Mechanisms of cell cycle control by calcium ion.

本文从遗传学、分子生物学和生理学等方面研究了钙离子在酿酒酵母钟形增殖、交配信息素反应和形态发生中的作用。研究主要集中在以下四个项目上。1)建立了适合于研究Ca^<2+>调控细胞周期的实验系统。利用该系统，我们发现Ca^<2+>是细胞周期中G_1和G_2/M事件所必需的，并调节cAMP水平。该系统将有助于分离条件致死性突变体，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)已经建立了测量单个酵母细胞胞质游离Ca^<2+>浓度（[Ca^<2+>]i）的技术。我们已经将fura-2作为钙特异性荧光探针与数字图像处理相结合。在尼康Microphot-FX显微镜下的fura-2荧光图像由…More的SIT相机获取，并传输到电视监视器和滨松ARGUS-100图像处理器。在S.cerevisiae中，单倍体细胞的交配过程是由交配信息素（α -和α -因子）控制的，这些信息素在相反交配类型的细胞中引起多种反应，是交配所必需的。利用该系统，我们已经证明，在a__-交配型细胞中添加α -因子可将[Ca^<2+>]i从100 nM的基础水平提高到500-800 nM，并且这种提高对于维持交配信息素反应途径后期酵母细胞的活力至关重要。3)为了建立另一种适合于检测细胞外刺激下[Ca^<2+>]i快速瞬态上升的测量系统，我们采用了发光蛋白aequorin作为钙特异性探针。我们已经构建了质粒，其中aequorin cDNA连接在GAL1启动子或GAP启动子的下游。我们成功地在完整的酵母细胞内再生了aequorin，并在细胞中引入Ca^<2+>后检测了aequorin的发光活性。4)将酵母钙调素基因的编码区与GAL1启动子融合，构建了一个半乳糖调控钙调素表达的酿酒酵母条件致死突变体。突变体在半乳糖培养基中正常生长，但在葡萄糖培养基中停止生长。生长停滞被发现与细胞内钙调素水平的降低有关。末端表型分析表明，当细胞停止生长时，它有一个芽，s期后有细胞核，有丝分裂纺锤体短。我们的结论是，缺陷主要是在核分裂。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