MOLECULAR BIOLOGY OF INTERCELLULAR COMMUNICATION

细胞间通讯的分子生物学

• 批准号: 3484272
• 负责人: DANIEL A. GOODENOUGH
• 金额: $ 20.89万
• 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/3484272
• 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.

这项提案将研究GAP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化的标本中获得。 渠道中涉及的动态部分(Connecexon) 门控和In Connecexon-Connexon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。 在连接蛋白结构中确定的结构域将是 相关的氨基酸序列由 重组DNA。还将使用Site-Site来研究通信- 使用合成的寡肽产生的特异性抗血清 对应于氨基酸序列中定义的结构域 从肝脏缝隙连接32kD的cDNA中预测。这些抗血清 将用于绘制缺口的拓扑结构图 用免疫细胞化学方法检测分离的完整和 尿素-裂开缝隙结。将对抗血清进行检测 AR4-2J胰腺细胞系的生理活性，以及 非洲爪哇卵母细胞。将建立卵巢颗粒细胞基因文库 孕马血清促性腺激素刺激大鼠卵巢 通过Northern分析进行预筛选，以显示阳性 与肝脏缝隙连接基因编码区进行杂交。 然后用相同的探针对这个文库进行筛选，以便 克隆颗粒细胞缝隙连接基因。合成的mRNA 由肝脏、卵巢颗粒和心肌间隙产生 结合部cNDA将被细胞内注射到非洲爪哇 被操控接触的卵母细胞。在这些 “重建”的系统，信道属性将在 电压钳制的细胞，以及肝脏、心脏和 在不变卵母细胞背景下测量的颗粒通道 细胞质。这些生理学上的差异将被探究。 进一步通过尝试构建肝脏/颗粒杂交GAP 连接：与组织特异性蛋白相对的连接 不对称注射的卵母细胞的缝隙。特定于站点和 组织特异性抗血清将用于提供严格的控制 这些实验。