A Drosophila Model for Angelman Syndrome

描述（由申请人提供）：天使人综合征是一种影响约1/ 15000人的神经系统疾病，导致严重的智力迟钝。这种疾病是由Ube3a的功能突变丧失引起的，Ube3a编码一种hect结构域泛素连接酶或E3蛋白。E3蛋白是泛素化的特异性因子；它们结合特定的底物并将泛素化机制带给它们。Angelman综合征的一个简单的生物化学模型是UbeSa活性的丧失导致E3正常底物的过度表达。果蝇的基因组包含一个名为CG6190的Ube3a同源物。这项研究的目标是建立一个天使综合症的果蝇模型，以验证这一假设，并确定与该疾病相关的底物。首先，将确定果蝇CG6190敲除突变体是否具有Angelman综合征样表型。突变蝇将分析其中枢神经系统的形态和功能缺陷。其次，阐述果蝇发育过程中CG6190 mRNA和蛋白的表达模式。原位杂交将用于检测整个生物体的mRNA，并产生抗cg6190抗体并用于检测蛋白质。第三，确定果蝇CG6190与人类UbeSa是否具有功能同源性。通过产生转基因，比较野生型CG6190和人UbeSa对CG6190突变型的补充能力和过表达时产生突变型的能力。此外，位点定向诱变将用于产生果蝇和人类基因的等位基因，这些等位基因包含在Angelman综合征患者中发现的突变。表达所产生的突变等位基因的效果将进行类似的测试。最后，将在果蝇中进行基因筛选，以确定候选的CG6190/Ube3a底物。野生型CG6190在果蝇眼睛中的过度表达导致严重的突变表型。将分离眼睛表型的修饰因子并鉴定突变基因。

DESCRIPTION (provided by applicant): Angelman syndrome is a neurological disorder affecting ~1/15,000 people that results in severe mental retardation. The disease is caused by loss-of-function mutations in Ube3a, which encodes a HECT-domain ubiquitin ligase or E3 protein. E3 proteins are specificity factors for ubiquitination; they bind specific substrates and bring the ubiquitination machinery to them. A simple model for the biochemistry of Angelman syndrome is that loss of UbeSa activity results in overexpression of the E3's normal substrates. The Drosophila genome contains a Ube3a homolog called CG6190. The goal of the research proposed is to generate a Drosophila model for Angelman syndrome that will allow a test of this hypothesis and the identification of the substrates relevant to the disease. First, it will be determined if Drosophila CG6190 knock-out mutants have an Angelman syndrome-like phenotype. Mutant flies will be analyzed for morphological and functional defects in their central nervous system. Second, the expression pattern of CG6190 mRNA and protein during Drosophila development will be described. In situ hybridization will be used to detect mRNA in whole organisms and anti-CG6190 antibodies will be generated and used to detect the protein. Third, it will be determined if Drosophila CG6190 and human UbeSa are functional homologs. By generating transgenes, the abilities of wild-type CG6190 and human UbeSa to complement the CG6190 mutant phenotype and to generate mutant phenotypes when overexpressed will be compared. In addition, site-directed mutagenesis will be used to generate alleles of the Drosophila and human genes that contain mutations identified in Angelman syndrome patients. The effects of expressing the resulting mutant alleles will be tested similarly. Finally, a genetic screen will be performed in Drosophila to identify candidate CG6190/Ube3a substrates. Overexpression of wild-type CG6190 in the Drosophila eye results in a severe mutant phenotype. Modifiers of the eye phenotype will be isolated and the mutant genes identified.