MOLECULAR BIOLOGY OF INTERCELLULAR COMMUNICATION

细胞间通讯的分子生物学

• 批准号: 3484276
• 负责人: DANIEL A. GOODENOUGH
• 金额: $ 24.98万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1975
• 资助国家: 美国
• 起止时间: 1975-01-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484276
• 关键词: X ray crystallography; Xenopus; autoradiography; complementary DNA; electrofocusing; electron microscopy; electron optics; freeze etching; gap junctions; gel electrophoresis; gel filtration chromatography; genetic library; histochemistry /cytochemistry; immunochemistry; immunocytochemistry; immunofluorescence technique; lens proteins; liver; membrane reconstitution /synthesis; membrane structure; molecular cloning; nucleic acid probes; protein biosynthesis; protein sequence; proteins; radiotracer; recombinant DNA; temperature sensitive mutant; thin layer chromatography; tissue /cell culture

This proposal will study the structure and function of the gap junction, and its role in intercellular communication. Electron image data will be obtained from frozen-hydrated specimens. Dynamic parts of the channel (connexon) involved in channel gating and in connexon-connexon interaction will be characterized by comparative electron microscopy and X-ray diffraction studies under a range of conditions which alter these flexible components. The domains indentified in the connexon structure will be correlated with the amino acid sequence determined from the recombinant DNA. Communication will also be studied using site- specific antisera generated using synthetic oligopeptides which correspond to defined domains within the amino acid sequence predicted from the liver gap junction 32kD cDNA. These antisera will be used for structural mapping of the topology of the gap junction protein by immunocytochemistry of isolated intact and urea-split gap junctions. The antisera will be tested for physiological activity in the AR4-2J pancreas cell line, and in Xenopus oocytes. Ovarian granulosa cDNA libraries will be made from pregnant mare serum gonadotrophin-stimulated rat ovaries prescreened by Northern analysis to demonstrate positive hybridization with the coding region of liver gap junction cDNA. This library will then be screened with the same probe in order to clone the granulosa gap junction cDNA. Synthetic mRNAs generated from liver, ovarian granulosa, and myocardial gap junction cNDAs will be intracellularly injected into Xenopus oocytes which have been manipulated into contact. In these "reconstructed" systems, channel properties will be measured in voltage-clamped cells, and differences in liver, heart and granulosa channels measured in the context of an invariant oocyte cytoplasm. These differences in physiology will be explored further by attempting to construct liver/granulosa hybrid gap junctions: junctions with tissue-specific proteins apposed across the gap in asymmetrically-injected oocytes. Site-specific and tissue-specific antisera will be used to provide rigorous control of these experiments.

本提案将研究间隙的结构和功能 连接及其在细胞间通讯中的作用。 电子 图像数据将从冷冻水合标本中获得。 通道涉及的通道动态部分（连接子） 门控和连接子-连接子相互作用将被表征 通过比较电子显微镜和 X 射线衍射研究 在一系列改变这些柔性组件的条件下。 连接子结构中识别的域将是 与从确定的氨基酸序列相关 重组DNA。 还将使用站点来研究通信 使用合成寡肽产生的特异性抗血清 对应于氨基酸序列内的定义域 从肝脏间隙连接预测32kD cDNA。 这些抗血清 将用于间隙拓扑的结构映射 通过免疫细胞化学分离的完整和连接蛋白 尿素分裂间隙连接。 抗血清将进行检测 AR4-2J 胰腺细胞系的生理活性，以及 非洲爪蟾卵母细胞。 将建立卵巢颗粒cDNA文库 来自怀孕母马血清促性腺激素刺激的大鼠卵巢 由北方分析预先筛选以证明积极 与肝间隙连接cDNA的编码区杂交。 然后将使用相同的探针筛选该文库，以便 克隆颗粒间隙连接 cDNA。 合成 mRNA 由肝脏、卵巢颗粒和心肌间隙产生 连接cNDAs将被细胞内注射到非洲爪蟾中 已被操纵接触的卵母细胞。 在这些 “重建”系统，通道属性将在 电压钳制细胞以及肝脏、心脏和 在不变卵母细胞的背景下测量颗粒通道 细胞质。 将探讨这些生理学差异 进一步尝试构建肝脏/颗粒混合间隙 连接点：与组织特异性蛋白质并列的连接点 不对称注射卵母细胞中的间隙。 特定地点和 组织特异性抗血清将用于提供严格的控制 这些实验。