IgH Enhancer Function in an Ectothermic Vertebrate

变温脊椎动物中的 IgH 增强子功能

• 批准号: 6228406
• 负责人: GREGORY W WARR
• 金额: $ 23.85万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6228406
• 关键词: B lymphocyte; biochemical evolution; catfish; gene expression; genetic enhancer element; immunogenetics; immunoglobulin genes; mutant; site directed mutagenesis; surface plasmon resonance; transcription factor; transfection

APPLICANT'S DESCRIPTION: Although transcriptional enhancers are essential for the expression of the immunoglobulin heavy chain (IgH) locus, they are also needed for the additional functions of V(D)J joining, V-region hypermutation and immunoglobulin (Ig) class switch recombination. This research is directed towards understanding the function of the enhancer that drives expression of the IgH locus in the channel catfish, a teleost fish and therefore lacking Ig class switching. Preliminary results suggest that the catfish IgH enhancer functions through a novel interaction of transcription factors bound to multiple interacting octamer and muE5 sites. The role of these sites in the function of the enhancer will be dissected by site-directed mutagenesis, and the function of the mutated enhancer will be assessed in transient expression assays upon transfection into catfish B-cells. Catfish muE5-binding factor(s) will be isolated and characterized. The interactions between Oct transcription factors, muE5binding factors and the DNA motifs that they recognize will be measured directly using the BiaCore 2000 instrument that utilizes surface plasmon resonance detection. Such studies will define whether or not cooperative binding reactions occur between the Oct and muE5-binding transcription factors. Gene expression driven by the interaction of octamer-binding (Oct) transcription factors with those binding to muE5 sites (typically bHLH or zinc-finger transcription factors) has not been reported in mammalian systems. Thus, these studies should shed light on novel' transcription factor interactions in Ig enhancers, and will also provide insight into how enhancer function has influenced the evolution of the IgH locus.

申请人简介：尽管转录增强剂对 免疫球蛋白重链(IgH)基因的表达，它们也是 V(D)J连接、V区超突变所需的附加功能 和免疫球蛋白(Ig)类开关重组。这项研究是针对 了解推动基因表达的增强子的功能 海峡中的IGH轨迹是一种硬骨鱼，因此缺乏Ig 班级转换。初步结果表明，叉尾鱼的免疫球蛋白增强剂 通过结合转录因子的一种新的相互作用发挥功能 多个相互作用的八聚体和muE5位点。这些网站在 增强子的功能将通过定点突变进行剖析，并且 突变的增强子的功能将在瞬时表达中进行评估 将其转入叉尾鱼B细胞后进行检测。鲶鱼MuE5结合因子(S) 将被隔离和定性。OCT转录之间的相互作用 它们识别的因子、muE5结合因子和DNA基序将是 使用利用表面的Biacore 2000仪器直接测量 等离子激元共振检测。这样的研究将确定 OCT和muE5结合之间发生协同结合反应 转录因子。基因的相互作用驱动的基因表达 八聚体结合(OCT)转录因子与muE5结合的转录因子 (通常是bHLH或锌指转录因子)在 哺乳动物的系统。因此，这些研究应该对小说的研究有所帮助。 转录因子在免疫球蛋白增强子中的相互作用，并将提供 深入了解增强子功能如何影响免疫球蛋白的演变 轨迹。