| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 94 === Loss or inactivation of p53 activity is tightly associated with tumor development in vertebrates, including zebrafish. As the guardian of the genome, p53 is usually activated when cells are under oxidative stress and upon DNA damage, and induces growth arrest or apoptosis. As an attempt to explore the apoptotic effects induced by p53 protein family on zebrafish neuronal development, we had previously expressed various zebrafish p53 family proteins fused to GFP under the control of neuron-specific HuC promoter in early stage of embryos. Interestingly, only p53-GFP, but not ���駛63-GFP nor p73-GFP, was specifically targeted to the nuclei of zebrafish YSL cells, a phenomenon has never been reported in mammalian cells and vertebrate embryos. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated and evaluated. Moreover, the nuclear localization signal in the C-terminal region of zebrafish p53 is required for this targeting to the YSL nuclei of zebrafish embryos. Using morpholino-mediated knockdown approach, we further provide evidence to show that the specific targeting of p53-GFP to the YSL nuclei is mediated by importin �� and ��. Knockdown of importin ��1, ��3, or ��, but not ��5 blocked the targeting to YSL. To ascertain that p53-GFP can serve as a tool to direct specific targeting to zebrafish YSL, the proapoptotic protein BAD as well as two transcription factors, Bozozok and casanova, were fused to p53-GFP and expressed in early zebrafish embryo. Our results showed that the expression of BAD-p53-GFP induces apoptosis of YSL cells, leading to incomplete YSL movement, whereas the expression of Boz-p53-GFP or cas-p53-GFP in YSL causes dorsalization, ventralization, suggesting that zebrafish p53 represents a bona fide YSL targeting molecule and can be a useful tool for the study of zebrafish development.�n On the other hand, we also compare the proapoptotic activity of BAP31 to BAP20 and endoG to �就1-48 endoG by fusing to p53-GFP. Taken together, we identified sequences represent a bona fide YSL targeting signal sequence and can be a useful tool for the study of zebrafish embryo development.
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