BICALMODULIN PARTITION & MOLECULAR GENETICS OF PARAMECIU

描述：孔敬博士请求继续提供五年的支持 他对钙调素在游泳行为中的作用的研究 纤毛虫，草履虫。 草履虫的游泳行为受到调节 通过膜离子通道的活动。 这些离子通道 转由钙和钙结合蛋白钙调素调节。 每一个钙离子动作电位都会引起纤毛运动的逆转 和一阵后仰游泳。 Na+电流维持了这种作用 潜力，并增加了向后游泳。 钾离子电流 趋向于终止动作电位。 1983年，孔博士和他的 合作者分离出一种草履虫突变体， “泛恐惧症”（pnt）;它向后游的时间比 野生型细胞 这种行为表型是一种突变的结果 在单一钙调素基因中。 具有延长或缩短的突变体 向后游泳也是孤立的。 令人惊讶的是， 向后游时间较短的突变体（快速-2）也在 钙调素基因 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 或具有降低的K+电流，并且N-末端突变体具有降低的Na+ 电流 突变表型和位置的这种分离是基于 13个突变中没有一个落在中心区域 蛋白质。 这些观察导致了这样的假设， 将在本提案中进一步测试钙调素分子是 双功能的 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 合作者已经测试了草履虫钙调蛋白是否可以激活 钙调磷酸酶，其是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N- 终端突变体不能。 这项建议有五个具体目标。 突变等位基因的分配 将通过分离额外的 pantophobic和fast-2突变体。 体外产生的突变也将 接受检查。 草履虫钙调蛋白已结晶 以1.8 A的分辨率，他们将检查突变体的位置 晶体结构的想法，具体的表面可能是 在两种类型的突变体中受到影响。 后来，变种人 钙调素将通过X射线晶体学和NMR进行检测。在 第二个目标，钙调蛋白的双功能性将在 与其他有钙调蛋白测定的实验室合作- 激活酶。 这些包括钙调神经磷酸酶，红细胞C++-ATP酶， 腺苷酸环化酶，肌球蛋白轻链激酶，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 试图克隆两种钙调素结合膜蛋白， 在最后一个补助期内确定的。 同样，他们将完成 分离和表征K+和Na+/Ca++通道基因的同源物。 在第四个目标中，将尝试改进转化， 行为突变体可以通过转化和互补来拯救。 在最终目标中，将使用称为PAJAMAS的程序来开发 一种耗尽特定序列的大核的方法， 可以在草履虫中进行反向遗传学。

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.