MOLECULAR GENETICS OF CALMODULIN IN PARAMECIUM

草履虫钙调蛋白的分子遗传学

• 批准号: 2185435
• 负责人: ROBERT D HINRICHSEN
• 金额: $ 16.87万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185435
• 关键词: Paramecium; antisense nucleic acid; calcium channel; calmodulin; gene expression; molecular genetics; potassium channel; protein structure; protein structure function; site directed mutagenesis; sodium channel; suppressor mutations

This research grant concerns the ubiquitous calcium-binding protein calmodulin and its role in the regulation of cellular processes in the ciliated protozoa Paramecium tetraurelia. Calmodulin has been shown to play a key role in the control of the behavioral process in Paramecium; calmodulin mutants inhibit the activity of the Ca++-dependent K+ and Na+ channels. The current project intends to continue the use of those unique aspects of the Paramecium system to investigate the role of calmodulin in the regulation of calmodulin-dependent cellular phenotypes. There are three sections to the grant. 1) Site-specific mutagenesis of the wildtype calmodulin gene, in combination with transformation of the existing calmodulin mutants, will be used to study those aspects of the calmodulin molecule that are required for the regulation of a Ca++-dependent ion channel. The calmodulin protein from those mutations that are shown to adversely affect the regulation of the ion channels will be analyzed using several in vitro enzyme assays to determine the ability of the mutated calmodulin to control other processes. 2) 3'-modified antisense oligodeoxynucleotides complementary to calmodulin mRNA will be used as a means to study mutagenized calmodulin genes in a wildtype background. The antisense oligodeoxynucleotides reduce the level of the endogenous wildtype calmodulin, and this will allow the altered transforming calmodulin gene to be the dominant calmodulin in the cell. 3) The isolation and characterization of intragenic suppressors of the existing calmodulin mutants will be undertaken. The intragenic suppressors will provide information as to the requirements of the calmodulin structure. The combination of these three sections of the grant will address the structure-function properties of calmodulin in regards to specific cellular phenotypes using an in vivo system.

这项研究经费涉及普遍存在的钙结合蛋白 钙调素及其在调节细胞过程中的作用 纤毛原生动物四脲草履虫。 钙调素已被证明 在草履虫的行为过程控制中起关键作用; 钙调素突变体抑制Ca++依赖的K+和Na+的活性， 渠道 目前的项目打算继续使用这些独特的 草履虫系统的各个方面，以研究钙调蛋白在 钙调素依赖性细胞表型的调节。 有 三是补助金。 1)野生型的位点特异性诱变 钙调素基因，结合转化现有的 钙调素突变体，将被用来研究这些方面的钙调素 调节Ca++依赖性离子所需的分子 频道 来自这些突变的钙调蛋白被证明 不利影响离子通道的调节将使用 几种体外酶测定法来确定突变的能力 钙调素来控制其他过程。2)3 '-修饰反义 与钙调蛋白mRNA互补的寡脱氧核苷酸将被用作 是指在野生型背景下研究突变的钙调素基因。 的 反义寡脱氧核苷酸降低内源性野生型 钙调素，这将允许改变的转化钙调素基因， 是细胞中占主导地位的钙调素。3)分离和 现有钙调素基因内抑制因子的表征 变种人将被带走。 基因内抑制因子将提供 关于钙调素结构的要求的信息。 的 这三个部分的赠款组合将解决 钙调素在特定细胞中结构-功能特性 使用体内系统的表型。