BICALMODULIN PARTITION & MOLECULAR GENETICS OF PARAMECIU

BICALMODULIN分区

• 批准号: 2415117
• 负责人: CHING KUNG
• 金额: $ 23.81万
• 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/2415117
• 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

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：龚静静博士请求五年的支持以继续 他对钙调素在小鼠游泳行为中作用的研究 纤毛虫、纤毛虫。草履虫的游泳行为受到调节 由膜离子通道的活性决定。这些离子通道在 转而受钙和钙结合蛋白钙调蛋白的调节。 每一个钙离子动作电位都会引起纤毛运动的逆转 以及一阵向后游的动作。钠电流维持这一作用 潜力和增加向后游的能力。外向K+电流 倾向于结束动作电位。1983年，孔博士和他的 合作者分离出了一株帕尔梅氏菌突变体，他们称之为 “长裤恐惧症”(PNT)；它向后游的时间比以前更长 通配单元格。这种行为表型是一种突变的结果 在单一的钙调蛋白基因中。延长或缩短的突变体 倒退游泳比赛也是孤立的。相当令人惊讶的是， 向后游较短时间的突变体(fast-2)也在 钙调蛋白基因。而PNT突变体在羧基末端 作为蛋白质的一部分，FAST-2突变体位于氨基末端叶。 当研究电流时，C-末端突变体缺失 或者降低了K+电流，N端突变体降低了Na+ 电流。这种突变表型和位置的分离是基于 在13个突变上，测试的13个没有落入中心区 蛋白质的含量。这些观察结果导致了这样的假设 将在这一提议中进一步测试钙调蛋白分子是 两种功能。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。孔博士和他的 合作者测试了草履虫钙调蛋白是否可以激活 钙调神经磷酸酶，是一种钙调素依赖的蛋白磷酸酶。他们 发现野生型和C末端突变体可以激活它，但N- 终末突变体则不能。 这项提议有五个具体目标。突变等位基因的划分 将通过分离出另外的 恐惧症和FAST-2变种人。在体外产生的突变也将 接受检查。草履虫钙调蛋白已结晶 在1.8A分辨率下，他们将检查突变体的位置 关于晶体结构的观点认为特定的表面可能是 在这两种类型的突变体中都有影响。在后来的几年里，变种人 钙调素将通过X射线结晶学和核磁共振进行检测。在 第二个目标，钙调蛋白的双功能将在 与其他拥有钙调蛋白检测方法的实验室合作- 被激活的酶。包括钙调神经磷酸酶、红细胞C++-ATPase、 腺苷环化酶、肌球蛋白轻链激酶、钙依赖钙调素 蛋白激酶和磷酸二酯酶。在第三个目标中，他们将 克隆两种钙调蛋白结合膜蛋白的尝试 在上一次授权期内确定的。在类似的情况下，他们将完成 分离和鉴定K+和Na+/Ca++通道基因的同源基因。 在第四个目标中，将努力改进转型，以便 行为突变可以通过转化和互补来拯救。 在最终目标中，将使用一种名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 草履虫可以进行反向遗传学。