The Evolution of Lymphocyte Recognition

小行星9406249 虽然在以前的研究中已经确定了编码癍点叉尾（Ictalurus punctatus）IgM抗体重链的基因的结构和表达途径，但是该基因的功能的许多方面仍然没有得到解决。 产生抗原受体（膜结合形式的IgM）的不寻常前mRNA加工途径的演变尚不清楚，驱动该基因表达的转录控制元件（特别是增强子）的性质也不清楚。 作为本申请的主要目的，提出根据其精确位置、结构和细胞类型表达的特异性来定义鲶鱼重链基因中的核心增强子元件。 鲶鱼重链基因的结构和表达的理解可以允许成功设计的遗传方法来操纵在商业上重要的鱼的抗体反应。 鲶鱼IgH增强子最初将通过功能标准进行研究：核心增强子将被定义为来自鲶鱼IG重链基因座的最小DNA片段，其将以B细胞特异性方式驱动报告基因（例如氯霉素乙酰转移酶）的表达。 将报告构建体转染到克隆的体外生长的B细胞、T细胞和巨噬细胞谱系的癍点叉尾鮰细胞系中，并在瞬时表达测定中测量它们的转录活性。 一旦定位了鲶鱼IgH基因座的核心增强子区域，则结合转录因子的特定序列基序将通过迁移率变化、足迹法和功能分析的组合来定义。 功能分析将包括在报告构建体中以各种组合利用增强子的片段，其中试图重建天然IgH增强子的组织特异性活性。 这些对鲶鱼抗体基因表达调控的分子水平研究将为深入了解免疫功能的进化提供依据，并为我们将来通过遗传手段增强鱼类免疫应答奠定基础。 ***

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. ***