The Evolution of Lymphocyte Recognition

9406249 Warr虽然通道鲶鱼IgM抗体重链编码基因的结构和表达途径在以往的研究中已经明确，但该基因功能的许多方面仍未得到明确。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。本文拟从鲶鱼重链基因的精确定位、结构和细胞型表达特异性等方面对其核心增强子元件进行定义，这是本研究的主要目的。对鲶鱼重链基因的结构和表达的理解可以允许成功设计遗传方法来操纵商业上重要的鱼类的抗体反应。鲶鱼的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重链位点的最小DNA片段，该片段将以B细胞特异性的方式驱动报告基因（例如氯霉素乙酰转移酶）的表达。报告基因构建体将被转染到克隆的、体外培养的通道鲶鱼B细胞、T细胞和巨噬细胞细胞系中，并通过瞬时表达测定它们的转录活性。一旦鲶鱼IgH基因座的核心增强子区域被定位，那么结合转录因子的特定序列基序将通过迁移转移、足迹和功能分析的组合来确定。功能分析将包括利用增强子的片段，以各种组合，在报告构建中，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提供对免疫功能进化的洞察，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的能力奠定基础。＊＊＊

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. ***