LENS METABOLIC COOPERATION, GAP JUNCTIONS & CATARACT

本申请提出鉴定透镜的蛋白质组分 纤维细胞间连接 确定身份的战略将 制备单克隆抗体和多克隆抗血清， 透镜纤维膜作为抗原。 这些抗体将通过 选择产生黄斑染色模式的试剂， 免疫荧光标记的整个透镜的冷冻切片，其识别 免疫复制品上的特异性多肽，并且其干扰透镜 纤维细胞间通讯测定细胞内 显微注射荧光染料荧光黄CH。 通过免疫电子显微镜观察以验证它们在透镜上的位置 纤维连接 成功筛选的抗血清将用于 从透镜上皮到透镜纤维的分子组成 连接和上皮细胞-上皮细胞连接。 这些 抗血清将用于研究体外合成的连接 蛋白质在无细胞系统，并确定连接的相互作用 分化和未分化细胞之间的差异。 抗血清 也将用于尝试克隆透镜纤维连接基因，使用 将透镜cDNA文库克隆到γ gtII表达载体中。 如果 成功的话，从该基因衍生的氨基酸序列将允许 产生另外的抗血清，以确定氨基酸序列内的 连接分子和蛋白质结构的拓扑映射 在连接膜内。 如果抗血清显示天然细胞 培养细胞之间的连接，白内障的直接影响， 将尝试关于细胞间通信的条件。

This application proposes to identify the protein components of the lens fiber intercellular junctions. The strategy for this identification will be to prepare monoclonal antibodies and polyclonal antisers using isolated lens fiber membranes as antigen. These antibodies will be screened by selecting for reagents which produce a macular staining pattern on immunofluorescently-labeled frozen sections of whole lens, which recognize specific polypeptides on immune replicas, and which interfere with lens fiber intercellular communication as assayed by intracellular microinjection of the fluorescent dye, Lucifer Yellow CH. Antigens will be visualized by immunoelectron microscopy to verify their location on lens fiber junctions. Successfully screened antisera will be used to demonstrate the molecular components of lens epithelium to lens fiber junctions, and epithelial cell-to-epithelial cell junctions. These antisera will be used to study the in vitro synthesis of junctional proteins in cell-free systems, and to define the junctional interactions between differentiated and undifferentiated cells in culture. The antisera will also be used to attempt cloning the lens fiber junction gene, using a lens cDNA library cloned into the Gamma gtll expression vector. If successful, the derived amino acid sequence from this gene will permit raising additional antisera to defined amino acid sequences within the junctional molecule, and topological mapping of the protein structure within the junctional membranes. If the antisera demonstrate native cell junctions between the cultured cells, direct effects of cataractogenic conditions on intercellular communication will be attempted.