Study on the phosphatidylcholine-synthetic enzyme genes in mammalian cells

在本研究中，我们试图从哺乳动物细胞中分离编码胆碱激酶和磷脂酰乙醇胺- n -甲基转移酶的CKI和PEM基因。首先，将放射性标记的酵母cki和PEM2基因片段与大鼠肝脏噬菌体cDNA文库的斑块进行杂交。虽然我们做了几次实验，改变了杂交条件，但我们都不能用这种方法得到基因。其次，在酵母表达载体上构建大鼠肝脏cDNA文库，分离出在酵母中抑制cki或pem2突变生长缺陷的基因。但该方法也未获得阳性克隆。因此，我们调查了这个系统不能工作的原因。我们发现cDNA插入的平均大小约为500个碱基对，不足以编码酶。第三，我们决定利用高频转化方法，利用Okazaki和Okayama开发的Shizosaccharomyces pombe的反式互补克隆哺乳动物cdna。人类肝脏cDNA文库由大阪大学冈山教授捐赠。利用甲磺酸乙酯诱变法从野生型菌株中分离出胆碱营养不良菌株SPC1和SPC2。通过磷脂中间体和甲基转移酶分析，鉴定菌株SPC2为pem2突变体。因此，我们已经为用这种方法克隆哺乳动物基因做好了准备。另一方面，我们使用酵母酿酒酵母作为哺乳动物磷脂合成的模型系统进行研究。比较编码磷脂酰乙醇胺甲基化途径酶的PEM1和PEM2基因的5‘边界调控区，发现存在一个共同的八聚体序列，5’-CATRTGAA-3'。该序列在肌肌醇胆碱调控PEM1和PEM2中起重要作用。

In the present study we tried to isolate the CKI and PEM genes encoding choline kinase and phosphatidylethanolamine-N-methyltransferase from mammalian cells. Firstly, hybridized radioactively labeled segments of the yeast-CKI and PEM2 genes to plaques of a pharge cDNA library of rat liver. Although we performed several experiments with changing the hybridization conditions, we could not get the genes by this method. Secondly, a rat liver cDNA library was constructed in a yeast expression vector and used to isolate genes that can function in yeast to suppress the grouth defect of the cki or pem2 mutation. But no positive clone was obtained by this method, too. Therefore we investigated the reason why this system could not work. We found that the avearage size of the cDNA insert was about 500 base pair and was insufficient to encode the enzymes. Thirdly, we decided to use the high-frequency transformation method for cloning mammalian cDNAs by trans-complementation of Shizosaccharomyces pombe which was developed by Okazaki and Okayama. A human liver cDNA library was donated by professor Okayama, Osaka University. Next the tow strains SPC1 and SPC2 were isolated as choline auxotroph from the wild-type strain by the use of of ethyl methanesulfonate mutagenesis. The strain SPC2 was identified as a pem2 mutant by the analysis of the phospholipid intermediates and by assaying the methyltrasferase. Thus we have been just ready for cloning the mammalian genes by this method. On the other hand we used a yeast Saccharomyces cerevisiae as a model system for the study on mammalian phospholipid synthesis. Comparison of the 5'boundary reguratoary regions pf PEM1 and PEM2 genes encoding the enzymes of phosphatidylethanolamine methylation pathway revealed the presence of a commmon octameric sequence, 5'-CATRTGAA-3'. The sequence was concluded to play an important role in the myo-inositol-choline regulation of PEM1 and PEM2.