Role of Membrane Phospholipid Biosynthesis in Cellular Responce to Enviornmental Stress and Start of Cell Division Cycle in Yeast.

(1)含氮磷脂在酵母细胞生长中的作用分析。(1)研究了一个缺乏磷脂酰丝氨酸（PS）合成的酵母突变体对不同环境条件的响应。通过孔雀石绿染色判断，即使在使突变体生长的胆碱或乙醇胺存在的情况下，只有10%的细胞在固定中期存活。对高浓度Ca^<2+>、Zn^<2+>、Mn^<2+>或碱性氨基酸的敏感性增加。喹啉染色细胞的诺马氏光学和荧光显微镜未观察到中心液泡。这些结果表明，PS的合成对于细胞内膜结构的功能和细胞对环境条件的适应是必要的。(2)构建了一株编码PS合成酶的染色体CHO1基因被破坏，但在着丝粒质粒上将其携带到GAL7启动子下游的菌株。当菌株从以半乳糖为碳源的培养基转移到以葡萄糖为碳源的培养基时，在改变c源后9小时，PS的含量下降到总磷脂的0.3%，磷脂酰乙醇胺的含量也有所下降，但磷脂酰肌醇的含量却增加了47%。培养基改变后，培养浊度的增加持续了9小时，但细胞数量的增加没有持续超过6小时，此时80% ~ 90%的细胞为未出芽细胞。这一结果表明，停止PS合成阻断了细胞分裂周期从G1期进入S期。(2) CHO1基因上游区分析(1)热休克后CHO1- lacz融合基因的表达。由于没有观察到报告酶活性的刺激，因此不清楚5'上游区域的hse样序列的作用。

(1) Analysis of role of nitrogen-containing phospholipids in yeast cell growth.(1) Response to various environmental conditions of a yeast mutant that were deficient in phosphatidylserine (PS) synthesis were examined. Even in the presence of choline or ethanolamine which enables the mutant to grow, only 10% cells were viable at mid-stationary phase, judging by Malachite green staining. Sensitivity to high concentration of Ca^<2+>, Zn^<2+>, Mn^<2+>, or basic amino acids was increased. Central vacuoles were not observed by Nomarski optics and fluorescence microscopy of quinacrine-stained cells. These results suggest that PS aynthesis is necessary for the function of intracellular membraneous structures and for cellular adaptation to environmental conditions.(2) A strain of which chromosomal CHO1 gene that encoded PS synthase was disrupted but that carried it downstream of GAL7 promoter on a centromere plasmid was constructed. When the strain was transferred from a medium with galactose to the one with glucose as a carbon source, the amount of PS was decreased to 0.3% of total phospholipids and that of phosphatidylethanolamine was also decreased, but that of posphatidylinositol was increased up to 47% at 9 hours after C-source change. Increase in culture turbidity was continued for 9 hours after medium change, but the increase in cell number did not continue over 6 hours, when 80% to 90% of cells were unbudded cells. This result suggests that the cessation of PS synthesis blocked entry from G1 to S phase of cell division cycle.(2) Analysis of upstream region of the CHO1 gene(1) Expression of a CHO1-lacZ fusion gene was examined after heat shock. Since stimulation of reporter enzyme activity was not observed, role of an HSE-like sequence in 5'-upstream region was not clear.