TRANSCRIPTION OF GENES CONTROLLING DEVELOPMENT

果蝇的发育是由一个等级网络控制的， 调节基因 正确的空间和时间表达 这些基因是指定水果的基本结构所必需的 飞翔紧紧 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， 调节一种最具特征的 发育控制基因，超双胸（Ubx）。 我们有 证明了含有蛋白质的同源结构域甚至跳过了 (eve)在体外抑制Ubx转录。 基因分析 以前认为eve是Ubx和其他 同源结构域蛋白作为Ubx的激活剂或抑制剂。 该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的转录 胚胎 我们将过度生产和净化夏娃和其他同源域 细菌和昆虫细胞中的蛋白质。 内源性同源结构域- 就像我们在果蝇胚胎中发现的DNA结合活性 还将纯化和表征核提取物。 这些 各种纯化的试剂将被用来比较和对比 相对DNA结合和体外转录活性 同源结构域蛋白质。 这应该能揭示 这些关键调节分子的特性。 我们亦建议 绘制Eve蛋白的功能结构域， 转录抑制，并确定哪些因素， 相互作用，以阐明转录机制， 真核生物中的阻遏。 最后，我们将采用共转染 这些蛋白质对基因表达的调节， 胚胎 我们的研究应该提供一个分子描述， 同源结构域蛋白质作为转录因子执行其功能， 发育调节功能。 我们还发现了三种激活Ubx的蛋白质 体外转录（zeste、GAGA和ddcITF蛋白）。 这三种蛋白质结合到一系列紧密排列的， Ubx RNA帽位点上游的重叠DNA元件。 我们 我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 在它们激活转录时彼此独立地起作用。 Zeste也是Ubx的基因定义，这些数据结合 通过我们使用zeste蛋白的生化实验， 提出它的作用是介导Ubx表达的控制， 通过位于距RNA帽位点10至60 kb的调节元件。 我们将使用共转染和P-元件转化试验， 测试这一想法，并研究GAGA和ddcITF的作用， 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，一个更完整的 应该推导出Ubx表达如何调节的描述。

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.