Pe. Olsson et al., STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE RAINBOW-TROUT (ONCORHYNCUS MYKISS) METALLOTHIONEIN-A GENE, European journal of biochemistry, 230(1), 1995, pp. 344-349
In the present study, the distal part of the 5'-flanking region of the
rainbow trout metallothionein-A promoter was sequenced in order to id
entify cis-acting regulatory elements. Analysis of this sequence combi
ned with that previously reported for the 5'-flanking region directly
proximal to the start of transcription revealed several putative regul
atory sequences. In total, six metal-responsive elements (MREs) were i
dentified; these sequences were organised into two clusters, one conta
ining two copies of MRE and located close to the predicted TATA box se
quence, and a second consisting of four MREs and lying 500-700 bp upst
ream from the start of transcription. In addition, the 5'-flanking reg
ion contained sequences sharing high similarity with the activator pro
tein 1 consensus sequence as well as one nuclear-factor-interleukin-6-
responsive element. Functional analysis of the promoter was performed
by introducing deletion mutants of the 5'-flanking region into the vec
tor pGL-2, directly upstream from the luciferase reporter gene. Both M
RE clusters were needed for maximal metal inducibility in both rainbow
trout hepatoma (RTH-149) and human hepatoblastoma (Hep G2) cell lines
. Furthermore, the distal region was found to be functional in promoti
ng gene transcription following exposure of RTH-149 cells to hydrogen
peroxide.