BIOCHEMICAL ANALYSIS OF PROMOTERS REGULATING DEVELOPMENT

一个复杂的多细胞生物体的发展需要精确的 调控基因表达的时空模式。 这是 部分通过序列特异性转录因子完成， 与启动子中的位点结合并调节起始速率。 因此 对发展的透彻理解需要详细了解 调控转录起始的分子机制。 果蝇 黑腹果蝇有两个特点，使其特别适用于 转录调控的生化分析。 首先，果蝇 胚胎提供了大量的材料来源， 忠实的无细胞转录提取物。 第二，基因 果蝇胚胎发生的研究提供了丰富的 有关监管网络的信息，可用于指导 生化分析 本项目的长期目标是了解 调节基因的表达，这些基因控制沿着 果蝇胚胎的背/腹轴。 这些基因对人类的生存起着至关重要的 在胚层建立中的作用，这是一个关键的过程， 所有复杂的多细胞生物的发展。 特别是 建议的实验将集中在两个合子的启动子上， 活性基因decapentaplegic和twist，它们在 外胚层和中胚层的分化。 这些 启动子，其在沿着背侧/腹侧的离散区域中有活性 胚胎的轴，在某种程度上受到调节，以响应位置线索， 发育中的有机体。 为了促进这些研究，体外检测方法 转录将被优化，使得无细胞转录系统 忠实地再现了在体内观察到的调节过程。 的in 体外转录系统将用于鉴定顺式作用 元件在decapentaplegic和扭曲的启动子和 与之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， 使用发育阶段的野生型和突变体的提取物测定 胚胎 接下来，将研究编码调控发育因子的基因。 分离并用作探针以确定 这些因素。 最后，将对这些因素进行生物化学表征 来阐明发育过程中 调节转录激活和抑制。 通过向一个 了解发展的生化基础，这些研究可能 最终阐明遗传和环境因素在 发育障碍的病因学。 此外，许多基因 调节果蝇发育的基因与脊椎动物的癌基因同源 因此这些研究应该有助于阐明 在肿瘤形成背后。

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.