Molecular Biology and Genetics of Calmodulin in Paramecium

本研究是对草履虫中钙调素的研究。这将扩大之前的观察结果，即钙调蛋白的点突变导致草履虫向后游泳。钙调蛋白参与钙调节钾通道和细胞活力的调节。还将制备其他突变体钙调蛋白，并检测它们对钡离子的敏感性；突变体Cam1已被证明对钡的敏感性增加，它可以取代其四个结合位点中的钙。我们将寻找钙调蛋白突变体cAM1和cam 11的基因间抑制因子，试图寻找与调节离子通道和细胞活力有关的其他基因。基因内和基因间的抑制物会影响钙依赖钾通道的激活，而不同于对钡的敏感性。将在哺乳动物细胞中寻找与野生型草履虫钙调蛋白结合得比其突变钙调蛋白更好的蛋白质，并分离出编码这些蛋白质的基因。钙调蛋白是一种蛋白质，存在于所有已检测的细胞类型中。它结合钙，并参与细胞功能的许多方面的调节。其中包括对允许离子进入细胞的通道的调节和对细胞周期的调节。在原生动物草履虫中，单一氨基酸的突变已被证明会导致有机体向后游。此外，这种突变增加了生物体对钡的敏感性，钡可以在特定结合部位取代钙。该建议包括通过各种技术寻找更多的钙调蛋白突变体，寻找蛋白质的基因间抑制物，以及在哺乳动物细胞中寻找与原生动物的野生型和突变型钙调蛋白结合不同的蛋白质。也将寻找不影响钙依赖的钾通道的激活，但确实影响钙调蛋白与钡结合的能力的蛋白质。这项研究应该会增加对钙调蛋白如何调节细胞过程的理解。

This proposal is an investigation of calmodulin in Paramecium. It will extend the previous observation that a point mutation in calmodulin causes the Paramecium to swim backward. Calmodulin is involved in calcium-regulated potassium channels and cell viability in this organism. Other mutant calmodulins will be prepared and their sensitivity to barium ions will be examined; the mutant cam1 has been shown to have increased sensitivity to barium, which can replace calcium in its four binding sites. Intergenic suppressors of the calmodulin mutants cam1 and cam 11 will be sought, to try to find other genes involved in the regulation of ion channels and cell viability. Intragenic and intergenic suppressors which affect activation of the calcium-dependent potassium channel differentially from the barium sensitivity will be sought. Proteins which bind wild-type Paramecium calmodulin better than its mutant calmodulins will be searched for in mammalian cells, and the genes coding for these proteins will be isolated. Calmodulin is a protein which is present in all cell types which have been examined. It binds calcium and is involved in regulation of many aspects of cellular function. Among these are the regulation of channels which permit entry of ions into cells and regulation of the cell cycle. In the protozoan Paramecium, a mutation in a single amino acid has been shown to cause the organism to swim backward. In addition, this mutation increases the sensitivity of the organism to barium, which can substitute for calcium at specific binding sites. The proposal involves a search for additional calmodulin mutants by a variety of techniques, a search for intergenic suppressors of the proteins, and a search for proteins in mammalian cells which bind differently to wild-type and mutant calmodulins from the protozoan. Proteins which do not affect the activation of the calcium-dependent potassium channel but do affect the ability of calmodulin to bind barium will also be sought. The study should increase understanding of how calmodulin regulates cell processes.