Isolation and characterization of ECT1 gene encoding CTP: phosphoethanolamine cytidylyltransferase of Saccharomyces cerevisiae.
Saccharomyces cerevisiae mutants that were unable to utilize extracellular ethanolamine for phosphatidylethanolamine synthesis were isolated. Two of them carried recessive chromosomal mutations in a same gene and were defective in CTP:phosphoethanolamine cytidylyltransferase (ECT) activity in vitro (Ect-). In an Ect- mutant that also carried the cho1 mutation, phosphatidylethanolamine accounted for less than 2% of total phospholipids, suggesting the importance of ECT in phosphatidylethanolamine synthesis. By screening a genomic library on a low copy number vector, three complementary clones of different size were isolated. A 2.8-kb common DNA region carried an open reading frame (ORF) of 969 bp in length, of which a truncated from failed to complement the Ect- mutation. This ORF was identical to the previously isolated MUQ1 gene of unknown function. Its deduced amino acid sequence had significant similarity to CTP: phosphocholine cytidylyl-transferases of yeast and rat. The entire ORF, when combined with the glutathione S-transferase gene and expressed in Escherichia coli, exhibited ECT activity. These results indicate that the cloned gene encodes a catalytic subunit of ECT of S. cerevisiae.
Min-Seok
R
R
Department of Biotechnology, University of Tokyo.
Kawamata
Y
Y
Nakamura
H
H
Ohta
A
A
Takagi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D012316:RNA Nucleotidyltransferases; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment
D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C504174:ECT1 protein, S cerevisiae; C020126:Ethanolamine-phosphate cytidylyltransferase