Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I- Sce I

Abstract

We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.

Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox)

“…Only cDNAs corresponding to AtrbohD were identified in this screening, the longest being 2852 bp. cDNA clones corresponding to AtrbohF were isolated from a Columbia cDNA library using a junction fragment probe derived from a transposed dAc-I-RS element recovered from a line exhibiting slow growth (Machida et al, 1997). The longest AtrbohF cDNA clone isolated was 3080 bp.…”

“…The position of AtrbohB on top of chromosome 1 has been confirmed by sequences from the BAC F7G19 (accession AC000106), from the A. thaliana genomic sequencing project. AtrbohF maps around position 120 of chromosome 1 (Machida et al, 1997). Homologues A, C and D are on chromosome 5, around positions 5:3, 5:74 and 5:88 (http://synteny.nott.ac.uk/agr/ri.html).…”

Trans-Kingdom Transposition of the Maize Dissociation Element

“…Hence their application is limited to only a few laboratories. Most importantly, unlike mutations introduced by ENU or retroviruses, transposon insertions can be reactivated in the presence of transposase, allowing mutagenesis of closely linked genes and inducible phenotype reversal experiments (Tower et al 1993;Preston et al 1996;Smith et al 1996;Machida et al 1997).…”

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

“…These plants were termed Germinals. The Ds elements present in the Germinals were mapped, first roughly by classical genetic linkage analysis and later physically by a combination of Pulsed Field Gel Electrophoresis of RsrII fragments and Southern analysis, analogous to Machida et al (1997). The relative orientations of the lox sites were determined using the probes and the method described by Stuurman et al (1998).…”