Miskiewicz P, Morrissey D, Lan Y, Raj L, Kessler S, Fujioka M, Goto T, Weir M
Department of Biology, Wesleyan University, Middletown, CT 06459, USA.
Development. 1996 Sep;122(9):2709-18. doi: 10.1242/dev.122.9.2709.
Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.
果蝇的配对基因是哺乳动物Pax - 3的同源物，在胚胎发育过程中是协调调控体节极性基因的关键。配对基因及其同源物在编码具有两个DNA结合结构域（一个配对结构域和一个同源结构域）的蛋白质方面很不寻常。我们正在使用一种体内试验来剖析这类分子各结构域的功能。特别地，我们感兴趣的是确定配对蛋白的个体体内功能是否需要一种或两种DNA结合活性。我们在每个结构域构建了旨在破坏DNA结合的点突变体，并在果蝇胚胎中通过异位表达试验对这些突变体进行了测试。任一结构域的突变都消除了对目标基因engrailed、刺猬蛋白、醋栗和间断翅脉的正常调控，这表明配对蛋白的这些体内功能需要通过两个结构域结合DNA，而不是单独通过任一结构域。然而，当将这两种突变蛋白置于同一个胚胎中时，配对蛋白的功能得以恢复，这表明两种DNA结合活性不必存在于同一个分子中。对这种效应的定量分析表明，配对结构域突变体具有显性负效应，这与配对蛋白可作为二聚体结合DNA的观察结果一致。
