MPS1 KINASE AND YEAST SPINDLE POLE CYCLE

拟议项目的目标是了解MPS1的作用 蛋白激酶在酿酒酵母纺锤体极体(SPB)的复制和 在纺锤体组件检查点，并确定该激酶是否 在中心体复制的控制中广泛保守。这个 负责染色体的有丝分裂和减数分裂纺锤体的形成 传递依赖于适当的中心体复制和 功能。了解中心体复制将与以下方面相关 染色体不分离机制的剖析 不平衡和隐性突变在肿瘤中的表达- 抑制基因。 Mps1基因突变导致SPB复制缺陷和 主轴组件检查点路径。调查员现在能够 监测从猪瘟病毒中分离的Mps1p(表位标记)蛋白激酶活性 内源水平的酵母。他将使用这种试剂来研究 Mps1p的激酶活性在细胞周期中受到调节。 分离Mps1p两种功能的诱变方法及分析 将进行Mps1p的磷酸化，目的是了解 Mps1p在细胞周期中如何执行其两个角色。要确定 他们计划识别其产物与Mps1p相互作用的基因 过度表达的不同剂量表型的抑制物 活性或非催化活性的MPS1。这些将被分析以 确定他们如何与MPS1互动并确定他们在SPB中的角色 复制或主轴组件检查点。 研究人员已经发现了不可能存在的突变 与Mps1基因的突变相结合。他将继续这样做 研究是一种新的、必不可少的MOE1基因，它似乎是 主轴功能正常。他们还将分离出相应的基因 与他们已经确认的其他几个合成致命突变有关。 对这些新发现的基因的分子分析应该表明 编码的基因产物与Mps1p相互作用，并证明如果 新的基因涉及SPB复制或纺锤体检查点。 Mps1的序列同源物已经在各种生物中被鉴定出来。 在小鼠中，一个潜在的同源物由Esk基因编码。他们提议 Esk在小鼠胚胎中的表达及活性研究 成纤维细胞。他还将过度表达活跃和不活跃的ESK 小鼠细胞，并确定这些细胞是否表现出指示 中心体复制或主轴组件检查点存在缺陷。 结果应该表明Mps1激酶是否编码一种保守的 在它的任何一个已知角色中发挥作用。

The proposed project's goals are to understand the role of the MPS1 protein kinase in S. cerevisiae spindle pole body (SPB) duplication and in the spindle assembly checkpoint, and to determine if this kinase is widely conserved in the control of centrosome duplication. The formation of mitotic and meiotic spindles responsible for chromosome transmission is dependent upon proper centrosome duplication and function. Understanding centrosome duplication will be pertinent to dissecting the mechanisms of nondisjunction involved in chromosome imbalance and in the expression of recessive mutations in tumor- suppressing genes. Mutations in the MPS1 gene cause defects in SPB duplication and the spindle assembly checkpoint pathway. The investigator's are now able to monitor Mps1p (epitope-tagged) protein kinase activity isolated from yeast at endogenous levels. He will use this reagent to study how the kinase activity of Mps1p is regulated during the cell cycle. Mutagenesis to separate the two functions of Mps1p and the analysis of Mps1p phosphorylation will be performed with the goal of understanding how Mps1p executes its two roles during the cell cycle. To identify genes whose products interact with Mps1p, they plan to identify suppressors of the distinct dosage phenotypes of overexpressing either active or catalytically inactive MPS1. These will be analyzed to determine how they interact with MPS1 and to determine their role in SPB duplication or the spindle assembly checkpoint. The investigator's have identified mutations that are inviable in combination with mutations in the MPS1 gene. One he will continue to study is the novel, essential MOE1 gene that appears to be required for normal spindle function. They also will isolate the genes corresponding to several other synthetic lethal mutations they have identified. Molecular analysis of these newly identified genes should indicate how the encoded gene product interacts with Mps1p, and demonstrate if the new genes is involved in SPB duplication or the spindle checkpoint. Sequence homologues of MPS1 have been identified in various organisms. In mice a potential homologue is encoded by the esk gene. They propose to study the expression and activity of esk in mouse embryonic fibroblasts. He also will overexpress both active and inactive esk in mouse cells and determine if the cells exhibit phenotypes indicative of defects in centrosome duplication or the spindle assembly checkpoint. The results should indicate if the MPS1 kinase encodes a conserved function in either of its known roles.