Cloning of CTP:phosphocholine cytidylyltransferase cDNA from Arabidopsis thaliana.
As one of the first steps to elucidate the relationship between the structure and function of CTP:phosphocholine cytidylytransferase (EC 2.7.7.15) in plants, the cytidylyltransferase cDNA of Arabidopsis thaliana was cloned and characterized. The A. thaliana cytidylyltransferase cDNA is 1447 bp long and contains an open reading frame of 993 bp coding for a protein of 331 amino acids. The deduced structure of the enzyme was composed of three main regions; the catalytic domain in the N-terminal half, the hydrophilic C-terminal region and the amphipathic domain in the middle. The catalytic domain region was relatively well conserved among different organisms, showing 76 and 72% homology with the rat and yeast protein sequences, respectively. The hydropathy profile revealed that the C-terminal non-catalytic portion of the protein was very hydrophilic, highly enriched in negatively charged aspartic acid and glutamic acid residues. In the region between the catalytic domain and the C-terminal region, there was an amphipathic alpha-helical domain, which was believed to bind the membrane surface in the active formation. Unlike animal counterparts, there was only one potential site of phosphorylation by protein kinase C and none by Ca2+/calmodulin protein kinase II in the C-terminal region. The identity of cytidylyltransferase cDNA was verified by successful transformation of a yeast mutant defective in the enzyme activity, using an expression vector inserted with the A. thaliana cytidylyltransferase cDNA. This was further confirmed by in vivo analysis of the enzyme reaction product after labeling the yeast transformants with radioactive phosphocholine. Southern analysis indicated the presence of a single copy of the citidylyltransferase gene in A. thaliana.
Choi
S B
SB
Department of Biology, Inha University, Inchon, Korea.
Lee
K W
KW
Cho
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D019916:Choline-Phosphate Cytidylyltransferase; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase