Controlled Deletional Mutagenesis and Gene Homing in Arabidopsis

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Deletional mutagenesis has not been used as extensively as insertional mutagenesis in Arabidopsis because large deletions are often not transmitted through gametes or are lethal early in development. The investigator will carry out a systematic study of small deletions by controlling their chromosomal location, developmental timing, and parent of origin. She will make deletions of known sizes using the site-specific bacteriophage P1-encoded Cre recombinase to delete chromosomal segments between a loxP-containing T-DNA donor site and a nearby loxP-containing transposon. Using an already tested transposon "launching pad" containing loxP recognition sites for the Cre recombinase, she will identify 3-4 transposons reinserted at different distances ranging from a few kilobases to a few megabases from each of several transposon donor sites to carry out these studies. She will use Cre recombinase genes expressed from a constitutive plant promoter, as well as from a chemically inducible promoter. She will detect deletions (and inversions) by both PCR-based and genetic methods. She will determine the genetic transmissibility of deletions and analyze the development of plants in which deletions are induced at different times after germination. This work will both increase the understanding of the detrimental effects of deletions and deletional heterozygosity and provide methods to control the size, chromosomal location, and timing of deletions.The second objective is to lay the groundwork for development of a transposon-based method to target genes to their original chromosomal locations. This technique, which the investigator calls gene "homing" to distinguish it from homology-based gene targeting, will use site-specific recombination to target a promoter-reporter cassette to a transposon-disrupted gene. If successful, this work will make it possible for the first time to study precise alterations in a plant gene's regulatory sequences in the gene's original chromatin context. The gene homing method will use transposons to target genes to their original chromatin environment by replacing a loxP-bracketed marker gene on the transposon with a promoter-reporter gene cassette. She will use existing plants with loxP transposons and transposon launching pads to determine whether an efficient "cassette replacement" technique developed in animal cells can also be used to integrate DNA segments efficiently between pairs of loxP sites in Arabidopsis. This work will establish the feasibility of replacing a transposon-borne marker gene cassette with a cassette carrying a reporter gene driven by the promoter of the disrupted gene.This work will provide basic information and techniques that will be widely applicable in contemporary plant genomic research. Both the constructs that will be made and the techniques that will be developed will be useful in other plants.