Genomic Regulation Dlx Bi-gene Cluster Simple Chordate

描述(由申请人提供)： 模式的肠索DII-A/DII-B双基因簇是已知的最紧凑和组织简单的发育基因簇的例子之一。虽然已经分析了基因簇内的一些调控相互作用，但肠杆菌中简单的DII-A/DII-B簇为获得整个发育基因簇的协调调控体系提供了一个机会。这个项目的第一个特定目标是构建一个双报告转基因来同时检测两个聚集性基因启动子中每一个的调节输出。然后，这个转基因将形成缺失实验的基础，以确定作用于其中一个或两个启动子的主要基因组调控元件。随着这项工作的进行，还将对单个报告基因进行测试，以评估非编码DMA内部部分(如基因间隔区)可能的调控活性。第二个目标将支持第一个目标，即使用整体安装原位杂交来确定内源转录表达模式。第三个具体目标是通过在整体结构中替换或移动选定的调控元件来检查它们的特异性和位置重要性。 一组实验将用另一种基本启动子的副本取代其中一种基本启动子 测试每个基本启动子对基因组环境的反应是否不同。另一组实验将在集群内移动监管模块，以测试位置对监管输出的重要性。肠杆菌被选为模式生物是因为它们拥有广泛的基因组和发育资源，而且非常适合在小规模研究环境中使用转基因报告器快速评估基因调控。长期目标是为基因组调控的类型和模式数据库做出贡献，这将有助于理解人类发育障碍的分子遗传学基础。

DESCRIPTION (provided by applicant): The DII-A/DII-B bi-gene cluster of the model chordate dona intestinalis is one of the most compact and organizationally simple examples of a developmental gene cluster known. While some of the regulatory interactions within gene clusters have been analyzed, the simple DII-A/DII-B cluster in C. intestinalis offers an opportunity to obtain a more complete picture of the coordinated regulatory architecture of a whole developmental gene cluster. The first specific aim of this project is to construct a dual reporter transgene to assay the regulatory output of each of the two clustered gene promoters simultaneously. This transgene will then form the basis for deletion experiments to identify the major genomic regulatory elements acting on either or both promoters. Along with this work will be tests of individual reporter transgenes that can assess the possible regulatory activity of internal portions of the non-coding DMA, such as the intergenic region. A second aim, which will support the first, is the determination of the endogenous transcript expression pattern using whole mount in-situ hybridization. The third specific aim is to examine the specificity and positional importance of selected regulatory elements by replacing or moving them within the overall construct. One set of experiments will replace one of the basal promoters with a copy of the other to test whether each basal promoter responds differently to the genomic environment. Another set of experiments will move regulatory modules within the cluster to test the importance of position on the regulatory output. C. intestinalis was chosen as the model organism because of extensive genomic and developmental resources, and because they are ideally suited to the rapid assessment of gene regulation using transgenic reporters in a small-scale research setting. The long-term objective is to contribute to the database of types and modes of genomic regulation, which will help in understanding the molecular genetic basis of human developmental disorders.