The Evolution of Lymphocyte Recognition

本文拟研究免疫球蛋白（Ig）重链基因在通道鲶鱼（Ictalurus punctatus）中的表达。解决的具体问题是：1)b细胞膜受体的差异表达和IgM重（mu）链分泌形式的调节。本实验室发现，在通道鲶鱼中，编码膜和mu链分泌形式的mrna的产生所涉及的交替外显子剪接模式与哺乳动物、板鳃目动物和两栖动物使用的模式不同。将含有通道鲶鱼mu链基因相关区域的表达载体转染到小鼠b前细胞或浆细胞中，研究鲶鱼mu链转录物剪接位点选择的影响因素。2)杂交鲶鱼/小鼠Ig重链在小鼠B细胞中的组装。本实验室观察到Igs的组装和分泌被阻断，其中重链由小鼠VH结构域融合到通道鲶鱼mu链恒定区域结构域。建议在生物合成途径中确定这种阻塞的位点。有待研究的潜在阻断位点包括二硫键形成缺陷、糖基化不当、重链与免疫球蛋白结合蛋白（BiP）的异常结合，以及无法与内源性小鼠轻链正确结合。3)通道鲶鱼Igh基因座表达的增强子鉴定。将通道鲶鱼的Igh位点置入合适的表达载体，寻求B细胞特异性增强子在异源（小鼠）B淋巴细胞中的活性。这些研究将有助于进一步了解抗体mu链基因在通道鲶鱼中的表达及其编码蛋白的功能。此外，它们将有助于我们全面了解免疫球蛋白及其基因在脊椎动物中的进化。

The proposed studies focus on the expression of the immunoglobulin (Ig) heavy chain gene in the channel catfish, Ictalurus punctatus. The specific issues addressed are: 1) Regulation of the differential expression of B-cell membrane receptor and secreted forms of the IgM heavy (mu) chain. This laboratory has found that the pattern of alternate exon splicing involved in the production of the mRNAs encoding the membrane and secreted forms of the mu chain in the channel catfish is different from that used by mammals, elasmobranchs and amphibia. Expression vectors containing relevant regions of the channel catfish mu chain gene will be transfected into mouse pre-B or plasma cells to investigate the factors influencing splice-site choice in the catfish mu chain transcript. 2) Assembly of hybrid catfish/mouse Ig heavy chains in mouse B cells. This laboratory has observed that the assembly and secretion of Igs in which the heavy chain consists of a mouse VH domain fused to the channel catfish mu chain constant region domains is blocked. It is proposed to identify the sites of this blockage in the biosynthetic pathway. Potential sites of blockade to be investigated include defects in disulfide bond formation improper glycosylation, abnormal association of the heavy chain with the immunoglobulin binding protein (BiP), and failure to associate correctly with the endogenous mouse light chains. 3) Identification of the putative enhancer driving expression of the channel catfish Igh locus. Regions of the channel catfish Igh locus will be put into appropriate expression vectors, and B-cell specific enhancer activity in heterologous (mouse) B lymphocytes will be sought. These studies should result in greater understanding of the expression of the antibody mu chain gene and the function of its encoded protein in the channel catfish. In addition, they will contribute to our overall understanding of the evolution of the immunoglobulins and their genes in the vertebrates.