title: Functional analysis of duplicated genes and N-terminal splice variant of phospholipase C-δ1 in Paralichthys olivaceus.
abstract: Phosphoinositide-specific phospholipase C δ (PLC δ) plays an important role in many cellular responses and is involved in the production of second messenger. Here, we describe the presence of novel N-terminal extended alternative splice form of PLC-δ1B in Paralichthys olivaceus, which differs from the reported mammalian PLC-δ1 isoform. The two variants PoPLC-δ1B-Lf and PoPLC-δ1B-Sf share exon 3 (including the PH domain) to exon 16, but differ at the exon 1 (Short form: Sf) and novel exon 2 (Long form: Lf) of the transcript. For the characterization of the novel duplicated gene variant of PLC-δ1B in P. olivaceus, tissue-specific expression with RT-PCR and real-time PCR, and purification and enzymatic characterization of native and recombinant proteins of all the three-types of PLC-δ1 isoforms (PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf) of P. olivaceus were studied. The PoPLC-δ1A was ubiquitously distributed in gill, kidney and spleen. The PoPLC-δ1B-Lf gene was widely detected in various tissues, especially in the digestive system, while PoPLC-δ1B-Sf was highly expressed in the stomach. The recombinant PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins were expressed as a histidine-tagged fusion protein in Escherichia coli. The PLC activity of the PoPLC-δ1 isoform proteins showed a concentration-dependent activity to phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). In addition, U73122, the PLC inhibitor, effectively inhibited PLC activities of PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins. However, PoPLC-δ1A and PoPLC-δ1B-Lf were sensitive at pH 7.5, while PoPLC-δ1B-Sf was relatively sensitive at pH 7. These results might be useful for the study of phospholipase C-mediated signal transduction in fish.
authors: Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, South Korea.
authors: Na Young
authors: NY
authors: Kim
authors: Moo-Sang
authors: MS
authors: Kim
authors: Sang Jung
authors: SJ
authors: Ahn
authors: Jung Soo
authors: JS
authors: Seo
authors: Hye Jin
authors: HJ
authors: Bak
authors: Bo Seong
authors: BS
authors: Kim
authors: Hyae In
authors: HI
authors: Jo
authors: Hee Young
authors: HY
authors: Jang
authors: Hee Sung
authors: HS
authors: Jo
authors: Hyung Ho
authors: HH
authors: Lee
authors: Joon Ki
authors: JK
authors: Chung
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D005432:Flounder / Q000235:genetics*; D020131:Genes, Duplicate / Q000235:genetics*; D054803:Phospholipase C delta / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D054803:Phospholipase C delta