Molecular cloning and expression of a phosphoinositide-specific phospholipase C of Dictyostelium discoideum.
A number of phosphoinositide-specific phospholipases C (PLC) of different species have recently been cloned. The predicted amino acid sequences of these isoforms contain two highly conserved domains. Here we report the identification of a PLC gene of Dictyostelium by using the polymerase chain reaction. Primers were designed coding for highly conserved amino acid regions located within one of the conserved domains of PLCs. Cloning and sequencing of the polymerase chain reaction product revealed one unique PLC-like sequence. This sequence was used to screen a library and isolate several overlapping cDNA clones. The complete cDNA was expressed in Dictyostelium cells resulting in increased basal levels of inositol 1,4,5-trisphosphate and enhanced PLC activity. The identified Dictyostelium PLC, DdPLC, encodes a protein with a calculated molecular mass of 91 kDa. The deduced amino acid sequence contains the two conserved domains found in other PLC isoforms, separated by a short variable region. The C-terminal part of the protein shows strong homology with the mammalian PLC-delta isoform. DdPLC is expressed at all stages of development, with an increase in transcription during starvation and in the culminating fruiting body.
Drayer
A L
AL
Department of Biochemistry, University of Groningen, The Netherlands.
van Haastert
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA; D004023:Dictyostelium / Q000201:enzymology*; D015870:Gene Expression; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase