two mode of rearrangement Figure 4.6 looping out and deletion : more common no deletion Enzymatic steps in the gene rearrangement - RAG (Recombination Activating Genes RAG-1,RAG-

5、2) protein complexes bind to RSSs make single-strand DNA breaks at sites 5 of each RSS - RAG are only expressed in developing lymphocytes - Other ubiquitously expressed DNA-modifying proteins DNA ligase IV : joins the processed ends together with XRCC4 DNA-dependent protein kinase (DNA-PK) Ku : a he

6、terodimer Ku70 : Ku80 Ku+DNA-PK+Artemis complex : a protein involved in the opening of covalently closed DNA hairpin structures - terminal deoxynucleotidyl transferase (TdT) randomly adding nucleotides - RAG genes, DNA-PK, TdT gene deficiency Severe combined immune deficiency(SCID) mouse Signal join

7、t and coding jointFigure 4.7Figure 4.7 The diversity of the Ig repertoire : four main process - combinatorial diversity multiple gene segments different combination different combination of H,L chain - Junctional diversity - Somatic hypermutation Combinatorial diversity kappa chain 40 5 = 200 lambda

8、 chain 30 4 = 120 H chain 40 25 6 = 6000 - Combinatorial diversity is likely to be less from the theoretical calculation3203201.91.9 10106 6 Junctional diversity - The diversity of CDR3 in H,L chains is significantly increased by the addition and deletion of nucleotides Figure 4.8 - The added nucleo

9、tide are known as P-nucleotides and N-nucleotides P-nucleotides : palindromic sequences at the end of the gene segment N-nucleotides : non template-encoded - Addition of P,N nucleotide is a random process Productive or nonproductive rearrangement TCR gene segments and rearrangement Figure 4.9 - The

10、same enzyme for Ig gene rearrangement can be used for TCR gene rearrangement Figure 4.10, Figure 4.11 - The numbers of human TCR gene segment Figure 4.12 TCR gene rearrangement Enzymatic steps in the gene rearrangement - RAG (Recombination Activating Genes RAG-1,RAG-2) protein complexes bind to RSSs

11、 make single-strand DNA breaks at sites 5 of each RSS - RAG are only expressed in developing lymphocytes - Other ubiquitously expressed DNA-modifying proteins DNA ligase IV : joins the processed ends together with XRCC4 DNA-dependent protein kinase (DNA-PK) Ku : a heterodimer Ku70 : Ku80 Ku+DNA-PK+A

12、rtemis complex : a protein involved in the opening of covalently closed DNA hairpin structures - terminal deoxynucleotidyl transferase (TdT) randomly adding nucleotides - RAG genes, DNA-PK, TdT gene deficiency Severe combined immune deficiency(SCID) mouse Signal joint and coding jointFigure 4.7Figure 4.7 TCR gene segments and rearrangement Fi