Cloning and expression of a novel, highly truncated phosphoinositide-specific phospholipase C cDNA from embryos of the brine shrimp, Artemia.
A novel, highly truncated form of a cDNA encoding Artemia phosphoinositide-specific phospholipase C (PLC), designated PLC-beta x, was isolated from a brine shrimp cDNA library. The full-length cDNA is of the beta-type, it is 2855 base pairs long, and it contains an open reading frame encoding 489 amino acids. The deduced amino acid sequence of PLC-beta c cDNA shows novel features. It lacks several hundred amino acids at the 5' end, as compared to PLC-beta s in the higher species. It contains conserved domains X and Y, but domain X is highly truncated at the 5' end (only 14-25 conserved amino acids as compared to about 150 amino acids in the higher eukaryotic organisms). Northern blot hybridization showed that the PLC-beta x cDNA corresponds to a 4.4-kilobase mRNA. Northern blot hybridization with a cDNA probe from the 5' end and PCR performed upstream from domain Y showed that PLC-beta x is not a cloning artifact due to fusion of an unrelated clone into the coding region of the PLC-beta homologue. A functional PLC and new protein bands on SDS-PAGE were observed after subcloning full-length PLC-beta x cDNA, as well as a fragment containing the conserved regions, into expression plasmid vectors and transfecting into Escherichia coli. 1 mM lithium markedly stimulated expression in E. coli.
Su
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Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Chen
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F
Hokin
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LE
D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia / Q000196:embryology / Q000201:enzymology*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase