BICALMODULIN PARTITION & MOLECULAR GENETICS OF PARAMECIU

BICALMODULIN分区

• 批准号: 2701494
• 负责人: CHING KUNG
• 金额: $ 24.62万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-15 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701494
• 关键词: Paramecium; X ray crystallography; alleles; binding proteins; calmodulin; cell membrane; cilium /flagellum motility; electrophysiology; gene complementation; gene expression; gene mutation; membrane channels; membrane potentials; membrane proteins; microinjections; molecular cloning; nuclear magnetic resonance spectroscopy; nucleic acid sequence; phenotype; site directed mutagenesis; Paramecium; X ray crystallography; alleles; binding proteins; calmodulin; cell membrane; cilium /flagellum motility; electrophysiology; gene complementation; gene expression; gene mutation; membrane channels; membrane potentials; membrane proteins; microinjections; molecular cloning; nuclear magnetic resonance spectroscopy; nucleic acid sequence; phenotype; site directed mutagenesis

DESCRIPTION: Dr. Ching Kung requests five years of support to continue his studies on the role of calmodulin in the swimming behavior of the ciliate, Parmecium. The swimming behavior of Paramecium is regulated by the activities of membrane ion channels. These ion channels are in turn regulated by calcium and by the calcium binding protein calmodulin. Each calcium action potential induces a reversal of the ciliary motion and a burst of backwards swimming. The Na+ current sustains the action potential and increases the backwards swimming. Outward K+ currents tend to end the action potential. In 1983, Dr. Kung and his collaborators isolated a Parmecium mutant that they termed "pantophobiac" (pnt); it swims backwards for longer periods than do wildtype cells. This behavioral phenotype was the result of a mutation in the single calmodulin gene. Mutants that have extended or short backward swimming bouts were also isolated. Rather surprisingly, mutants that swim backwards for shorter periods (fast-2) also were in the calmodulin gene. While the pnt mutants are in the carboxy terminal part of the protein, the fast-2 mutants are in the amino terminal lobe. When the currents are investigated, the C-terminal mutants are missing or have reduced K+ current and the N-terminal mutants have reduced Na+ current. This segregation of mutant phenotypes and location is based on 13 mutations and none of the 13 tested fell into the central region of the protein. These observations have lead to the hypothesis that will tested further in this proposal that the calmodulin molecule is bifunctional. One end can interact with one set of molecules and the other end can interact with different molecules. Dr. Kung and his collaborators have tested whether Paramecium calmodulin can activate calcineurin, which is a calmodulin dependent protein phosphatase. They find that wildtype and C-terminal mutants can activate it, but N- terminal mutants cannot. This proposal has five specific aims. The partitioning of mutant alleles to the two ends will be examined further by the isolation of additional pantophobic and fast-2 mutants. Mutations generated in vitro will also be examined. The Paramecium calmodulin protein has been crystallized at 1.8 A resolution and they will examine the location of the mutants on the crystal structure with the idea that specific surfaces may be affected in the two types of mutants. In later years, mutant calmodulins will be examined by X-ray crystallography and NMR. In the second aim, the bifunctionality of calmodulin will be tested in collaboration with other laboratories that have assays for calmodulin- activated enzymes. These include calcineurin, red cell C++-ATPase, adenylate cyclase, myosin light chain kinase, Ca-calmodulin dependent protein kinase, and phosphodiesterase. In the third aim, they will attempt to clone two calmodulin-binding membrane proteins that they have identified in the last grant period. In a similar vein, they will finish isolating and characterizing homologs of K+ and Na+/Ca++channel genes. In a fourth aim, attempts will be made to improve transformation so that behavioral mutants can be rescued by transformation and complementation. In the final goal, a procedure known as PAJAMAS will be used to develop a method for depleting the macronucleus of a particular sequence so that reverse genetics can be done in Paramecium.

描述：Ching Kung博士请求五年的支持继续 他对钙调蛋白在游泳行为中的作用的研究 纤毛，parmecium。 甲虫的游泳行为受到调节 通过膜离子通道的活动。 这些离子频道在 由钙和钙结合蛋白钙调蛋白调节的转弯。 每个钙动作电位都会引起睫状运动的逆转 和一阵向后游泳。 NA+电流维持动作 潜力并增加向后游泳。 向外K+电流 倾向于结束动作潜力。 1983年，功夫博士及其 合作者隔离了他们称之为的parmecium突变体 “ Pantophobiac”（PNT）；它比向后游泳的时间比 野生型细胞。 这种行为表型是突变的结果 在单个钙调蛋白基因中。 延长或短的突变体 向后游泳也被隔离了。 令人惊讶的是， 较短时间后退的突变体也在 钙调蛋白基因。 虽然PNT突变体在羧基终端 蛋白质的一部分，快速2突变体位于氨基末端叶中。 当研究电流时，C末端突变体缺失 或减少K+电流，N末端突变体减少了Na+ 当前的。 突变表型和位置的这种隔离是基于的 在13个突变中，未经测试的13个突变落入了中部地区 蛋白质。 这些观察结果导致了以下假设 将在该提议中进一步测试，即钙调蛋白分子是 双功能。 一端可以与一组分子相互作用， 另一端可以与不同的分子相互作用。 功博士和他的 合作者已经测试了黑甲钙调蛋白是否可以激活 钙调蛋白，这是一种钙调蛋白依赖性蛋白磷酸酶。 他们 发现野生型和C末端突变体可以激活它，但是n- 末端突变体不能。 该提案具有五个具体目标。 突变等位基因的分区 通过隔离额外 恐惧症和快速2突变体。 体外产生的突变也将 被检查。 甲虫钙调蛋白蛋白已结晶 分辨率为1.8，他们将检查突变体的位置 在晶体结构上，特定表面可能是 在两种类型的突变体中受到影响。 在后来的几年中 将通过X射线晶体学和NMR检查钙调蛋白。在 第二个目的，将在 与其他实验室合作，这些实验室有钙调蛋白的测定法 活化的酶。 其中包括钙调神经酶，红细胞C ++ ATPase， 腺苷酸环化酶，肌球蛋白轻链激酶，Ca-钙调蛋白依赖性 蛋白激酶和磷酸二酯酶。 在第三个目标中，他们将 尝试克隆两个钙调蛋白结合膜蛋白 在最后一个赠款期间确定。 同样，他们会完成 分离和表征K+和Na+/Ca ++通道基因的同源物。 在第四个目标中，将尝试改善转型，以便 行为突变体可以通过转化和互补来挽救。 在最终目标中，将使用称为睡衣的程序来开发 一种耗尽特定序列的大核的方法，以便 反向遗传学可以在甲虫中进行。