MOLECULAR GENETICS APPROACH TO GAP JUNCTIONS

间隙连接的分子遗传学方法

• 批准号: 3782533
• 负责人: THADDEUS BARGIELLO
• 金额: --
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3782533
• 关键词: Drosophilidae; Nematoda; Xenopus oocyte; affinity chromatography; complementary DNA; cytogenetics; electrophysiology; gap junctions; gel electrophoresis; gene deletion mutation; gene expression; genetic regulatory element; homozygote; immunocytochemistry; in situ hybridization; messenger RNA; molecular cloning; monoclonal antibody; nucleic acid sequence; protein purification; salivary glands; transposon /insertion element

Gap junctions are found in virtually all animal cells and have evolved to comprise a multigene family, with a multiplicity of properties and regulatory mechanisms that are expressed in a tissue specific manner. The characterization of the genetic elements that underlie gap junction expression may aid in the definition of the differences, as well as similarities, in gap junction properties, regulation and function of the various members of the connexin gene family. The goal of this proposal is to apply a genetic approach to the study of gap junctions by using two organisms particularly amenable to genetic and molecular manipulations; the fruit fly, Drosophila melanogaster and the nematode, Caenorhabditis elegans. These organisms by virtue of their strengths in classical and molecular genetics provide numerous alternate strategies for the identification and isolation of gap junction mutations. Since fundamental molecular and biophysical descriptions of gap junctions are lacking in these organisms, we will generate the necessary immunological and molecular probes and undertake electrophysiological analyses to identify the type, distribution and biophysical properties of gap junctions in different tissues and developmental stages. Due to the inaccessibility of many of the cell types in both organisms to standard electrophysiological recording techniques, the biophysical characterizations will be accomplished by the incorporation of isolated gap junctions into planar lipid bilayers and by the expression of purified mRNA in pairs of Xenopus oocytes. Antibodies directed against connexins and agents known to modulate gap junction gating will be used to confirm the identity of the channels recorded in the artificial systems. The information obtained from the immunological and biophysical approaches provides a means of defining gap junction phenotypes and consequently identifying and recovering potential gap junction mutants. Our strategies for the recovery of mutants include the study of phenotypes that are likely to result as a consequence of changes in the structure or regulation of gap junctions and the construction of segmental aneuploids to identify regions of the Drosophila genome that display dosage sensitive regulation. The recovery of genetic munitions affecting gap junction mediated communication should lead to the cloning of connexin genes from C. elegans and Drosophila and will help to define the role gap junctions play in complex physiological and behavioral functions. The combination of immunological, biophysical, molecular and genetic approaches in Drosophila and C. elegans offers the opportunity to address questions related to the roles of gap junction that are not possible in other organisms lacking powerful genetic tools. Also, since it appears that there is strong conservation between ion channels in vertebrates and invertebrates, studies in Drosophila and C. elegans are likely to provide information applicable to gap junction communication in general.

差距连接几乎在所有动物细胞中都发现，并且已经演变为 包括一个具有多种属性的多基因家族， 以组织特定方式表达的调节机制。这 基于间隙连接的遗传元素的表征 表达可能有助于差异的定义 相似性，在间隙连接属性中，调节和功能 连接蛋白基因家族的各种成员。该提议的目的是 通过使用两个 有机体特别适合遗传和分子操纵；这 果蝇，果蝇黑色素果司机和线虫，Caenorhabditis 秀丽隐杆线。这些生物是由于它们在古典和 分子遗传学为 间隙连接突变的识别和隔离。自基本 缺乏间隙连接的分子和生物物理描述 这些生物，我们将产生必要的免疫和分子 探针并进行电生理分析以识别类型， 间隙连接的分布和生物物理特性不同 组织和发育阶段。由于许多 两种生物的细胞类型符合标准电生理记录 技术，生物物理特征将由 将孤立的间隙连接纳入平面脂质双层和通过 纯化的mRNA在成对的异爪蟾卵母细胞中的表达。抗体 针对已知调节间隙连接门的连接蛋白和剂 将用于确认记录的通道的身份 人工系统。从免疫学获得的信息和 生物物理方法提供了定义间隙连接表型的手段 因此，识别和恢复潜在的间隙连接突变体。 我们恢复突变体的策略包括对表型的研究 由于结构变化或 间隙连接的调节和分段非整倍体的构造 识别果蝇基因组的区域，显示剂量敏感 规定。遗传弹药的回收 介导的通信应导致C. Connexin基因的克隆。 秀丽隐杆线虫和果蝇，将有助于定义差距连接的角色 在复杂的生理和行为功能中。结合 果蝇中的免疫，生物物理，分子和遗传方法 秀丽隐杆线虫提供了解决与该问题有关的问题的机会 间隙连接处的作用在其他缺乏的生物中是不可能的 强大的遗传工具。另外，由于似乎有强大 脊椎动物和无脊椎动物中的离子通道之间的保护，研究 在果蝇和C. c.c。可能会提供适用的信息 一般而言，间隙连接通信。