Studies on the regulatory mechanisms and the biological significance of metabolisms of membrane phospholipids in mammalian cells by using technics of biochemical genetics.

本研究主要是利用生物化学遗传学技术，对哺乳动物细胞膜磷脂代谢的变异体进行研究。1.本文报道了一个PS（磷脂酰丝氨酸）调控突变株的分离和鉴定，并提出了PS生物合成调控因子的存在。2.利用PS合成酶Ⅰ缺陷突变体成功地克隆了PS合成酶Ⅰ和PS脱羧酶基因，并表明该酶在调节PS和PE（磷脂酰乙醇胺）的合成以及细胞生长和分裂中具有重要意义。3.进一步.通过辛德毕斯病毒感染该突变株的实验，证实了PS和PE在膜融合中的重要性。4.还分离出了一种在Na'依赖性肌醇转运（这是磷脂酰肌醇合成的重要过程）中有缺陷的CHO变体，并提出存在两种肌醇转运系统。5.从CHO细胞中分离到一个对温度敏感的突变体，该突变体具有鞘脂合成的限速酶丝氨酸-帕米酰转移酶的缺陷，并且鞘脂在哺乳动物细胞生长中是必需的。6.从CHO细胞中还分离到PG（磷脂酰甘油）合酶的缺陷突变体，并提出PG和心磷脂在细胞生长中的重要性。7.从J774.1巨噬细胞中分离到一株耐高浓度脂多糖（LPS）的突变株，经LPS处理后，花生四烯酸释放和O_2 ~-产生均出现缺陷。此外，来自磷脂酶激活的调节途径被认为参与了干扰素γ对LPS诱导的O_2 ~-产生的控制。8.钙调素在细胞外ATP控制膜通透性中的重要性。在小鼠3 T3细胞中，GLUT-1（一种葡萄糖转运蛋白基因）通过TGF-β的表达和GLUT-1蛋白糖基化的改变被提出。

This research was performed mainly by using technics of biochemical genetics with variants of mammalian cells concerning metabolisms of membrane phospholipids. 1. A CHO(Chinese hamster ovary)cell mutant with altered regulation of PS(phosphatidylserine)was isolated and the existence of a regulatory factor of PS biosynthesis was suggested. 2. Gene cloning of PS synthase I and PS decarboxylase was succeeded by using a variant defective in PS synthase I, and significance of the enzyme was suggested in regulation of PS and PE(phosphatidylethanolamine)synthesis and in growth and division of cells. 3. Further. importance of PS and PE in the membrane fusion was suggested by the experiment of Sindbis virus infection to this mutant. 4. A CHO variant defective in the Na' dependent inositol transport, which is an important process in phosphatidylinositol synthesis, was also isolated, and the presence of two inositol transport systems was suggested. 5. A temperature sensitive mutant with a defect in serine-parmitoyl transferase, the rate limiting enzyme of the synthesis of sphingolipids, was isolated from CHO cells, and sphingolipids were suggested essential in the mammalian cell growth. 6. A defective mutant of PG(phosphatidylglycerol)synthase was also isolated from CHO cells and importance of PG and cardiolipin in cellular growth was also suggested. 7. A resistant mutant to high concentrations of LPS(lipopolysaccharide)was isolated from J774.1 macrophage cells, and found a defect in both arachidonate release and O_2^- generation after LPS treatment. Further, regulatory pathways from phospholipases activation was suggested to be involved in the control of LPS-induced O_2^- generation by interferon gamma. 8. Importance of calmodulin in control of the membrane permeability with extra-cellular ATP was suggested. In mouse 3T3 cells, the expression of GLUT-1, a glucose transporter gene, by TGF-beta and an altered glycosylation of GLUT-1 protein were suggested.