The Evolution of Lymphocyte Recognition

拟议的研究重点是斑点叉尾鮰免疫球蛋白（Ig）重链基因的表达。 解决的具体问题是： 1) B 细胞膜受体的差异表达和 IgM 重 (mu) 链的分泌形式的调节。 该实验室发现，斑点叉尾鮰中编码膜和分泌形式的 mu 链的 mRNA 的产生所涉及的交替外显子剪接模式与哺乳动物、软骨鱼和两栖动物所使用的模式不同。 将含有斑点叉尾鮰mu链基因相关区域的表达载体转染至小鼠前B细胞或浆细胞中，以研究影响鲶鱼mu链转录本中剪接位点选择的因素。 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，重链由与斑点叉尾鮰μ链恒定区融合的小鼠VH结构域组成的Igs的组装和分泌被阻断。 建议确定生物合成途径中这种阻断的位点。 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) 的异常关联，以及未能与内源性小鼠轻链正确关联。 3) 驱动斑点叉尾鮰 Igh 基因座表达的推定增强子的鉴定。 将斑点叉尾鮰 Igh 基因座的区域放入适当的表达载体中，并寻找异源（小鼠）B 淋巴细胞中的 B 细胞特异性增强子活性。 这些研究应该可以使人们更好地了解斑点叉尾鮰抗体μ链基因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脊椎动物免疫球蛋白及其基因的进化。

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.