The Evolution of Lymphocyte Recognition

本研究的重点是免疫球蛋白(Ig)重链基因在斑点叉尾鱼中的表达。具体问题包括：1)调节B细胞膜受体和分泌型IgM重链的差异表达。该实验室发现，斑点叉尾鱼在编码膜和mU链分泌形式的mRNAs的生产中所涉及的交替外显子剪接模式与哺乳动物、棘鳃和两栖动物所使用的模式不同。将含斑点叉链基因相关区域的表达载体导入小鼠Pre-B细胞或浆细胞，研究影响斑点叉尾转录体剪接位点选择的因素。2)在小鼠B细胞中组装杂交叉尾鱼/小鼠Ig重链。本实验室观察到，重链由小鼠VH结构域与斑点叉叉鱼Mu链恒定区融合而成的IGs的组装和分泌被阻断。有人建议确定这种阻塞在生物合成途径中的位置。需要研究的潜在阻断部位包括二硫键形成缺陷、糖基化异常、重链与免疫球蛋白结合蛋白(BiP)的异常结合以及未能与内源性小鼠轻链正确结合。3)斑点叉尾叉尾轮鱼IgH基因表达增强子的鉴定。我们将把斑点叉尾鱼IgH基因的区域放入合适的表达载体中，并在异源(小鼠)B淋巴细胞中寻找B细胞特异的增强子活性。这些研究将有助于更好地了解抗体Mu链基因在斑点叉尾鱼中的表达及其编码蛋白的功能。此外，它们还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.