Cloning and expression of a novel, highly truncated phosphoinositide-specific phospholipase C cDNA from embryos of the brine shrimp, Artemia

• Published in: The Journal of biological chemistry 1994-04, Vol.269 (17), p.12925-12931
• Main Authors: Su, X, Chen, F, Hokin, L E
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Artemia; Artemia - embryology; Artemia - enzymology; Base Sequence; Biological and medical sciences; Blotting, Northern; Brackish; Cloning, Molecular; DNA, Complementary; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Hydrolases; Molecular Sequence Data; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases - genetics; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)99964-3

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.