Transient expression assays with the proximal promoter of a newly characterized actin gene from the oyster Crassostrea gigas

We undertook the characterization of an actin gene and its proximal promote r in the oyster Crassostrea gigas. A complete actin cDNA,vas identified, se quenced and its amino acid sequence deduced. Comparative analysis showed a high homology with actin of other species and that this gene is closer to t he cytoplasmic form of actins than to the muscle type. A probe derived from the 5'-untranslated region of the cDNA was then used to isolate the actin gene from a genomic library. The gene was sequenced and shown to contain a single 643 bp intron, A 1670 bp fragment upstream from the open reading fra me was isolated and sequenced. This upstream region displays typical featur es of actins such as a serum response element (CarG box). This fragment was cloned into the promoterless vector pGL3-basic and the resulting construct was transfected into cells of dissociated oyster heart primary cultures. I ts capacity to express the luciferase in this in vitro homologous system wa s monitored and showed high expression levels. This is the first complete a ctin sequence reported so far for the oyster C.gigas and its promoter is th e first available among bivalves, (C) 1999 Federation of European Biochemic al Societies.