Capovilla M, Eldon E D, Pirrotta V
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Development. 1992 Jan;114(1):99-112. doi: 10.1242/dev.114.1.99.
The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Krüppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Krüppel and knirps segmentation gap genes. The analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Krüppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.
来自果蝇巨基因的cDNA序列表明，其产物有一个碱性结构域，后面跟着一个亮氨酸拉链基序。这两个特征都包含DNA结合蛋白b-ZIP家族的特征性保守元件。该基因在细菌中表达或通过体外翻译产生的蛋白质，其迁移速度比从果蝇胚胎中提取的蛋白质快得多。用磷酸酶处理表明，这种差异是由于胚胎中巨蛋白的多重磷酸化所致。该蛋白在前细胞胚盘胚胎中的异位表达产生异常表型，其节段缺失模式与Krüppel突变体胚胎非常相似。免疫染色显示，从hsp70启动子异位表达的巨蛋白抑制了Krüppel和knirps节段间隙基因的表达。对Krüppel、knirps和巨蛋白之间相互作用的分析揭示了一个负调控网络。我们表明，Krüppel对knirps的明显正调控实际上是由Krüppel对巨蛋白的负效应和巨蛋白对knirps的负效应介导的。在细菌或胚胎中产生的巨蛋白在体外与Krüppel调控元件CD1和CD2结合，并识别一个类似于其他b-ZIP蛋白结合位点的序列。
