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

真核系统中转录调控的共同特征是基因调控区域中顺式作用DNA元件的模块化和组合结构以及相应的反式作用蛋白，即转录（tf）。目前正在作出重大努力，以建立信托基金与相应的信托基金之间的相互作用。然而，一旦确定，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，因为无法在植物基因组中进行基因替换或位点定向突变。这就是这个项目想要填补的基本知识空白。PI认为CREMA （Cis Regulatory Element Mutational Analysis，顺式调控元件突变分析）是一种高通量鉴定拟南芥CREs点突变的新策略。在这个SGER项目中，CREMA将与一小部分已充分表征的拟南芥启动子进行测试。为了鉴定大型ems突变群体中的启动子，CREMA利用突变修复检测（MRD）方法和定制的重测序阵列来富集突变。如果成功，这种组合有望将其他点突变检测系统（例如TILLING）的吞吐量提高两个数量级或更多。CREMA的长期目标是提供一种定位于特定启动子区域的定义突变资源，供研究人员使用。更广泛的影响CREMA将遗传学、分子生物学和生物信息学整合在一个策略中，如果成功，将允许在启动子和其他调控区域中大量识别点突变。这些突变体的可用性将有助于建立cre的体内功能。从这些分析中获得的突变体将通过公共AGRIS （http://arabidopsis.med.ohio-state.edu/）数据库提供给社区。该项目将有助于PI正在进行的将研究和教育联系起来的活动。活动包括让本科生参与研究，以及参加暑期研究研讨会。

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.