Mtf-1 Mediated Zinc Enhances Metallothionein Gene Expression during Early Development in Zebrafish
| Autor: | Wen-Ya Chen, 陳玟雅 |
|---|---|
| Rok vydání: | 2005 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 93 Metallothioneins (Mts) are small cysteine-rich multifunctional stress proteins. The mt promoter containing highly conserved MRE sequences, activated by the binding of metal-responsive element transcription factor-1 (Mtf-1), in response to heavy metals. Mt proteins can sequestrate, up to seven bivalent of heavy metal ions, and release as required for the biological activities in heavy metal ion homeostasis and detoxification. We found Zn deprivation have an effect on zebrafish embryonic development. However, excess of Zn (up to 1 mM) resulted in high mortality, indicating the deleterious effect of zinc on development. Less is known about the expression pattern and characteristic of mtf-1 and mt gene in embryogenesis. In the present study, we cloned, sequenced and studied the expression pattern of mtf-1 and mt gene during embryonic and early larval stages in zebrafish, previously (Chen et al., 2002; 2004). The unique N-terminal region of zebrafish Mtf-1 is followed by a DNA binding site containing six zinc fingers of C2H2 type. The C terminal region of the protein contains a transactivation domain comprised of a series of three putative distinct activation domains, including an acidic-, a proline- and a serine/threonine-rich regions. We isolated the gene encoding zebrafish Mtf-1 and analyzed the amino acid sequence similarity with that of mouse, human, fugu and drosophila. RT-PCR and whole-mount in situ hybridization results show the maternal expression of Mtf-1 transcripts. Stronger and ubiquitous expression of Mtf-1 transcripts was observed during the embryonic stages whereas, specific expression, especially in the neural parts, was observed throughout the stages studied after hatching. The zebrafish mt gene consists of three exons encoding 60 amino acids with 20 conserved cysteine residues. The deduced amino acid sequence of zebrafish Mt was compared with that of mouse, snake, chicken and xenopus revealed high homology. The maternal contribution of Mt transcripts are also, stronger as well as ubiquitous expression of mt gene during early embryonic stages narrowed to specific expression after 26 somite stages. The mt promoter region contains seven copies of putative metal-responsive elements (MREs), which are shown to be important for the high level activity by deletion analysis. The expression of mtf-1and mt gene during embryogenesis implies their significant roles on development. Whole-mount in situ hybridization showed the Zn-induced specific expression of mt in olfactory pit, chloride cells and liver of 1, 2 and 3-days post-hatched zebrafish, whereas Zn could not induce mtf-1 expression in all the tested stages. By fluorescence techniques, the Zn and Cd mediated translocation of Mtf-1-EGFP fusion protein from cytoplasm to nucleus of zebrafish cell line and embryo is demonstrated. The rMtf-1 protein was expressed using couple transcript/translation system or using pET system in E. coli and purified. Electrophoretic mobility shift assay revealed the binding of the recombinant Mtf-1 in response to Zn but not Cd at the putative metal responsive elements found in the promoter region of mt gene. These data suggest that although Zn and Cd are efficiently involved in Mtf-1 nuclear translocation, only Zn, and not Cd, plays an important role on the enhanced Mt expression in zebrafish through the activation of Mtf-1 binding activity at the MREs. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|