Expression pattern of metallothionein, MTF-1 nuclear translocation, and its dna-binding activity in zebrafish (Danio rerio) induced by zinc and cadmium.
Metallothionein is a small (6-kDa), cysteine-rich protein expressed by a six-zinc finger protein called metal-responsive element-binding transcription factor-1 (MTF-1) in response to Zn and Cd. Our previous reports have shown the basal expression of metallothionein (mt) and MTF-I (mtf-1) genes in embryo and early larval stages of zebrafish (Danio rerio). In the present study, we investigated the mt expression in zebrafish early larvae induced by exposure to Cd and Zn (48, 72, 96, and 120 h postfertilization). Whole-mount in situ hybridization showed that Zn induced mt expression in the olfactory pit, cerebellum, ceratobranchials, liver, chloride cells, and neuromasts of the lateral line. Cadmium also induced mt expression in all the above regions except the cerebellum. Using fluorescence techniques, we have shown that Zn and Cd mediate cytoplasmic and nuclear translocation of MTF- 1-enhanced green fluorescent protein fusion protein in zebrafish liver cell line. The MTF-1 protein was produced recombinantly by inserting zebrafish mtf-1 cDNA (1.8 kb) into pET-20b(+) expression vector and expressing in Escherichia coli BL21 (DE3) pLysS host strain competent cell on induction with isopropyl-beta-D-thiogalactopyranoside. The protein was then purified by affinity chromatography on a nickel-nitrilotriacetic acid column. Electrophoretic mobility shift assay revealed binding of the recombinant MTF-1 in response to Zn and Cd at the putative metal-responsive elements (MREs) in the promoter region of the mt gene. Taken together, these results suggest that Zn and Cd are efficiently involved with mt expression induced in zebrafish embryos and with MTF-1 nuclear translocation and that this induction is achieved through the activation of MTF-1 binding at the MREs.
Chen
Wen-Ya
WY
Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan.
John
Joseph Abraham Christopher
JA
Lin
Cheng-Hui
CH
Chang
Chi-Yao
CY
D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000633:toxicity*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D008668:Metallothionein / Q000235:genetics*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish; D015032:Zinc / Q000633:toxicity*; D000097809:Transcription Factor MTF-1
D002104:Cadmium; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008668:Metallothionein; D011994:Recombinant Proteins; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1