Very Different Outcomes of Inaccurate DS Break Repair in Four Grass Species

Our lab has been using chromosome breakage at I-SceI sites in transgenic plants as an assay for the outcomes of inaccurate double strand (ds) break repair.   We engineer the 18 bp recognition and cutting site for I-SceI into one plant, then make a second transgenic plant that expresses the I-SceI enzyme driven by an constitutive promoter.  Crossing these two plants yields a chromosome cutting and repair cycle, where both will continue until the recognition site is destroyed by an inaccurate repair event.   This inaccurate repair event is recovered by PCR across the restriction site, and is subsequently characterized by DNA sequence analysis.  Deletions, insertions and mixed events are observed, with a number of distinctive properties like (1) the same boundaries of deletions often being found, (2) insertions often containing DNA from near the I-SceI site, and (3) preferential directions for both insertion source and deletion.  These experiments were performed in comparable manners in maize, pearl millet, rice and sorghum genomes.  The results are quite different depending on the genome studied.  In addition, we are expanding our work to look at the effects of mutations in various DNA repair and chromatin remodeling genes, and studying the effects of various environmental stresses.