de Vetten N C, Lu G, Feri R J
Horticultural Sciences Department, University of Florida, Gainesville 32611.
Plant Cell. 1992 Oct;4(10):1295-307. doi: 10.1105/tpc.4.10.1295.
The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize. We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex. Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay. Immunoprecipitation experiments suggested that the protein recognized by the antibody is not a DNA binding protein in and of itself, but rather is associated with the DNA binding complex. These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14. Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic alpha-helix similar to known transcriptional activators such as VP16 and GAL4. Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GF14; the protein is also present predominantly in the kernel and root. The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases. GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast. This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes.
G盒元件是许多诱导型基因启动子中一个中度保守的组成部分，包括拟南芥和玉米的乙醇脱氢酶基因。我们使用针对部分纯化的G盒结合因子（GBF）活性产生的单克隆抗体来表征作为DNA结合复合物一部分的玉米蛋白。抗体与部分纯化的玉米GBF复合物相互作用，在凝胶阻滞试验中产生迁移较慢的复合物。免疫沉淀实验表明，抗体识别的蛋白质本身不是DNA结合蛋白，而是与DNA结合复合物相关。这些单克隆抗体被用于分离编码一种我们命名为GF14的蛋白质的cDNA克隆。玉米GF14包含一个类似于亮氨酸拉链的区域以及酸性的羧基末端和氨基末端，其中后者可以形成一个类似于已知转录激活因子如VP16和GAL4的两亲性α螺旋。细胞培养提取物的蛋白质凝胶印迹分析表明，抗GF14识别出一种单一的、主要的约30kD的蛋白质；该蛋白质也主要存在于籽粒和根中。玉米GF14推导的氨基酸序列与拟南芥GF14和月见草PHP - O的序列一致性超过80%，与一类被描述为蛋白激酶C抑制剂以及酪氨酸和色氨酸羟化酶激活剂的哺乳动物脑蛋白的序列一致性超过60%。GF14存在于多种单子叶植物和双子叶植物、裸子植物以及酵母中。这表明与许多基因启动子区域中发现的核心序列相关的潜在调节蛋白具有深度的进化保守性。
