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545906 
Journal Article 
Cloning and characterization of a tilapia (Oreochromis aureus) metallothionein gene promoter in Hepa-T1 cells following the administration of various heavy metal ions 
Chan, WWL; Chan, KM 
2008 
Yes 
Aquatic Toxicology
ISSN: 0166-445X
EISSN: 1879-1514 
86 
1 
59-75 
English 
18023887 
Metallothioneins (MTs) are highly conserved intracellular metal-binding proteins that contribute to the homeostasis of essential metals and the detoxification of non-essential heavy metals. MT gene expression is induced by various heavy metal ions, and Zn super(2) super(+) is able to bind and activate a transcription factor associated with the MT gene that is known as the metal responsive element (MRE) binding transcription factor-1 (MTF-1). Heavy metals other than Zn super(2) super(+), such as Cd super(2) super(+) and Cu super(2) super(+), fail to activate the binding of MTF-1 to MREs despite their ability to induce the transcription of the MT gene. To study how different metal ions regulate MT gene expression, a tilapia (ti)-MT gene promoter was cloned and its responses to activation by various metal ions measured using a Hepa T1 cell culture model. The tiMT gene promoter contains six functional MREs within 2118bp 5' of the translational start site. A transient gene expression study showed the tiMT gene promoter fragment to be responsive to Cd super(2) super(+), Cu super(2) super(+), Hg super(2) super(+), Pb super(2) super(+), and Zn super(2) super(+). Deletions from the 5' end and the site-directed mutagenesis of individual MREs in the tiMT gene promoter confirmed that both proximal and distal clusters of MREs were required for the maximal metal induction of the tiMT gene. The distal cluster of MREs greatly enhanced the induction of tiMT gene expression by several of the heavy metal ions, and especially the non-Zn super(2) super(+) ions. Individual MREs showed a different responsiveness to metal ions, with MREe being the most potent, MREb being responsive to Zn super(2) super(+) but not to other metal ions, and MREa being mainly for the basal expression of the tiMT gene. Electrophoretic mobility shift assay (EMSA) identified a transcription factor that was able to bind most of the MREs, with the exception of MREd, but the binding was only activated by the in vivo administration of Zn super(2) super(+), not the administration of Cd super(2) super(+) or Cu super(2) super(+). In conclusion, the results of this study on a Hepa T1 cell model suggest that the mechanism of MT gene activation by non-Zn super(2) super(+) metal ions is different from that of activation by Zn super(2) super(+), and that different MREs may be involved in the activation of the tiMT gene by different metal ions without enhancing the binding of MTF-1 to MREs. 
Article Subject Terms: Administration; Cadmium; Cell culture; Cloning; Copper; Detoxification; Electrophoretic mobility; Freshwater fish; Gene deletion; Gene expression; Heavy Metals; Heavy metals; Homeostasis; Ions; Lead; Metallothionein; Metals; Mobility; Model; Studies; Mutagenesis; Pollution effects; Promoters; Proteins; Regulatory sequences; Site-directed mutagenesis; Tilapia; Toxicants; Transcription activation; Transcription factors; Translation; Zinc; heavy metals; metal ions; metallothioneins; Article Taxonomic Terms:; Oreochromis aureus; X 24360 Metals; P 6000 TOXICOLOGY AND HEALTH; Q5 01504 Effects on; organisms; AQ 00008 Effects of Pollution; SW 3030 Effects of; pollution; G 07730 Development & Cell Cycle; EE 40 Water; Pollution: Monitoring, Control & Remediation