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

真核系统转录调控的共同特征是基因调控区中顺式作用的DNA元件和相应的反式作用蛋白转录(TF)的模块化和组合式结构。目前正在投入大量的努力，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的Cre对体内基因整体表达的贡献，因为无法在植物基因组中进行基因替换或定点突变。这是这个项目打算填补的基本知识空白。PI将CREMA(顺式调控元件突变分析)设想为高通量鉴定拟南芥Cres点突变的一种新策略。在这个SGER项目中，CREMA将用一小部分特征良好的拟南芥启动子进行测试。为了在EMS诱变的大种群中识别启动子，CREMA利用突变修复检测(MRD)方法和定制的重新测序阵列来丰富突变。如果成功，这种组合有望将其他点突变检测系统的吞吐量增加两个数量级或更多，例如TILING。CREMA的长期目标是提供一个明确的突变资源，位于特定的启动子区域，供研究人员使用。Broader Impacts CREMA整合了遗传学、分子生物学和生物信息学，如果成功，将能够识别大量启动子和其他调控区域的点突变。这些突变体的获得将有助于建立Cres的体内功能。从这些分析中获得的突变将通过公共AGIS(http://arabidopsis.med.ohio-state.edu/)数据库)提供给社区。该项目将有助于国际和平研究所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，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.