BIOCHEMICAL ANALYSIS OF PROMOTERS REGULATING DEVELOPMENT

复杂的多细胞生物体的发展恰恰需要 调控基因表达的时间和空间模式。这是 部分是由序列特异性转录因子完成的，这些转录因子 与启动子中的位点结合并调节起始率。因此，一个 透彻地了解发展需要详细了解 调控转录启动的分子机制。果蝇 黑素胃膏有两个特点，使其对 转录调控的生化分析。首先，果蝇 胚胎提供了丰富的材料来源，为高度活跃的 忠实的细胞自由转录精华。第二，基因 对果蝇胚胎发生的研究为果蝇胚胎发育提供了丰富的 有关可用于指导 生化分析。 这个项目的长期目标是了解这些机制 调控调控细胞分化的基因表达 果蝇胚胎的背轴/腹轴。这些基因起着至关重要的作用 在胚层建立中的作用，这是一个关键过程 所有复杂的多细胞生物体的发育。尤其是， 拟议的实验将集中在这两个合子的启动子上。 活性基因截瘫和扭曲，它们在早期发挥关键作用 外胚层和中胚层的分化。这些 启动子，它们活跃在背侧/腹侧的离散区域 胚胎的轴，以某种方式调节对位置线索的反应 发育中的有机体。为了促进这些研究，体外培养的方法 转录将得到优化，从而使无细胞转录系统 忠实地再现了在体内观察到的调控过程。入内 然后使用体外转录系统来鉴定顺式作用。 十指肠麻痹和扭转的启动子中的元素和 与它们相互作用的转录因子。这些因素的作用 在产生基因表达的空间和时间模式时 利用发育阶段野生型和突变型的提取物进行测定 胚胎。接下来，编码调节发育的因素的基因将是 分离并用作探针以确定其表达模式 这些因素。最后，我们将对这些因素进行生化表征。 阐明发育相关的分子机制 受调控的转录激活和抑制。通过为 了解发育的生化基础，这些研究可能 最终弄清遗传和环境因素在 发育障碍的病因学。此外，许多基因 调控果蝇发育与脊椎动物癌基因同源 因此，这些研究应该有助于阐明分子机制。 在肿瘤发生的背后。

The development of a complex multicellular organism requires precisely regulated temporal and spatial patterns of gene expression. This is accomplished, in part, by sequence specific transcription factors which bind to sites in promoters and modulate the rate of initiation. Thus, a thorough understanding of development requires a detailed knowledge of the molecular mechanisms regulating transcriptional initiation. Drosophila melanogaster has two characteristics that make it particularly useful for a biochemical analysis of transcriptional regulation. First, Drosophila embryos provide a plentiful source of material for highly active and faithful cell free transcription extracts. Second, the genetic investigation of Drosophila embryogenesis has provided a wealth of information about regulatory networks which can be used to guide a biochemical analysis. The long term goal of this project is to learn about the mechanisms regulating the expression of genes that govern differentiation along the dorsal/ventral axis of the Drosophila embryo. These gene play an essential role in germ layer establishment, which is a critical process in the development of all complex multicellular organisms. In particular, the proposed experiments will focus on the promoters of the two zygotically active genes decapentaplegic and twist, which play critical early roles in the differentiation of ectoderm and mesoderm, respectively. These promoters, which are active in discrete regions along the dorsal/ventral axis of the embryo, are somehow regulated in response to positional cues in the developing organism. To facilitate these studies, methods for in vitro transcription will be optimized so that the cell-free transcription system faithfully reproduces the regulatory processes observed in vivo. The in vitro transcription system will then be used to identify cis-acting elements in the promoters of decapentaplegic and twist and the transcription factors which interact with them. The roles of these factors in generating spatial and temporal patterns of gene expression will then be determined using extracts of developmentally staged wild-type and mutant embryos. Next, the genes encoding factors regulating development will be isolated and used as probes to ascertain the patterns of expression of these factors. Finally, these factors will be biochemically characterized to elucidate the molecular mechanisms responsible for developmentally regulated transcriptional activation and repression. By contributing to an understanding of the biochemical basis of development, these studies may ultimately clarify the role of genetic and environmental factors in the etiology of developmental disorders. In addition, many of the genes regulating Drosophila development are homologous to vertebrate oncogenes and thus these studies should help to illuminate the molecular mechanisms behind oncogenesis.