TRANSCRIPTION OF GENES CONTROLLING DEVELOPMENT

决定受精卵如何发育成成体的关键机制 动物是基因表达的时空调控。这 过程可能在果蝇身上理解得最好，在那里，一个暂时的 发育控制基因的有序层级作用于控制模式 关于基因表达的。我们的长期目标是了解生物化学 调控这些发育控制基因转录的机制， 尤其是超双胸(UBX)基因。 许多发育控制基因，包括许多调节Ubx的基因， 共享一个同源DNA结合域，称为同源结构域。先前 已经不可能严格确定结合在一起的目标DNA 胚胎中的同源结构域蛋白。为了克服这一困难，我们有 开发了一种改进的体内紫外光交联法。这种方法揭示了 同源结构域蛋白甚至跳过(EVE)在整个 它的目标基因的长度只有它的十倍低 最初预计不会有约束力。我们现在建议，使用这种方法来 探索EVE和其他同源结构域结合的靶基因范围 体内的蛋白质，并评估不同机制的重要性， 包括合作捆绑，实现这么大范围的占用 所有的DNA站点。因为我们的实验表明EVE蛋白结合到 启动子区域以前没有被认为是被调控的，我们 将利用基因和分子实验来检测 与这些区域结合的同源结构域蛋白。生化分析和 电子显微镜也将被用来关联体外性质 Eve和其他同源结构域蛋白与广泛的DNA结合 活着。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Eve是如何 蛋白质在体外抑制转录。EVE蛋白含有一种 抑制域，我们现在建议确定这是如何与 一般转录因子抑制其功能。这些研究 应该提供关于同源结构域蛋白如何 执行它们的生物功能。 UBX转录的最终模式也需要协调动作 许多其他不包含同源结构域的调节器。我们是 研究其中三个因子，zust，Gaga和NTF-1是如何调节的 UBX转录的特定方面。Zust和NTF-1似乎是 冗余的Ubx调节器，我们建议使用基因实验来 检查这种冗余的性质。有人建议，泽斯特 介导远距离增强元件的作用，并在体内进行紫外线交叉 链接将用于确定这是否通过以下位置的协作绑定发生 一段距离。最后，Gaga可能在调解镇压 将对多梳UBX进行检查。

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.