CLONING AND CHARACTERIZATION OF A NOVEL ZINC-FINGER PROTEIN THAT ASSOCIATES WITH NUCLEAR MATRIX

We have recently reported that the nuclei of B16 melanoma cells are in tensely 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 nu clei 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, Screen ing of an NIH3T3 cDNA library resulted in the isolation of a nearly fu ll-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 zi nc finger moths and glutamic acid-rich domains in the C-terminal regio n, When a fusion protein of green fluorescence protein and p75 was exp ressed in NIH3T3 cells, fluorescence was preferentially observed in th e nuclei, demonstrating that the protein has a nuclear localization si gnal. 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 clo sely 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 wa s low in the G(0)/G(1) phase, increased during the G(1) phase, and per sisted during the S and G(2)/M phases, suggesting that ZAN75 plays a r ole in regulating cell growth.