Study on the phosphatidylcholine-synthetic enzyme genes in mammalian cells

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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.