No abstract

In the present study, the distal part of the 5′‐flanking region of the rainbow trout metallothionein‐A promoter was sequenced in order to identify cis‐acting regulatory elements. Analysis of this sequence combined with that previously reported for the 5′‐flanking region directly proximal to the start of transcription revealed several putative regulatory sequences. In total, six metal‐responsive elements (MREs) were identified; these sequences were organised into two clusters, one containing two copies of MRE and located close to the predicted TATA box sequence, and a second consisting of four MREs and lying 500–700 bp upstream from the start of transcription. In addition, the 5′‐flanking region contained sequences sharing high similarity with the activator protein 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 vector pGL‐2, directly upstream from the luciferase reporter gene. Both MRE 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 promoting gene transcription following exposure of RTH‐149 cells to hydrogen peroxide.

No abstract

In the present study, the distal part of the 5'-flanking region of the rainbow trout metallothionein-A promoter was sequenced in order to identify cis-acting regulatory elements. Analysis of this sequence combined with that previously reported for the 5'-flanking region directly proximal to the start of transcription revealed several putative regulatory sequences. In total, six metal-responsive elements (MREs) were identified; these sequences were organised into two clusters, one containing two copies of MRE and located close to the predicted TATA box sequence, and a second consisting of four MREs and lying 500-700 bp upstream from the start of transcription. In addition, the 5'-flanking region contained sequences sharing high similarity with the activator protein 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 vector pGL-2, directly upstream from the luciferase reporter gene. Both MRE 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 promoting gene transcription following exposure of RTH-149 cells to hydrogen peroxide.