TRANSCRIPTION OF GENES CONTROLLING DEVELOPMENT

决定受精卵如何发育成成虫的关键机制 动物是基因表达的时空调控。这 这个过程可能在果蝇中得到最好的理解， 发育控制基因的有序层次控制模式 的基因表达。我们的长期目标是了解 调节这些发育控制基因转录的机制， 特别是超双胸（Ubx）基因。 许多发育控制基因，包括许多调节Ubx的基因， 共有一个同源DNA结合域，称为同源域。先前 不可能严格地确定与DNA结合的靶DNA， 胚胎中的同源域蛋白质。为了克服这一困难，我们 开发了一种改进的体内UV交联方法。该方法揭示了 同源结构域蛋白甚至跳过（eve）结合整个 它的目标基因的长度，并在只有十倍低的水平，基因， 最初并没有被预期会结合。我们现在建议，使用这种方法， 探测Eve和其他同源结构域结合的靶基因的范围 蛋白质在体内，并评估不同机制的重要性， 包括合作绑定，以实现如此广泛的范围内的占用 的DNA位点。因为我们的实验表明eve蛋白质与 启动子区域以前没有被认为是调节，我们 将使用遗传和分子实验来研究 同源结构域蛋白结合到这些区域。生化分析和 电子显微镜也将被用来与体外性质的 eve和其他同源结构域蛋白与在细胞中观察到的广泛DNA结合有关。 vivo.为了更好地了解详细的生化机制， 同源结构域蛋白控制转录，我们正在研究 蛋白质在体外抑制转录。Eve蛋白质含有 抑制结构域，我们现在建议确定它如何与 一般转录因子抑制其功能。这些研究 应该提供一个清晰的分子描述，同源结构域蛋白质 执行其生物功能。 Ubx转录的最终模式也需要协调作用 许多其他不含同源结构域的调节子。我们 研究这些因子中的三个，zeste，GAGA和NTF-1， Ubx转录的特定方面。zeste和NTF-1似乎 Ubx的冗余调节器，我们建议使用遗传实验， 检查这种冗余的性质。有人认为，zeste 介导远端增强子元件的作用，并且体内UV交叉 链接将被用于确定这是否通过在 远处最后，GAGA可能在介导抑制 将检查polycomb的Ubx。

A key mechanism determining how a fertilized egg develops into an adult animal is the spatial and temporal regulation of gene expression. This process is probably best understood in Drosophila, where a temporally ordered hierarchy of developmental control genes act to control patterns of gene expression. Our long term goal is understand the biochemical mechanisms regulating transcription of these developmental control genes, particularly the Ultrabithorax (Ubx) gene. Many of the developmental control genes, including many that regulate Ubx, share a homologous DNA binding domain, termed the homeodomain. Previously it has been impossible to rigorously determine the target DNAs bound by homeodomain proteins in embryos. To overcome this difficulty, we have developed an improved in vivo UV cross linking method. This method reveals that the homeodomain protein even skipped (eve) binds throughout the length of its target genes and at only ten fold lower levels to genes it was not initially expected to bind. We now propose, to use this method to probe the range of target genes bound by eve and other homeodomain proteins in vivo and to assess the importance of different mechanisms, including cooperative binding, to achieve occupancy of such a broad range of DNA sites. Since our experiments indicate that eve protein binds to promoter regions it had not previously been considered to regulate, we will use genetic and molecular experiments to examine the function of homeodomain proteins bound to these regions. Biochemical assays and electron microscopy will also be used to relate the in vitro properties of eve and other homeodomain proteins to the broad DNA binding observed in vivo. To better understand detailed biochemical mechanisms by which homeodomain proteins control transcription, we are studying how eve protein represses transcription in vitro. eve protein contains a repression domain, and we now propose to determine how this interacts with the general transcription factors to inhibit their function. These studies should provide a clear molecular description of how homeodomain proteins execute their biological function. The final pattern of Ubx transcription also requires the coordinate action of many other regulators which do not contain a homeodomain. We are studying how three of these factors, zeste, GAGA and NTF-1, regulate specific aspects of Ubx transcription. zeste and NTF-1 appear to be redundant regulators of Ubx, and we propose to use genetic experiments to examine the nature of this redundancy. It has been suggested that zeste mediates the action of distant enhancer elements, and in vivo UV cross linking will be used to determine if this occurs by cooperative binding at a distance. Finally, a possible role for GAGA in mediating repression of Ubx by polycomb will be examined.