TRANSCRIPTION OF GENES CONTROLLING DEVELOPMENT

控制发育的基因转录

• 批准号: 3300886
• 负责人: Mark D BIGGIN
• 金额: $ 28.12万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3300886
• 关键词: DNA binding protein; DNA footprinting; Drosophilidae; bacteria; chromatography; developmental genetics; gene expression; genetic mapping; genetic promoter element; genetic transcription; nonmammalian vertebrate embryology; protein structure; regulatory gene; transcription factor

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.

果蝇开发受到一个层次网络的控制 调节基因。 正确的空间和时间表达 这些基因需要指定水果的基本身体计划 飞。 我们的长期目标是提供分子描述 如何控制这些调节基因的表达。 因此 到目前为止，我们的研究集中于蛋白质的生化分析 调节最佳特征之一的转录 发育控制基因，超脑术（UBX）。 我们有 证明含有蛋白质的同源域均匀 （EVE）在体外抑制UBX转录。 遗传分析具有 先前确定的前夕是UBX和其他 同源域蛋白作为UBX的激活剂或阻遏物。 该提案的主要部分旨在研究分子 夏娃和其他同源域蛋白调节的机制 UBX和其他发育控制基因的转录 胚胎。 我们将过量生产和净化前夕和其他同源域 细菌和昆虫细胞中的蛋白质。 内源性同源域 - 像DNA结合活性一样，我们在果蝇胚胎中发现 核提取物也将被纯化和表征。 这些 然后将使用各种纯化的试剂比较和对比 相对DNA结合和体外转录活性 同源域蛋白。 这应该揭示基本的生化 这些关键调节分子的特性。 我们还建议 绘制夏娃蛋白的功能域 转录抑制并确定哪些因素 与之相互作用，以阐明转录的机制 桉树中的抑制作用。 最后，我们将使用共转染 这些蛋白质以调节基因表达 胚胎。 我们的研究应提供分子描述 同源域蛋白充当转录因素，以执行其 发展调节功能。 我们还确定了激活UBX的三种蛋白质 体外转录（Zeste，Gaga和DDCITF蛋白）。 这三种蛋白质与一系列紧密间隔的结合，并且 UBX RNA CAP位点上游的重叠DNA元件。 我们 建议研究这三个之间的蛋白质/DNA相互作用 因素和UBX启动子，并确定他们是否 在激活转录时彼此独立起作用。 Zeste也是一个遗传定义的UBX，此数据结合了 使用我们使用Zeste蛋白的生化实验，已LED UT 提出它可以介导UBX表达的控制 通过RNA CAP位点10到60 KB的调节元素。 我们将使用共转染和P元素转换测定法 测试这个想法，并研究GAGA和DDCITF在 控制胚胎中的UBX表达。 通过研究这三个 蛋白质，除了同源域蛋白外，更完整 应得出如何调节UBX表达的描述。