TRANSCRIPTION OF GENES CONTROLLING DEVELOPMENT

果蝇的发育由一个等级网络控制 调控基因。正确的时空表达 这些基因是指定果实的基本身体计划所必需的 飞。我们的长期目标是提供一个分子描述 这些调控基因的表达是如何被控制的。因此， 到目前为止，我们的研究主要集中在蛋白质的生化分析上。 调控转录的最具特点的一种 发育控制基因，超双胸(UBX)。我们有 证明含有同源结构域的蛋白质甚至跳过 (Eve)在体外抑制Ubx转录。基因分析有 之前发现EVE是UBX和其他 同源结构域蛋白作为UBX的激活物或抑制物。 这项提案的主要部分旨在研究分子 EVE和其他同源结构域蛋白调节 Ubx和其他发育控制基因在小麦中的转录 胚胎。我们将过度生产和净化EVE和其他同源区域 细菌和昆虫细胞中的蛋白质。一个内源性的同源结构域- 与我们在果蝇胚胎中发现的DNA结合活性类似 核提取物也将被提纯和鉴定。这些 然后使用各种纯化的试剂进行比较和对比 相对DNA结合和体外转录活性 同源结构域蛋白。这应该揭示出基本的生物化学 这些关键调控分子的性质。我们还建议 绘制EVE蛋白的功能结构域 转录抑制和确定哪些因子 与之相互作用，以阐明转录的机制 真核生物中的抑制。最后，我们将采用共转染法 这些蛋白质对基因表达的调控 胚胎。我们的研究应该提供一个分子描述如何 同源结构域蛋白作为转录因子执行其 发育调节功能。 我们还鉴定了三种激活Ubx的蛋白质 体外转录(ZAST、GagA和ddcITF蛋白)。 这三种蛋白质结合在一系列紧密相连的 在Ubx RNA帽部位上游重叠的DNA元件。我们 建议研究这三者之间的蛋白质/DNA相互作用 因子和UBX启动子，并确定它们是否 当它们激活转录时，它们相互独立地起作用。 ZEST也是UBX的基因定义，这些数据结合在一起 通过我们使用ZEST蛋白的生化实验，已经导致了 提出它在调节Ubx基因表达调控中的作用 通过位于RNA帽位置10-60kb的调控元件。 我们将使用共转染法和P-元件转化试验 测试这一想法，并研究Gaga和ddcITF在 控制Ubx在胚胎中的表达。通过研究这三个 蛋白质，除了同源结构域蛋白质外，还有一个更完整的 应该推导出Ubx表达是如何调控的描述。

Drosophila development is controlled by a hierarchical network of regulatory genes. The correct spatial and temporal expression of these genes is required to specify the basic body plan of the fruit fly. Our long term goal is to provide a molecular description of how the expression of these regulatory genes is controlled. Thus far, our studies have focused on a biochemical analysis of proteins regulating the transcription of one of the best characterized developmental control genes, Ultrabithorax (Ubx). We have demonstrated that the homeodomain containing protein even-skipped (eve) represses Ubx transcription in vitro. Genetic analysis has previously identified eve as a repressor of Ubx and other homeodomain proteins as either activators or repressors of Ubx. The major portion of this proposal aims to study the molecular mechanisms by which eve and other homeodomain proteins regulate the transcription of Ubx and other developmental control genes in the embryo. We will overproduce and purify eve and other homeodomain proteins in bacteria and insect cells. An endogenous homeodomain- like DNA binding activity we have discovered in Drosophila embryo nuclear extracts will also be purified and characterized. These various purified reagents will then be used to compare and contrast the relative DNA binding and in vitro transcriptional activities of homeodomain proteins. This should reveal the basic biochemical properties of these key regulatory molecules. We also propose to map the functional domains of eve protein required for transcriptional repression and to identify which factors eve interacts with, to elucidate mechanisms of transcriptional repression in eucaryotes. Finally, we will employ cotransfection of these proteins to their regulation of gene expression in the embryo. Our studies should provide a molecular description of how homeodomain proteins act as transcription factors to execute their developmental regulatory function. We have also identified three proteins that activate Ubx transcription in vitro (the zeste, GAGA and ddcITF proteins). These three proteins bind to a series of closely spaced and overlapping DNA elements upstream of the Ubx RNA cap site. We propose to study the protein/DNA interactions between these three factors and the Ubx promoter and to determine whether or not they act independently of each other as they activate transcription. Zeste is also a genetically defined of Ubx, and this data combined with our biochemical experiments using zeste protein, has led ut to propose that it acts to mediate the control of Ubx expression by regulatory elements located 10 to 60 kb from the RNA cap site. We will use cotransfection and P-element transformation assays to test this idea and also to study the role of GAGA and ddcITF in controlling Ubx expression in the embryo. By studying these three proteins, in addition to homeodomain proteins, a more complete description of how Ubx expression is regulated should be derived.