BIOCHEMICAL ANALYSIS OF PROMOTERS REGULATING DEVELOPMENT

描述：该提案解决了生物学最基本的问题之一 问题：一组未分化的细胞是如何转化为 有序排列的分化组织 重点放在最初的 细胞分裂：这种转变的一个阶段，在这个阶段中，一个区域的细胞被分成 几个离散的领域。 为了研究这一过程，生物化学和 将结合遗传学方法来研究分子相互作用 控制果蝇胚胎发育的基因 背侧形态原是启动子选择性转录因子， 决定细胞命运的功能位置沿着背/腹 果蝇胚胎的中轴。 在发育早期，这种蛋白质 分布在一个活动梯度，与高活性的 胚胎的腹侧和背侧的低活性。 这 Dorsal的分级分布导致空间调节 表达对胚层建立至关重要的基因。 例如，由于Dorsal是twi基因的激活剂，twi 只在位于腹侧的假定中胚层中转录， 背部活动度很高。 与此相反，Dorsal是一个阻遏物， decapentaplegic（DPP）和zerknllt（zen）基因，因此它们仅 转录在背侧的假定外胚层， 活动很低。 这项研究的目的是确定如何背 与其他核因子相互作用以激活一些启动子， 压制别人。 这些因素包括某些基本的螺旋-环-螺旋 （BHLH）转录因子，与Dorsal相互作用， 增强背侧介导激活结构域以及辅阻遏物 蛋白质，其有助于Dorsal介导的转录抑制。 通过对动物发育的理解，这些研究 应该有助于阐明人类发育障碍的基础。 具体目标是：1）分析 Dorsal和bHLH转录因子。 体外转录和 将采用瞬时转染测定来检测分子水平。 使Dorsal与bHLH因子协同作用的相互作用 由双胞胎、无子女和阿刻忒盾形复合体编码。 在 此外，负责协同作用的蛋白质结构域将被绘制 并进行分析，以确定它们是否介导了 因素 利用从该分析中获得的信息，显性阴性 这些因子的等位基因将被发育并引入胚胎 以确定Dorsal/BHLH因子协同作用的发育作用。 （二） 描述Dorsal和辅阻遏物之间的相互作用， 阐明腹侧抑制的机制。 首先是生殖细胞 将采用转化测定来定位Dorsal中的结构域， 是抑制所必需的，因此可能直接接触 辅阻遏蛋白。 第二，通过使用基因镶嵌， NTF-1是一种推定的共阻遏物，将被分析以确定这是否 因子对于正常的背侧/腹侧图案形成是必不可少的。 第三、 将表征其它推定的共阻遏蛋白。 第四，在背/腹图案形成中的作用， 将评估Dorsal和辅阻遏物之间的相互作用。

DESCRIPTION: This proposal addresses one of biology's most basic questions: how is a group of undifferentiated cells transformed into an ordered array of differentiated tissues? The focus is on the initial phase of this transformation in which a field of cells is divided into a few discrete domains. To examine this process, biochemical and genetic approaches will be combined to study the molecular interactions that control Drosophila embryogenesis. The dorsal morphogen is a promoter selective transcription factor that determines cell fate as a function of position along the dorsal/ventral axis of the Drosophila embryo. Early in development, this protein comes to be distributed in an activity gradient, with high activity on the ventral side and low activity on the dorsal side of the embryo. This graded distribution of Dorsal results in the spatially regulated expression of genes that are essential for germ layer establishment. For example, since Dorsal is an activator of the twist (twi) gene, twi is only transcribed in the ventrally- situated presumptive mesoderm where Dorsal activity is high. In contrast, Dorsal is a repressor of the decapentaplegic (dpp) and zerknllt (zen) genes, which are therefore only transcribed in the dorsally-situated presumptive ectoderm where Dorsal activity is low. The goal of this research is to determine how Dorsal interacts with other nuclear factors to activate some promoters and repress others. These factors include certain basic helix-loop- helix (BHLH) transcription factors, which interact with Dorsal to broaden and intensify domains of Dorsal-mediated activation, as well as co-repressor proteins, which assist in Dorsal-mediated transcriptional repression. By contributing to an understanding of animal development, these studies should help to elucidate the basis of human developmental disorders. The specific aims are to 1) Analyze the synergistic interactions between Dorsal and bHLH transcription factors. In vitro transcription and transient transfection assays will be employed to examine the molecular interactions that allow Dorsal to synergize with the bHLH factors encoded by twi, daughterless, and the Achaete Scute complex. In addition, the protein domains responsible for the synergy will be mapped and analyzed to determine if they mediate direct contacts between the factors. Using information gained from this analysis, dominant negative alleles of the factors will be developed and introduced into the embryo to determine the developmental role of Dorsal/BHLH factor synergy. 2) Characterize the interactions between Dorsal and co- repressors to elucidate the mechanisms of ventral repression. First the germline transformation assay will be employed to map the domains in Dorsal that are required for repression and which may, therefore, directly contact co-repressor proteins. Second, through the use of genetic mosaics, NTF-1, a putative co-repressor, will be analyzed to determine if this factor is essential for normal dorsal/ventral pattern formation. Third, additional putative co-repressor proteins will be characterized. Fourth, the role in dorsal/ventral pattern formation of direct interactions between Dorsal and co- repressors will be assessed.