DISSECTION OF DROSOPHILA PAIRED PROTEIN FUNCTION

转录因子是调控模式形成的核心 在发育中，果蝇分节基因，其中许多编码 转录因子参与调节级联反应， 决定了胚胎中细胞的发育命运配对（PRD） 分割基因，这个建议的主题，功能组合 与其他配对规则基因一起调节片段的转录， 极性基因，它们在级联反应中处于下游。prd基因是 特别令人感兴趣的是，与其哺乳动物同源物一样，Pax 3（相关 与人Waardenburg综合征），它编码几个保守的基序， 包括同源结构域和配对结构域，每个结构域具有DNA 约束力活性和富含脯氨酸的转录激活结构域。 通过分析Prd蛋白的DNA和蛋白质相互作用， 几种补充方法，这项研究应该提供重要的 深入了解细胞命运的组合机制， 在开发过程中指定。具体目标是： (1)为了研究两个DNA结合结构域的要求， Prd.配对结构域的特异性和相对位置， Prd蛋白内的同源结构域将被改变， 构建体将使用异位表达和拯救测定进行测试， 果蝇胚胎 (2)为了鉴定和表征Prd的蛋白质相互作用物， 酵母双杂交系统筛选果蝇胚胎cDNA文库。 将使用体外和体内方法表征假定的相互作用物 测定。 (3)使用缺失鉴定Prd的核定位信号 在果蝇Schneider细胞中的分析。将检测缺失构建体 在体外与一组Prd相互作用物形成蛋白质相互作用， 确定核定位机制的假定组件。 (4)为了评估富含脯氨酸的激活结构域对于 组合调节Prd嵌合体的功能，其中 富含脯氨酸的结构域已被其他类型的激活所取代 将在胚胎中进行测试。 (5)目的：通过报告基因分析，确定Prd的DNA靶点， 胚胎将对已识别目标的更改版本进行测试， 确定Prd功能的顺序要求。 (6)通过分析比较小鼠Pax 3和果蝇Prd的功能， Pax 3/Prd嵌合体在果蝇胚胎中的功能。从进化的角度来看- Pax 3的保守分子特性也可能对 将分析哺乳动物细胞中的Pax 3功能。

Transcription factors are central to the regulation of pattern formation in development Drosophila segmentation genes, many of which encode transcription factors, participate in a regulatory cascade that ultimately specifies the developmental fates of cells in the embryo. The paired (prd) segmentation gene, the subject of this proposal, functions in combination with other pair-rule genes to regulate the transcription of segment- polarity genes, which are downstream in the cascade. The prd gene is of particular interest because like its mammalian homolog, Pax3 (associated with human Waardenburg's syndrome), it encodes several conserved motifs, including a homeodomain and a paired domain, each of which has DNA binding. activity, and a proline-rich transcriptional activation domain. By analyzing the DNA and protein interactions of the Prd protein using several complementing approaches, this study should provide significant insights into the combinatorial mechanisms by which cell fates are specified during development. The specific aims are: (1) To investigate the requirements for the two DNA-binding domains of Prd. The specificities and relative positions of the paired domain and homeodomain within the Prd protein will be changed, and the resulting constructs will be tested using ectopic expression and rescue assays in the Drosophila embryo. (2) To identify and characterize protein interactors of Prd using the yeast two-hybrid system to screen a Drosophila embryonic cDNA library. Putative interactors will be characterized using in vitro and in vivo assays. (3) To identify nuclear localization signals of Prd using a deletion analysis in Drosophila Schneider cells. Deletion constructs will be tested in vitro for protein interactions with a panel of Prd interactors to identify putative components of the nuclear localization machinery. (4) To assess the importance of the proline-rich activation domain for combinatorial regulation. The functions of Prd chimeras in which the proline-rich domain has been replaced with other classes of activation domain will be tested in embryos. (5) To identify DNA targets of Prd using a reporter gene analysis in embryos. Altered versions of identified targets will be tested to determine the sequence requirements for Prd function. (6) To compare the functions of mouse Pax3 and Drosophila Prd by analyzing the functions of Pax3/Prd chimeras in Drosophila embryos. Evolutionarily- conserved molecular properties of Pax3 that might also be important for Pax3 function in mammalian cells will be analyzed.