A 717-bp 5′-flanking regulatory sequence of the metallothionein gene in Crassostrea hongkongensis (ChMT) was isolated using anchored polymerase chain reaction. Several regulatory elements embedded in the flanking sequence, including the central promoter region, five putative metal-responsive elements (MRE), and an octamer-binding site (OBS), were analyzed using online prediction software. Then, the effects of element-based promoter truncations in the upstream regulatory region of ChMT on the metal stress response were examined. The expression levels of the promoters of various lengths were determined individually using a luciferase reporter gene in HEK293T cells. Transfection assays revealed that MRE1- and MRE5-containing DNA fragments inhibited transcription, whereas MRE4-containing DNA sequences activated promoter expression under both metal-stressed and unstressed conditions. A 39-bp DNA fragment containing the OBS was shown to respond positively to metal stress induced by cadmium and copper, but not to mercury. MRE2- and MRE3-containing DNA fragments comprised a portion of the central promoter region, the loss of which resulted in a significant reduction in promoter activity. Interestingly, the loss of promoter activity due to MRE3 deletion could be rescued by copper-induced stress. This study demonstrates the importance of MRE and additional regulatory elements in the regulation of ChMT promoter expression in response to metal stress.
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