博士 / 國防醫學院 / 生命科學研究所 / 91 / In these studies, we cloned three types of the pro-apoptotic genes from zebrafish (Danio rerio), and investigated their functions in vitro and in vivo.
Firstly, we cloned a Bad cDNA from zebrafish, which encodes a protein of 147 amino acid residues. zBAD contains a BH3 domain and a putative 14-3-3 binding site, and shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis shown that zBAD was found in various tissues. The treatment with z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zBAD protein, indicating that zebrafish BAD protein may be cleaved by caspase(s). zBAD was shown to induce apoptosis when overexpressed in COS-1 cells. In addition, when zBAD was expressed in the muscle under a muscle-specific promoter from zebrafish a-actin gene, abnormal skeletal muscles were observed. Taken together, we have cloned a zebrafish BAD gene, whose over-expression could induce apoptosis in vivo and in vitro.
Secondly, four hypoxia-induced apoptotic-related genes were cloned from zebrafish and named as zNIP3a, zNIP3b, zNIP3c and zNIX. They encode proteins with 213, 179, 183, and 233 amino acid residues, respectively. By amino acid sequence analysis, these membranes of NIP3/NIX gene family in the zebrafish could divided into three groups: zNIP3a/zNIX, zNIP3b, and zNIP3c. When transfected into the COS-1 cells, the expression of zNIP3a and zNIX increased at different time interval after transfection, while the expression of zNIP3b and zNIP3c was very low and could not be detected by Western blot analysis. The cleavage of zNIP3b and zNIP3c was inhibited by the treatment of MG132, but not by zVAD-fmk, suggesting that caspase(s) may not involve in the cleavage process. When transfected into the COS-1 cells and under normal growth condition, the subcellular localization of zNIP3a, zNIP3b, and zNIP3c was show to localize in the ER while zNIX was in mitochondria. However, after serum starvation, zNIP3a, zNIP3b, and zNIX could translocate to the mitochondria. All zNIP3a, zNIP3b, zNIP3c and zNIX could trigger apoptosis when were they were transfected into the COS-1 cells.
Thirdly, a novel zebrafish gene, zFT27, was cloned and it encoded a protein of 305 amino acids. This protein shows 69% identity to that of human and mouse FT27 proteins at the level of amino acid sequence. Comparison of the genomic structure indicates that all human, mouse and zebrafish FT27 genes are organized into 6 exons and 5 introns with conserved exon/intron junction site. Through the Northern blot and RT-PCR analysis, both mFT27 and zFT27 are ubiquitously expressed in various tissues. The treatment with z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zFT27 protein, indicating that zebrafish FT27 protein may be cleaved by caspase(s). zFT27 was expressed in the COS-1 cells mainly in the Golgi apparatus. Overexpression of zFT27 in the COS-1 cells could induce the fragmentation of the Golgi complex during apoptosis. In addition, the FT27 protein is an evolutionarily conserved protein, because its ortholog is found not only in vertebrates, but also in yeast, fly and worm.
| Identifer | oai:union.ndltd.org:TW/091NDMC0105022 |
| Date | January 2003 |
| Creators | Yueh-Chun Hsieh, 謝岳峻 |
| Contributors | Chang-Jen Huang, 黃銓珍 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 164 |
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