CLONING OF A HUMAN CDNA FOR CTP-PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE BY COMPLEMENTATION IN-VIVO OF A YEAST MUTANT

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that c atalyzes the formation of CDP-ethanolamine in the phosphatidylethanola mine biosynthetic pathway from ethanolamine. We constructed a Saccharo myces cerevisiae mutant of which the ECT1 gene, putatively encoding ET , was disrupted. This mutant showed a growth defect on ethanolamine-co ntaining medium and a decrease of ET activity. A cDNA clone was isolat ed from a human glioblastoma cDNA expression library by complementatio n of the yeast mutant, Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylet hanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology w ith the yeast, rat, and human CTP-phosphocholine cytidylyl-transferase s, as well as yeast ET. The cDNA gene product was expressed as a fusio n with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained he re encodes human ET.