IgTCR基因重排原理及多样性实用教案

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页/共26页第一页，共27页。第2页/共26页第二页，共27页。第3页/共26页第三页，共27页。- 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 fami

3、ly 第4页/共26页第四页，共27页。第5页/共26页第五页，共27页。第6页/共26页第六页，共27页。 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

4、sequence-heptamer-space-nonamer (12/23) - 12/23 rule - exception : D-D joining (fusion) in human 5% looping out and deletion : more common no deletion 第7页/共26页第七页，共27页。第8页/共26页第八页，共27页。第9页/共26页第九页，共27页。第10页/共26页第十页，共27页。第11页/共26页第十一页，共27页。 Enzymatic steps in the gene rearrangement - RAG (Recombinati

5、on Activating Genes RAG-1,RAG-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 pro

6、tein kinase (DNA-PK) Ku : a heterodimer 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 defi

7、ciency(SCID) mouse Signal joint and coding joint第12页/共26页第十二页，共27页。第13页/共26页第十三页，共27页。第14页/共26页第十四页，共27页。第15页/共26页第十五页，共27页。 The diversity of the Ig repertoire : four main process - combinatorial diversity multiple gene segments different combination different combination of H,L chain - Junctional d

8、iversity - Somatic hypermutation Combinatorial diversity kappa chain 40 5 = 200 lambda chain 30 4 = 120 H chain 40 25 6 = 6000 - Combinatorial diversity is likely to be less from the theoretical calculation320106第16页/共26页第十六页，共27页。 Junctional diversity - The diversity of CDR3 in H,L chains is signif

9、icantly 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 rearrangeme

10、nt第17页/共26页第十七页，共27页。第18页/共26页第十八页，共27页。第19页/共26页第十九页，共27页。第20页/共26页第二十页，共27页。 TCR gene segments and rearrangement - The same enzyme for Ig gene rearrangement can be used for TCR gene rearrangement - The numbers of human TCR gene segment TCR gene rearrangement第21页/共26页第二十一页，共27页。第22页/共26页第二十二页，共27页。第23页/共26页第二十三页，共27页。第24页/共26页第二十四页，共27页。第25页/共26页第二十五页，共27页。感谢您的观看(gunkn)！第26页/共26页第二十六页，共27页。NoImage内容(nirng)总结Ig g