Pe. Olsson et P. Kille, FUNCTIONAL COMPARISON OF THE METAL-REGULATED TRANSCRIPTIONAL CONTROL REGIONS OF METALLOTHIONEIN GENES FROM CADMIUM-SENSITIVE AND TOLERANT FISH SPECIES, Biochimica et biophysica acta, N. Gene structure and expression, 1350(3), 1997, pp. 325-334
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.