Structure of an Ectodermally Expressed Sea Urchin Metallothionein Gene and Characterization of Its Metal-Responsive Region

Abstract

The metallothionein-A gene in the metallothionein gene family of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) was sequenced and found to contain three coding exons plus a 3′ entirely noncoding exon. Putative a and β MT domains were encoded, by its exons 2 and 3, respectively, in reverse of the order in vertebrate metallothionein genes. The SpMTA promoter was characterized through the expression of recombinant constructs containing various portions of the proximal 678-base-pair (bp) 5′-flanking region of the SpMTA gene. Zygotes injected with constructs were cultured to the blastula stage in the presence of a heavy-metal chelator and then incubated in the presence or absence of cadmium. The longest constructs were expressed only when heavy-metal ion was present. Two putative metal-responsive elements (MREs a and b) within 240 bp of the transcription start site resembled mammalian MREs in their critical 8-bp cores (TGCRCNCS) and in their locations relative to each other and to the TATA box. Elimination of activity by site-specific mutations in MREs a and b, separately or in both, identified them as metal regulatory elements. Thus, MRE recognition in this invertebrate resembles that in vertebrates. Upstream sites with single-mismatched MREs neither acted as MREs nor amplified the activity of MREs a and b. The SpMTA, Specl, and CylHa actin genes, which have the same ectodermal specificity, have common DNA elements at relatively similar locations in their promoter regions; however, these elements are insufficient in themselves to promote gene expression.