BIOCHEMICAL ANALYSIS OF PROMOTERS REGULATING DEVELOPMENT

描述：这项建议解决了生物学中最基本的 问题：一群未分化的细胞是如何转化为 有序的分化组织阵列？重点放在最初的 这种变换的一个阶段，在这个阶段中，一场单元格被分成 几个离散的域。为了检查这一过程，生化和 遗传学方法将结合起来研究分子间的相互作用 控制果蝇胚胎发生的物质。 背部形态原是一种启动子选择性转录因子， 确定细胞命运作为背侧/腹侧位置的函数 果蝇胚胎的轴。在发育早期，这种蛋白质来自 以活动梯度分布，在 胚胎的腹侧和背侧的活动较低。这 背侧的渐变分布导致空间调节 胚层建立所必需的基因的表达。 例如，由于背部是twist(Twi)基因的激活因子，twi 仅在位于腹侧的推定中胚层转录 背部活动度高。相反，背侧是一种抑制 十足瘫痪(DPP)和零点(Zenkllt)基因，因此只有 在位于背部的推定外胚层中转录 活跃度很低。这项研究的目标是确定背部 与其他核因子相互作用，激活一些启动子和 压抑他人。这些因子包括某些基本螺旋-环-螺旋 (BHLH)转录因子，它与背部相互作用，扩大和 加强背部介导的激活结构域以及辅助抑制物 蛋白质，这有助于背部介导的转录抑制。 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)分析企业与企业之间的协同作用 背部转录因子和bHLH转录因子。体外转录和 我们将使用瞬时转基因技术来检测分子。 允许背部与bHLH因子协同作用的相互作用 由双胞胎、无女和Achaete Scute复合体编码。在……里面 此外，负责协同作用的蛋白质结构域将被绘制出来 并进行分析以确定它们是否在 各种因素。利用从这一分析中获得的信息，显性负面 这些因子的等位基因将被开发并引入到胚胎中 探讨背侧/BHLH因子协同作用在发育中的作用。2) 描述背部和共抑制物之间的相互作用 阐明腹侧抑制的机制。首先是生殖系 转化实验将被用来定位背部的结构域 是镇压所必需的，因此可能会直接与 共抑制蛋白。第二，通过使用基因马赛克， 将对NTF-1进行分析，以确定这是否是一种假定的辅助抑制物 因子对于正常的背侧/腹侧模式的形成是必不可少的。第三, 其他假定的共抑制蛋白将被鉴定。 第四，DIRECT在背侧/腹侧模式形成中的作用 背部和共抑制物之间的相互作用将被评估。

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.