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 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.
Olsson
P E
PE
Department of Cellular and Developmental Biology, Umeå University, Sweden.
Kling
P
P
Erkell
L J
LJ
Kille
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D011401:Promoter Regions, Genetic; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000494:pharmacology
D005938:Glucocorticoids; D008668:Metallothionein; D015032:Zinc