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Characterization of a Zinc Finger Protein ZAN75: Nuclear Localization Signal, Transcriptional Activator Activity, and Expression during Neuronal Differentiation of P19 Cells

机译:锌指蛋白ZAN75的表征:核定位信号,转录激活活性和P19细胞神经元分化过程中的表达。

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摘要

The ZAN75 cDNA was first identified in NIH 3T3 cells and codes for a DNA-binding protein with two zinc finger motifs. In this study, we characterized the nuclear localization signal of ZAN75, tested if ZAN75 regulates transcription, and examined its expression during embryonic development and neuronal differentiation of P19 mouse embryonal carcinoma cells. By examining the cellular localization of deletion mutants of ZAN75 fused to green fluorescence protein, ZAN75 was revealed to have a bipartite nuclear localization signal sequence upstream of the zinc finger domains. The N-terminal region of ZAN75, when fused to the GAL4 DNA-binding domain, strongly activated transcription. The expression of ZAN75 mRNA was found to be developmentally regulated, showing the highest expression in E11.5 embryos. In situ hybridization experiments using E11.5 embryos showed a high expression of the transcripts in neuronal tissues such as brain and neural tube. The expression of ZAN75 was transiently increased at both the mRNA and the protein levels when P19 cells were treated with retinoic acid to induce neuronal differentiation. Taken together, these results indicate that ZAN75 is a transcriptional activator with a bipartite nuclear localization signal and may play a role in neuronal differentiation.
机译:ZAN75 cDNA首先在NIH 3T3细胞中鉴定,并编码具有两个锌指基序的DNA结合蛋白。在这项研究中,我们表征ZAN75的核定位信号,测试ZAN75是否调节转录,并检查其在P19小鼠胚胎癌细胞的胚胎发育和神经元分化过程中的表达。通过检查与绿色荧光蛋白融合的ZAN75缺失突变体的细胞定位,发现ZAN75在锌指结构域上游具有二分核定位信号序列。当与GAL4 DNA结合域融合时,ZAN75的N端区域会强烈激活转录。发现ZAN75 mRNA的表达受发育调节,显示在E11.5胚胎中最高表达。使用E11.5胚胎的原位杂交实验表明,转录本在神经组织(如脑和神经管)中高表达。当视黄酸处理P19细胞以诱导神经元分化时,ZAN75的表达在mRNA和蛋白水平上均瞬时升高。综上所述,这些结果表明ZAN75是具有两部分核定位信号的转录激活因子,并且可能在神经元分化中起作用。

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