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GrainGenes Reference Report: TPJ-24-253

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Reference	TPJ-24-253
Title	An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function
Journal	Plant Journal
Year	2000
Volume	24
Pages	253-263
Author	Koprek T McElroy D Louwerse J Williams-Carrier R Lemaux PG
Abstract	Summary: To devise a method for function-based gene isolation and characterization in barley, we created a plasmid containing the maize Activator (Ac) transposase (AcTPase) gene and a negative selection gene, codA, and a plasmid containing Dissociation (Ds) inverted-repeat ends surrounding the selectable herbicide resistance gene, bar. These plasmids were used to stably transform barley (Hordeum vulgare). In vitro assays, utilizing a Ds-interrupted uidA reporter gene, were used to demonstrate high-frequency excisions of Ds when the uidA construct was introduced transiently into stably transformed, AcTPase-expressing plant tissue. Crosses were made between stably transformed plants expressing functional transposase under the transcriptional control of either the putative AcTPase promoter or the promoter and first intron from the maize ubiquitin (Ubi1) gene, and plants containing Ds-Ubi-bar. In F1 plants from these crosses, low somatic and germinal transposition frequencies were observed; however, in F2 progeny derived from individual selfed F1 plants, up to 47% of the plants showed evidence of Ds transposition. Further analyses of F3 plants showed that approximately 75% of the transposed Ds elements reinserted into linked locations and 25% into unlinked locations. Transposed Ds elements in plants lacking the AcTPase transposase gene could be reactivated by reintroducing the transposase gene through classical genetic crossing, making this system functional for targeted gene tagging and studies of gene function. During the analysis of F3 plants we observed two mutant phenotypes in which the transposed Ds elements co-segregate with the new phenotype, suggesting the additional utility of such a system for tagging genes
Keyword	[ Hide all but 1 of 40 ] ac ac transposase activator activator ac arabidopsis thaliana assay bar barley dissociation excision ds transposition elements functional genomics gene gene tagging genome germinal transposition herbicide resistance hordeum vulgare in vitro insertional mutagen intron inverted repeat maize messenger rna mutant phenotypes plasmids preferential transposition progeny promoter reporter gene resistance resistance gene selection t-dna tissue tool transgenic rice plants transposition ubiquitin

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