IgTCR基因重排原理及多樣性PPT課件

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 V region of light chain : V gene segment (VL protein) (VL gene) J gene segment (JL gene) V region of heavy chain : V gene segment (VH

2、 protein) (VH gene) D gene segment (DH gene) J gene segment (JH gene)第1頁/共26頁第2頁/共26頁第3頁/共26頁- The numbers of functional gene segments for the V region of human heavy and light chains pseudogenes : not functional protein- The germline organization of the Ig genes gene family 第4頁/共26頁第5頁/共26頁第6頁/共26頁

3、 Rearrangement of V,D,J gene segments - DNA rearrangements are guided by conserved (recombination signal sequence, RSS) heptamer : contiguous with the coding sequence space : 12 or 23 nucleotides long nonamer : second conserved sequence - coding sequence-heptamer-space-nonamer (12/23) - 12/23 rule -

4、 exception : D-D joining (fusion) in human 5% looping out and deletion : more common no deletion 第7頁/共26頁第8頁/共26頁第9頁/共26頁第10頁/共26頁第11頁/共26頁 Enzymatic steps in the gene rearrangement - RAG (Recombination Activating Genes RAG-1,RAG-2) protein complexes bind to RSSs make single-strand DNA breaks at sit

5、es 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+Artemis complex : a protein involved i

6、n 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 joint第12頁/共26頁第13頁/共26頁第14頁/共26頁第15頁/共26頁 The diversity o

7、f 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 chain 30 4 = 120 H chain 40 25 6 = 6000 - Combinatori

8、al diversity is likely to be less from the theoretical calculation320106第16頁/共26頁 Junctional diversity - The diversity of CDR3 in H,L chains is significantly increased by the addition and - The added nucleotide 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第17頁/共26頁第18頁/共26頁第19頁/共26頁第20頁/共26頁 TCR gene segments and rearrangement - The same enzyme for Ig gene rearrangement can be used for TCR g