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

Ohio State University Research Foundation -Do Not Use, Columbus OH

Investigators

Abstract

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 researchers Broader Impacts CREMA 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.