Mechanisms of cell cycle control by calcium ion.

本论文从遗传学、分子生物学和生理学的角度研究了钙离子在酿酒酵母钟状细胞增殖、交配信息素反应和形态建成中的调控作用。研究重点是以下四个项目。1)我们建立了一个适合于研究Ca^<2+>调控细胞周期的实验系统。利用该系统，我们发现Ca^（2+）对细胞周期中的G_1和G_2/M期事件是必需的，并调节cAMP水平。该系统将用于分离条件致死突变体，这些突变体只有在培养基中存在足够浓度的Ca^2+时才能生长。2)测定单个酵母细胞胞浆游离Ca^2+浓度（[Ca^2+]i）的技术已经建立。我们采用fura-2作为钙特异性荧光探针，结合数字图像处理。在Nikon Microphot-FX显微镜下的fura-2荧光图像通过以下方式获得： ...更多信息 SIT摄像机，并将其中继到电视监视器和Hamamatsu ARGUS-100图像处理器。在酿酒酵母中，单倍体细胞的交配过程由交配信息素，α__和α-因子控制，它们在相反交配类型的细胞中诱导几种反应，并且是交配所必需的。使用这个系统，我们已经表明，添加α-因子到α-交配型的细胞中使[Ca^<2+>]i从100 nM的基础水平升高到500-800 nM，并且这种升高对于在交配信息素反应途径的后期维持酵母细胞的活力是必需的。3)为了建立另一种适合于检测细胞外刺激引起的[Ca^2+]i快速和短暂升高的测量系统，我们采用了发光蛋白水母发光蛋白作为钙特异性探针。我们构建了质粒，其中水母发光蛋白cDNA连接在GAL 1启动子或差距启动子的下游。我们成功地在完整的酵母细胞内再生了水母发光蛋白，并检测到当Ca^2+导入细胞后水母发光蛋白的发光活性。4)将酵母钙调素基因编码区与GAL 1启动子融合，构建了半乳糖调控钙调素表达的酿酒酵母条件致死突变株。该突变株在半乳糖培养基中生长正常，而在葡萄糖培养基中则停止生长。发现生长停滞与细胞内钙调素水平的降低有关。终末表型分析表明，当细胞停止生长时，它有一个芽，S期后有一个核和一个短的有丝分裂纺锤体。我们认为这种缺陷主要存在于核分裂中。少

Roles of calcium ion in regulation of bell proliferation, the mating pheromone response and morphogenesis of Saccharomyces cerevisiae have been investigated using genetic, molecular biological and physiological approaches. The research is particularly focused on the following four projects. 1) We have established an experimental system suitable for study of cell cycle regulation by Ca^<2+>. Using this system, we have shown that Ca^<2+> is essential for G_1 and G_2/M events during the cell cycle and regulates cAMP level. This system will be useful to isolate conditional lethal mutants which can grow only when sufficient concentrations of Ca^<2+> are present in media. 2) Techniques for measuring the cytosolic free Ca^<2+> concentration ([Ca^<2+>]i) in individual yeast cells have been established. We have employed fura-2 as a calcium specific fluorescent probe in conjunction with digital lmage processing. Images of fura-2 fluorescence under a Nikon Microphot-FX microscope are acquired by … More a SIT camera and relayed into a TV monitor and a Hamamatsu ARGUS-100 image processor. In S.cerevisiae, the mating process of haploid cells is controlled by the mating pheromones, a__- and alpha-factors, that induce several responses in cells of opposite mating type and are essential for mating. Using this system, we have shown that addition of alpha-factor to cells of a__- mating type raises [Ca^<2+>]i to 500-800 nM from a basal level of 100 nM and that this rise is essential for maintaining viability of yeast cells late in the mating pheromone response pathway. 3) To establish another measurement system suitable for detecting a rapid and transient rise in [Ca^<2+>]i in response to extracellular stimuli, we have employed a luminescent protein, aequorin, as a calcium specific probe. We have constructed plasmeds in which the aequorin cDNA is joined downstream of either the GAL1 promoter or the GAP promoter. We could successfully regenerate aequorin inside intact yeast cells and detect luminescent activity of it when Ca^<2+> was introduced into the cells. 4) The coding region of a yeast calmodulin gene was fused to the GAL1 promoter and a conditional-lethal mutant of S.cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, it ceased growing. The growth arrest was found to be associated with a decrease in intracellular calmodulin levels. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. We concluded that the defect is mainly in nuclear division. Less