STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE RAINBOW-TROUT (ONCORHYNCUS MYKISS) METALLOTHIONEIN-A GENE

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.