The Evolution of Lymphocyte Recognition

9406249 WARR虽然先前的研究已确定斑点叉尾斑点叉尾鱼(Ictalurus Marktatus)免疫球蛋白M抗体重链基因的结构和表达途径，但该基因的许多功能仍未解决。产生抗原受体、膜结合形式的IgM的不寻常的前mRNA加工途径的进化还不清楚，也不知道驱动该基因表达的转录控制元件(特别是增强子)的性质。作为这一应用的主要目的，建议根据其精确的位置、结构和细胞类型表达的特异性来确定该重链基因中的核心增强子元件。对叉尾鱼重链基因的结构和表达的了解可以使成功地设计遗传方法来操纵具有重要商业价值的鱼类的抗体反应。最初的研究将以功能标准进行：核心增强子将被定义为重链基因的最小片段，它将以B细胞特异性的方式驱动报告基因(如氯霉素乙酰转移酶)的表达。将构建的报告基因导入克隆的、体外培养的斑点叉尾轮鱼细胞系B细胞、T细胞和巨噬细胞系，并通过瞬时表达检测其转录活性。一旦该基因的核心增强子区域被定位，结合转录因子的特定序列基序将通过迁移率转移、足迹和功能分析来确定。功能分析将包括在尝试重建天然IgH增强子的组织特异性活性的报告构建体中以各种组合利用增强子片段。这些分子水平的研究将深入了解免疫功能的进化，并产生基本信息，为我们未来通过遗传手段增强鱼类免疫反应奠定基础。***

9406249 Warr Although the structure and pathways of expression of the gene encoding the heavy chain of the IgM antibody of the channel catfish (Ictalurus punctatus) have been defined in previous studies, many aspects of the function of this gene remain unresolved. The evolution of the unusual pre-mRNA processing pathways which produce the antigen receptor, membrane-bound form of the IgM is not understood, and neither is the nature of the transcriptional control elements (particularly the enhancer) that drive the expression of this gene. As the major objective of this application, it is proposed to define the core enhancer element in the catfish heavy chain gene in terms of its precise location, structure and specificity of cell-type expression. An understanding of both the structure and expression of the catfish heavy chain gene could permit the successful design of genetic approaches to manipulating the antibody response in a commercially important fish. The catfish IgH enhancer will be investigated initially by functional criteria: the core enhancer will be defined as that minimal segment of DNA from the catfish Ig heavy chain locus that will drive expression of a reporter gene (e.g. chloramphenicol acetyltransferase) in a B cell-specific manner. Reporter constructs will be transfected into cloned, in vitro grown channel catfish cell lines of B cell, T cell and macrophage lineage and their transcriptional activity measured in transient expression assays. Once the core enhancer region of the catfish IgH locus has been located, then the specific sequence motifs that bind the transcription factors will be defined by a combination of mobility- shift, footprinting and functional analyses. The functional analysis will consist of utilizing fragments of the enhancer, in various combinations, in reporter constructs in which it is attempted to reconstruct the tissue specific activity of the native IgH enhancer. %%% These molecular level studies of the regulati on of catfish antibody gene expression will provide insight into the evolution of immune function and generate fundamental information that will lay the groundwork for our future ability to enhance fish immune responses by genetic means. ***