DISSECTION OF DROSOPHILA PAIRED PROTEIN FUNCTION

转录因子是模式形成调控的中心 在发育中的果蝇片段基因，其中许多编码 转录因子，参与调控级联反应，最终 指定胚胎中细胞的发育命运。结对的(珠江三角洲) 片段基因，这一提议的主题，在组合中发挥作用 与其他配对规则基因一起调节片段的转录- 位于级联下游的极性基因。PRD基因是 特别感兴趣是因为像它的哺乳动物同源物，Pax3(相关的 人类瓦登堡综合征)，它编码几个保守的基序， 包括一个同源结构域和一个配对结构域，每个结构域都有DNA 有约束力的。活性，以及一个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 几种互补的方法，这项研究应该提供有意义的 对细胞命运的组合机制的洞察 在开发过程中指定。具体目标是： (1)研究对两个DNA结合域的要求 珠江三角洲。配对结构域和配对结构域的特性和相对位置 PRD蛋白中的同源结构域将发生变化，从而导致 将使用异位表达和救援分析对构建进行测试 果蝇胚胎。 (2)鉴定和鉴定PRD的蛋白质相互作用因子 酵母双杂交系统筛选果蝇胚胎cdna文库。 可能的相互作用因素将使用体外和体内的方法进行表征 化验。 (3)使用缺失来识别PRD的核定位信号 果蝇施耐德细胞的分析。将测试删除构造 体外研究蛋白质与一组PRD相互作用因子的相互作用 确定核定位机器的假定部件。 (4)评估富含脯氨酸的激活结构域对 组合调控。珠江三角洲嵌合体的功能 富含脯氨酸的结构域已被其他类别的激活所取代 DOMAIN将在胚胎中进行测试。 (5)利用报告基因分析鉴定PRD的DNA靶点 胚胎。已识别目标的更改版本将进行测试，以 确定PRD功能的顺序要求。 (6)通过分析比较小鼠Pax3和果蝇PRD的功能 Pax3/PrD嵌合体在果蝇胚胎中的功能从进化的角度来看- Pax3保守的分子性质，这可能对 Pax3在哺乳动物细胞中的功能将被分析。

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.