CLONING & EXPRESSION OF GAP JUNCTION CHANNELS

克隆

• 批准号: 3293409
• 负责人: DAVID L PAUL
• 金额: $ 20.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293409
• 关键词: Drosophilidae; Xenopus oocyte; antisense nucleic acid; complementary DNA; gap junctions; gel electrophoresis; gene expression; genetic library; growth /development; heart cell; immunofluorescence technique; in situ hybridization; laboratory rat; lens proteins; membrane channels; messenger RNA; molecular cloning; myocardium; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein biosynthesis; protein structure; protein structure function

Intracellular communication mediated by gap junctions is thought to underli a number of crucial cell behaviors. These include the spread of electrical and hormonal signals among populations of cells and regulation of growth an development. DNA cloning indicates that there are a number of related gap junction proteins, which we generically call connexins. 1) The functional properties of gap junctions formed from many of the identified connexins have not been described. We will use an in vitro expression system consisting of pairs of voltage clamped Xenopus oocytes to study those properties. Hybrid proteins will be functionally expressed to identify protein domains that control different behavior. 2) The identity of gap junction proteins in the lens remains unclear. There are reports suggestin that a 26 kD protein (MP26) and a 70 kD protein (MP70 are gap junction structural proteins. In addition, we have cloned CDNA for what may be another gap junction protein in lens. We will make antibodies to this new protein and determine its distribution. We will clone cDNA for MP70 to determine its relationship to MP26 and other possible gap junction proteins 3) We have identified a gap junction protein whose mRNA is present only in Xenopus early embryos. We will determine the spatial and temporal expression of the protein and attempt to modulate its expression in early Xenopus embryos. 4) We will clone cDNAs coding for gap junction proteins in Drosophila. The powerful techniques available for the manipulation of gene expression in this organism will permit close examination of possible roles of gap junctional communication in growth and development.

由缝隙连接介导的细胞内通讯被认为是 一系列重要的细胞行为。 其中包括电力的普及 以及细胞群之间的激素信号和生长的调节， 发展 DNA克隆表明，有一些相关的差距， 连接蛋白，我们一般称之为连接蛋白。 1)功能 由许多已鉴定的连接蛋白形成的缝隙连接的性质 还没有被描述。 我们将使用体外表达系统 由成对的电压钳爪蟾卵母细胞组成， 特性. 杂合蛋白将被功能性表达以鉴定 控制不同行为的蛋白质结构域。 2)gap的身份 透镜中的连接蛋白仍不清楚。 有报道称， 26 kD蛋白（MP26）和70 kD蛋白（MP70）是间隙连接蛋白 结构蛋白 此外，我们还克隆了cDNA， 透镜中的另一种间隙连接蛋白。 我们将制造针对这种新的 蛋白质，并确定其分布。 我们将克隆MP70的cDNA， 确定其与MP26和其他可能的间隙连接蛋白的关系 3)我们已经鉴定了一种间隙连接蛋白，其mRNA仅存在于 非洲爪蟾早期胚胎。 我们将确定空间和时间 表达的蛋白质，并试图调节其表达的早期 非洲爪蟾胚胎 4)我们将克隆编码间隙连接蛋白的cDNA 在果蝇中。 强大的技术可用于操纵 这种生物体中的基因表达将允许仔细检查可能的 缝隙连接通讯在生长发育中的作用。