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 catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.
Nakashima
A
A
Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka 820, Japan.
Hosaka
K
K
Nikawa
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D012316:RNA Nucleotidyltransferases; D051381:Rats; D012441:Saccharomyces cerevisiae
D018076:DNA, Complementary; D004983:Ethanolamines; D019856:Ethanolamine; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C504174:ECT1 protein, S cerevisiae; C020126:Ethanolamine-phosphate cytidylyltransferase