MOLECULAR GENETICS OF CALMODULIN IN PARAMECIUM

这项研究经费涉及普遍存在的钙结合蛋白。 钙调蛋白及其在细胞过程调控中的作用 纤毛虫草履虫。钙调蛋白已被证明可以 在草履虫行为过程的控制中起关键作用； 钙调素突变体抑制钙依赖的K+和Na+的活性 频道。目前的项目打算继续使用这些独特的 草履虫系统研究钙调蛋白在体内的作用 钙调素依赖的细胞表型的调节。确实有 拨款有三个部分。1)野生型的定点突变 钙调蛋白基因，结合转化现有的 钙调蛋白突变体，将用于研究钙调蛋白的这些方面 调节依赖钙离子所需的分子 频道。来自这些突变的钙调蛋白被证明是 对离子通道的调节产生不利影响的分析将使用 几种确定突变株能力的体外酶试验 钙调蛋白控制其他过程。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 在野生型背景下研究突变的钙调蛋白基因的方法。这个 反义寡核苷酸降低内源性野生型水平 钙调蛋白，这将使改变后的转化钙调素基因 是细胞内主要的钙调蛋白。3)与世隔绝 已有钙调蛋白基因内抑制物的特性 变种人将被接受。基因内抑制子将提供 关于钙调蛋白结构要求的信息。这个 赠款的这三个部分的组合将解决 钙调素针对特定细胞的结构-功能特性 使用活体系统的表型。

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.