MOLECULAR BIOLOGY OF INTERCELLULAR COMMUNICATION

细胞间通讯的分子生物学

• 批准号: 3484275
• 负责人: DANIEL A. GOODENOUGH
• 金额: $ 22.49万
• 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/3484275
• 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; radiotracer; 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.

本研究将对差距的结构和功能进行研究 连接及其在细胞间通讯中的作用。 电子 图像数据将从冷冻-水合试样获得。 参与通道的通道动力学部分（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 在改变这些柔性部件的一系列条件下。 在连接子结构中鉴定的结构域将是 与从氨基酸序列中确定的氨基酸序列相关。 重组DNA 通信也将研究使用网站- 使用合成寡肽产生的特异性抗血清，所述合成寡肽 对应于氨基酸序列内的限定结构域 从肝间隙连接32 kD cDNA预测。 这些抗血清 将用于差距拓扑结构的结构映射 通过免疫细胞化学方法， 尿素分裂间隙连接。 将对抗血清进行检测 在AR 4 - 2 J胰腺细胞系中的生理活性，以及在 非洲爪蟾卵母细胞。 卵巢颗粒细胞cDNA文库的构建 孕马血清促性腺激素刺激大鼠卵巢 通过北方分析进行预筛选，以证明阳性 与肝间隙连接cDNA编码区杂交。 然后用相同的探针筛选该文库，以便 克隆颗粒层缝隙连接cDNA。 合成mrna 由肝脏、卵巢颗粒层和心肌间隙产生 将连接cNDA细胞内注射到非洲爪蟾中 卵母细胞已经被操纵到接触。 在这些 “重建”系统，信道特性将在 电压钳位细胞，以及肝脏，心脏和 在不变卵母细胞中测量的颗粒通道 细胞质 这些生理上的差异将被探讨 进一步通过尝试构建肝脏/颗粒层杂种间隙 连接：组织特异性蛋白质并列的连接 不对称注射卵母细胞中的差距。 位点特异性和 组织特异性抗血清将用于提供严格的控制 这些实验。