Lee J Y, Kambe M, Hayashi M, Takenaga K
Division of Chemotherapy, Chiba Cancer Center Research Institute, Japan.
DNA Cell Biol. 1998 Oct;17(10):849-58. doi: 10.1089/dna.1998.17.849.
We have recently reported that the nuclei of B16 melanoma cells are intensely stained with anti-rat vitronectin (Vn) antibody, which reacts with both mouse and rat Vn. In the present study, we characterized the protein immunoreactive with the antibody using NIH3T3 cells, whose nuclei were also stained with the antibody. Western blot analysis showed that a protein with an approximate molecular weight of 75 kDa (p75), which was distinct from Vn, existed in the nuclear fraction, and, more specifically, in the nuclear matrix fraction, of NIH3T3 cells. Screening of an NIH3T3 cDNA library resulted in the isolation of a nearly full-length cDNA clone encoding p75. A database search revealed that the cDNA represents a novel gene. The deduced amino acid sequence showed that the protein is 580 amino acids long and contains two C2H2-type zinc finger motifs and glutamic acid-rich domains in the C-terminal region. When a fusion protein of green fluorescence protein and p75 was expressed in NIH3T3 cells, fluorescence was preferentially observed in the nuclei, demonstrating that the protein has a nuclear localization signal. The p75 protein, termed ZAN75, exhibited DNA-binding activity in a zinc-dependent manner. Southern blot analysis demonstrated that the ZAN75 gene exists in a single copy in the mouse genome and that a closely related gene is also present in chicken, rat, and human. Northern blot analysis showed that the ZAN75 gene is ubiquitously expressed in adult mouse tissues. In the cell cycle of NIH3T3 cells, expression was low in the G0/G1 phase, increased during the G1 phase, and persisted during the S and G2/M phases, suggesting that ZAN75 plays a role in regulating cell growth.
我们最近报道，B16黑色素瘤细胞的细胞核被抗大鼠玻连蛋白（Vn）抗体强烈染色，该抗体可与小鼠和大鼠的Vn发生反应。在本研究中，我们使用NIH3T3细胞对与该抗体发生免疫反应的蛋白质进行了表征，其细胞核也被该抗体染色。蛋白质印迹分析表明，在NIH3T3细胞的核组分中，更具体地说是在核基质组分中，存在一种分子量约为75 kDa（p75）的蛋白质，它与Vn不同。对NIH3T3 cDNA文库的筛选导致分离出一个编码p75的近乎全长的cDNA克隆。数据库搜索显示该cDNA代表一个新基因。推导的氨基酸序列表明该蛋白质长580个氨基酸，在C末端区域包含两个C2H2型锌指基序和富含谷氨酸的结构域。当绿色荧光蛋白与p75的融合蛋白在NIH3T3细胞中表达时，荧光优先在细胞核中观察到，表明该蛋白质具有核定位信号。p75蛋白，称为ZAN75，以锌依赖的方式表现出DNA结合活性。Southern印迹分析表明ZAN75基因在小鼠基因组中以单拷贝形式存在，并且在鸡、大鼠和人类中也存在一个密切相关的基因。Northern印迹分析表明ZAN75基因在成年小鼠组织中普遍表达。在NIH3T3细胞的细胞周期中，G0/G1期表达较低，G1期增加，并在S期和G2/M期持续存在，这表明ZAN75在调节细胞生长中起作用。
