Isolation and Characterization of an Actin Promoter from the Red Abalone (Haliotis rufescens)

Abstract:

We have isolated and characterized the 5′-flanking and part of the coding region of an actin gene from the red abalone Haliotis rufescens. There is high sequence homology between the abalone actin coding region and actins from other species. The sequence of this abalone actin is more closely related to vertebrate cytoplasmic actins than to muscle actins. RNase protection assays located the position of the transcription start point 66 bp upstream of the initiation codon. Promoter prediction by neural network located a TATA box 30 bp upstream of the transcription start point. A search with the SIGNAL SCAN program identified several potential transcription factor binding sites in the abalone sequence. These sites include sequences highly conserved in other actin promoters, like several putative CAAT and E boxes and a modified CArG box. Transfection assays with a construct containing the 5′ flanking region of the abalone actin coupled to a luciferase reporter gene showed that the promoter is functional in mammalian and fish cell lines, as well as in abalone gonad tissue. Expression vectors constructed with the abalone actin promoter will be useful for gene transfer studies into abalone and other mollusks.