FUNCTIONAL COMPARISON OF THE METAL-REGULATED TRANSCRIPTIONAL CONTROL REGIONS OF METALLOTHIONEIN GENES FROM CADMIUM-SENSITIVE AND TOLERANT FISH SPECIES

The promoter region of teleost metallothioneins (MTs) contains multipl e metal-responsive elements (MREs) organized in proximal and distal cl usters which together mediate gene induction by heavy metals. This arr angement of MREs is found both in cadmium-sensitive species, such as t he rainbow trout, and in cadmium-tolerant species such as the pike and the stone leach. On comparison of the putative regulatory elements id entified within the 5'-flanking region of these genes the major differ ences are that the number of MREs differ between the different species and that, while both the stone loach and rainbow trout MT genes conta in TATA boxes, the pike MT gene has a TTTA box. In order to investigat e if the metal sensitivity of a species is correlated to the regulator y potential of the putative MT detoxification system the promoter regi ons of MT genes from all three species were assessed for their ability to enhance transcription in response to the heavy metals Zn, Cd and C u. The polymerase chain reaction was used to produce nested deletion s ets of each promoter region and these were cloned into the mammalian e xpression vector pGL-2 upstream of the firefly luciferase gene. The in ducibility of the different constructs in response to heavy metal chal lenge was tested in two cell lines, one fish cell line (homologous to rainbow trout and heterologous to the two other species), the rainbow trout hepatoma, RTH-149, cell line and one cell line that was heterolo gous to all studied species, the human hepatoblastoma; HepG2, cell lin e. Maximum inducibility of each gene was achieved with constructs cont aining both the proximal and the distal MRE clusters. Both the rainbow trout and the stone leach MT genes showed inducibility of comparable amplitude whilst the pike MT gene on the other hand was less inducible , partly due to fewer MREs and partly due to the TTTA box: These data indicate that more than one mechanism-is responsible for the differenc es in cadmium sensitivity of these three teleost species. Although MT is the main heavy-metal detoxifying system in most vertebrates and app ears to be contributing to the differences seen between rainbow trout and pike, the present study shows that the relative sensitivity of the se species is not primarily due to MT.