Analysis of B cell memory and activation
B 细胞记忆和激活分析
基本信息
- 批准号:16043223
- 负责人:
- 金额:$ 16.9万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research on Priority Areas
- 财政年份:2004
- 资助国家:日本
- 起止时间:2004 至 2006
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
We previously revealed that activation induced cytidine deaminase (AID) is essential and control two genetic alteration systems namely immunoglobulin class switch recombination and somatic hypermutation. Still it is elusive how AID undergoes and differentially controls such two events. In order to understand how class switch recombination and somatic hypermutation are regulated by AID, we are trying to identify cofactors for AID. We took two approaches to raise possible candidates for AID binding protein. One is two hybrid screening using AID and AID mutants as baits. After cDNA screening of libraries, we isolated 30 candidates that are expressed in 8 cells and physically interact with AID protein at the level of two-hybrid assay. The other approach was co-immunoprecipitation using mammalian cell lines that express AID tagged with flag peptide. AID complex was enriched by the immunoprecipitation and resolved by SDS-PAGE, and then AID or AID mutant-specific precipitants were decided by mass analysis. Previous studies provided two kinds of AID mutants (class switch defective-and somatic hypermutation defective mutants). Class switch defective AID mutants do not have 16 amino acids at C-terminus. On the other hand, somatic hypermutation defective AID mutants do have missense mutation an N-terminus. The two mutants were applied to take AID binding protein. Based on expression in B cells, mode of binding with AID/mutants and cDNA information, we took eight candidates as reasonable and possible AID cofactors. Now to see functional significance for the candidates, we limit expression of candidates by transduction of siRNA. When we knock down expression of one particular candidate, we observed 90% reduction of class switch inducing activity. Further study of this candidate should be done to know why class switch inhibition is observed by reducina expression level of the candidate.
我们以前发现,激活诱导的胞苷脱氨酶(AID)是必不可少的,并控制两个遗传改变系统,即免疫球蛋白类转换重组和体细胞超突变。然而,艾滋病署如何经历和区别控制这两个事件,仍是一个谜。为了了解类转换重组和体细胞超变是如何被AID调节的,我们试图鉴定AID的辅因子。我们采用了两种方法来提高可能的候选人AID结合蛋白。一种是以AID和AID突变体为诱饵的双杂交筛选。通过对文库的cDNA筛选,我们分离到了30个在8个细胞中表达的候选基因,并在双杂交水平上与AID蛋白发生了物理相互作用。另一种方法是使用表达标记有flag肽的AID的哺乳动物细胞系进行免疫共沉淀。通过免疫沉淀富集AID复合物,并通过SDS-PAGE进行解析,然后通过质量分析确定AID或AID突变体特异性沉淀物。以往的研究提供了两种AID突变体(类转换缺陷型和体细胞超突变缺陷型)。类转换缺陷型AID突变体在C-末端不具有16个氨基酸。另一方面,体细胞超突变缺陷型AID突变体确实在N-末端存在错义突变。将这两个突变体用于提取AID结合蛋白。基于在B细胞中的表达、与AID/突变体的结合方式以及cDNA信息,我们将8个候选物作为合理且可能的AID辅因子。现在为了观察候选物的功能意义,我们通过siRNA的转导来限制候选物的表达。当我们敲低一种特定候选物的表达时,我们观察到类转换诱导活性降低90%。对该候选物的进一步研究应通过降低候选物的表达水平来了解为什么观察到类转换抑制。
项目成果
期刊论文数量(54)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Identification of a specific domain required for dimerization of activation-induced cytidine deaminase
活化诱导胞苷脱氨酶二聚化所需的特定结构域的鉴定
- DOI:
- 发表时间:2006
- 期刊:
- 影响因子:0
- 作者:Wang;J. et al.
- 通讯作者:J. et al.
Target selection of somatic hypermutations is regulated similarl : between T and B cells upon activation-induced cytidine deaminasi expression
体细胞超突变的目标选择受到类似的调节:在激活诱导的胞苷脱氨表达后,T 细胞和 B 细胞之间
- DOI:
- 发表时间:2005
- 期刊:
- 影响因子:0
- 作者:Kotani A;Okazaki IM;Muramatsu M;Kinoshita K;Begum NA Nakajima T;Saito H;Honjo T.
- 通讯作者:Honjo T.
RNA-editing cytidine deaminse Apobec-1 is unable to induce somatic hypermutation in mammalian cells.
RNA 编辑胞苷脱氨酶 Apobec-1 无法诱导哺乳动物细胞体细胞超突变。
- DOI:
- 发表时间:2003
- 期刊:
- 影响因子:0
- 作者:Alugupalli KR;Leong JM;Woodland RT;Muramatsu M;Honji T;Gerstein RM.;Eto T
- 通讯作者:Eto T
Evolution of class switch recombination function in fish activation-induced cytidine deaminase, AID
鱼类激活诱导胞苷脱氨酶 AID 中类别转换重组功能的进化
- DOI:
- 发表时间:2006
- 期刊:
- 影响因子:0
- 作者:Wakae;K.et al.
- 通讯作者:K.et al.
Activation-induced cytidine deaminase (AID) promotes B cell lymphomagenesis in Emu-cmyc transgenic mice
- DOI:10.1073/pnas.0610732104
- 发表时间:2007-01-30
- 期刊:
- 影响因子:11.1
- 作者:Kotani, Ai;Kakazu, Naoki;Honjo, Tasuku
- 通讯作者:Honjo, Tasuku
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MURAMATSU Masamichi其他文献
MURAMATSU Masamichi的其他文献
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{{ truncateString('MURAMATSU Masamichi', 18)}}的其他基金
Trial of isolation and functional analysis of AID-regulatory elements
AID调节元件的分离和功能分析试验
- 批准号:
19390138 - 财政年份:2007
- 资助金额:
$ 16.9万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
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