Xiao X, Marzluf G A
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Genetica. 1996 Mar;97(2):153-63. doi: 10.1007/BF00054622.
The nit-2 gene of Neurospora crassa encodes the major nitrogen regulatory protein which acts in a positive fashion to activate the expression of many different structural genes during conditions of nitrogen limitation. An E. coli-expressed NIT2/beta-Gal fusion protein binds specifically to DNA in vitro by recognizing GATA core elements. Nuclear extracts prepared from a wild-type N. crassa strain contain a protein factor which displays all of the properties expected for the native NIT2 protein. The native NIT2 protein in nuclear extracts binds with high affinity to DNA fragments which contain two GATA elements, weakly to fragments with a single GATA element, and fails to bind to DNAs which lack these sequences. The DNA binding ability of the protein factor in nuclear extracts is efficiently blocked by a polyclonal antibody developed against the zinc-finger region of NIT2 protein. Western blot analysis with the anti-NIT2 antiserum revealed a specific protein with a size of approximately 110,000 daltons, in excellent agreement with the predicted size of NIT2. Both the specific NIT2 DNA binding activity and the protein detected by Western blot are totally lacking in nuclear extracts of a nit-2 rip mutant strain. These results all support the conclusion that the native NIT2 protein in Neurospora cells has been identified. The NIT2 protein is localised in nuclei and could not be detected in the cytoplasmic fraction of cells subjected to nitrogen derepression or nitrogen repression, indicating that the nuclear import of NIT2 is not regulated.
粗糙脉孢菌的nit-2基因编码主要的氮调节蛋白，该蛋白在氮限制条件下以正向方式激活许多不同结构基因的表达。一种在大肠杆菌中表达的NIT2/β-半乳糖苷酶融合蛋白通过识别GATA核心元件在体外与DNA特异性结合。从野生型粗糙脉孢菌菌株制备的核提取物中含有一种蛋白质因子，它表现出天然NIT2蛋白所预期的所有特性。核提取物中的天然NIT2蛋白与含有两个GATA元件的DNA片段高亲和力结合，与含有单个GATA元件的片段弱结合，并且不与缺乏这些序列的DNA结合。针对NIT2蛋白锌指区域产生的多克隆抗体有效地阻断了核提取物中蛋白质因子的DNA结合能力。用抗NIT2抗血清进行的蛋白质免疫印迹分析显示一种大小约为110,000道尔顿的特异性蛋白质，与预测的NIT2大小非常一致。在nit-2 rip突变菌株的核提取物中完全缺乏特异性的NIT2 DNA结合活性和蛋白质免疫印迹检测到的蛋白质。这些结果都支持已鉴定出粗糙脉孢菌细胞中天然NIT2蛋白的结论。NIT2蛋白定位于细胞核，在经历氮去阻遏或氮阻遏的细胞的细胞质部分中未检测到，这表明NIT2的核输入不受调节。
