SGER-Development of CREMA: A High Throughput Method for the Identification of Promoter Mutations

真核生物系统中转录调控的共同特征是基因调控区中顺式作用DNA元件和相应的反式作用蛋白质转录因子（transcription，TF）的模块化和组合结构。 正在作出重大努力，建立工作队与相应的克雷斯的互动关系。然而，一旦被鉴定，由于不能在植物基因组中进行基因置换或定点突变，目前还没有可用的方法来研究特定CRE对体内基因总体表达的贡献。这是本项目打算填补的基本知识空白。PI已将克雷马（Cis Regulatory Element Mutational Analysis）作为一种新的策略，用于高通量鉴定拟南芥克雷斯中的点突变。在这个SGER项目中，克雷马将用一小部分特征明确的拟南芥启动子进行测试。为了鉴定大的EMS诱变群体中的启动子，克雷马利用突变修复检测（MRD）方法和定制的重测序阵列富集突变的组合。如果成功的话，这种组合预计将增加其他点突变检测系统（例如TILLING）的通量的两个数量级或更多。克雷马的长期目标是提供一个确定的突变资源，定位到特定的启动子区域，以提供给研究人员。更广泛的影响克雷马整合遗传学，分子生物学和生物信息学的战略，如果成功的话，将允许在启动子和其他调控区域的大量点突变识别。这些突变体的可用性将有助于建立克雷斯的体内功能。将通过公共AGRIS（http：//arabidopsis.med.ohio-state.edu/）数据库向社区提供从这些分析中获得的突变体。该项目将有助于PI正在开展的将研究与教育联系起来的活动。 活动包括本科生参与研究，并参加夏季研究讲习班。

Common characteristics of transcriptional regulation in eukaryotic systems are the modular and combinatorial structure of cis-acting DNA elements in the regulatory regions of genes and the corresponding trans-acting proteins, the transcription (TFs). Significant efforts are being invested in establishing the interactions of TFs with the corresponding CREs. Once identified, however, there is no currently available method to investigate the contribution of a particular CRE to the overall expression of a gene in vivo, because of the inability to carry out gene replacements or site directed mutations in plant genomes. This is the fundamental knowledge gap that this project intends to fill. The PI has conceived CREMA (Cis Regulatory Element Mutational Analysis) as a novel strategy for the high-throughput identification of point mutations in Arabidopsis CREs. In this SGER project, CREMA will be tested with a small subset of well characterized Arabidopsis promoters. To identify promoters in large EMS-mutagenized populations, CREMA utilizes a combination of enrichment of mutations using the Mutation Repair Detection (MRD) method and custom-made re-sequencing arrays. If successful, this combination is expected to increase two-orders of magnitude or more the throughput of other point mutation detection systems, such as for example TILLING. The long-term goal of CREMA is to provide a resource of defined mutations, located to specific promoter regions, to be made available to researchersBroader ImpactsCREMA integrates genetics, molecular biology and bioinformatics in a strategy that, if successful, will permit identifying in large numbers point mutations in promoters and other regulatory regions. The availability of these mutants would facilitate establishing the in vivo function of CREs. Mutants derived from these analyses will be made available to the community through the public AGRIS (http://arabidopsis.med.ohio-state.edu/) database. The project will contribute to ongoing activities of the PI linking research and education. Activities include the involvement of undergraduate students in research, and participation of summer research workshops.