Worm Breeder's Gazette 16(3): 27 (June 1, 2000)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705|
|2||Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602|
|3||Department of Environmental Cell Responses, Gifu International Institute of Biotechnology, Mitake, Gifu 505-0116, Japan|
Phospholipase D (PLD) hydrolyzes phospholipids, especially phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. PA acts as a second messenger and can be converted to other messenger molecules, such as 1,2-diacylglycerol and lysophosphatidic acid. In lipid-mediated signal transducing enzymes, cDNAs encoding protein kinase Cs, phospholipases A2 and C have been demonstrated in Caenorhabditis elegans. Although PLD cDNAs have been cloned from a wide variety organisms from bacteria to mammalians, C. elegans PLD cDNA has not been yet isolated. We have recently isolated the gene encoding C. elegans PLD (pld-1). The isolated cDNA encodes a 1,427 amino acid protein which contains four conserved regions defined by the primary structures of bacterial, plant, yeast and mammalian PLDs. Furthermore, HKD motifs (HxKxxxxD) in regions II and IV, which are critical for PLD activity, are completely conserved. The corresponding genomic sequence was covered by the cosmid clone C04G6. Comparison of the sequences between C04G6 and the cloned cDNA revealed that the pld-1 gene has 18 exons and that the transcription unit is 7.8 kb. An extensive screening of the complete C. elegans genome by the BLAST algorithm has revealed that PLD-1 is the sole member of the PLD family which contains four conserved regions including two catalytic HKD motifs. The protein expressed in COS-7 cells catalyzed transphosphatidylation reaction to generate phosphatidylbutanol (PBut) in the presence of 0.3% butanol. It also released [3H]choline from [3H]phosphatidylcholine, indicating that expressed protein exhibited PLD activity. This PLD activity was dependent on phosphatidylinositol 4,5-bisphosphate and weakly stimulated by ADP-ribosylation factor. However, it was strongly inhibited by oleic acid. These properties are similar to those of mammalian PLD2. To determine where pld-1 is expressed, we constructed a translational fusion between pld-1 and the green fluorescent protein (GFP) to generate pld-1::GFP. This construct contains about 4.7 kb pld-1 promoter regions in addition to first 668 amino acids. pld-1 was expressed in pharyngeal muscles and most of neurons. Weak expression was also observed in epithelial cells, body-wall muscles, and excretory canal.