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

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.

Figure 1

(A) Schematic diagram of the (I-RS/dAc-I-RS)T-DNA construct in plasmid pGAHΔNI-RS/dAc-I-RS. dAc-I-RS indicates the non-autonomous derivative of the Ac element that contained I-SceI sites and RSs (see text). (I-RS/dAc-I-RS)T-DNA indicates the T-DNA that containes a site for I-SceI, RS, and the dAc-I-RS element. LB and RB indicate left and right border sequences on the T-DNA, respectively. I-SceI, the cleavage site for endonuclease I-SceI; 1′ promoter, the 1′ promoter of the octopine Ti plasmid TR-DNA; P35S, the promoter for 35S RNA from cauliflower mosaic virus; HPT, the coding sequence of the gene for hygromycin phosphotransferase; NPTII, the coding sequence of the gene for neomycin phosphotransferase II; Tnos, the terminator of the gene for nopaline synthase; Tocs, the terminator of the gene for octopine synthase; IR, the terminal inverted repeat sequences of Ac; RS, the recombination site that is recognized by the R protein from Zygosaccaromyces rouxii. Thick lines indicate the probes (probes 1 and 2) used for Southern blot analysis. (B) The strategy for measurement of the distance of transposition. I-SceI indicates the site of cleavage by endonuclease I-SceI. A dark grey box indicates dAc-I-RS. Light grey boxes indicate regions of T-DNA other than dAc-I-RS in (I-RS/dAc-I-RS)T-DNA.

Figure 2

Southern blot analysis for measurement of the distance of transposition of dAc-I-RS of the pulse-field gel electrophoresis. High-molecular-weight genomic DNA was prepared from Arabidopsis plants that had a transposed dAc-I-RS that originated from line #14, as described. Isolated DNA was digested with I-SceI, and then fractionated by PFGE in a 1% agarose gel for 18 hr at 170 V with a switch interval of 80 sec and then for 3 hr at 170 V with a switch interval of 110 sec (A and B; for analysis of fragments of 5–1,000 kb), and on a 0.6% agarose gel for 3 hr at 50 V with a switch interval of 5 sec and for 7 days at 50 V with a switch interval of 1,800 sec (C; for analysis of fragments of 1–5 Mb). After transfer of the DNA to nitrocellulose membrane filters, filters were probed with probe 1 (A and C) and probe 2 (B), respectively. Smears that were observed around 100 kb (A and B) and 1,000 kb (C) were nonspecific, because they were also found with digests of the line #14 genomic DNA before transposition. Standards are shown with size in kb. Lanes: 1, 6, and 11, the I-RS/dAc-I-RS#14 line (before transposition); 2 and 7, plant 14-20.2.2; 3 and 8, plant 14-52.2.3; 4 and 9, plant 14-52.4.1; 5 and 10, plant 14-58.3.1; and 12, plant 14-68.1.4.

Figure 3

(A) Summary of distances of transposition in the I-RS/dAc-I-RS#14 line. LB and RB indicate left and right border sequences on the T-DNA, respectively. Each bar represents the distance of transposition in each plant that had a transposed dAc-I-RS. Some of the names of F₃ progeny plants are indicated in parentheses. (B) Alignment of the genetic map around position 80. Relative positions of the transposed dAc-I-RS elements in plant 14-58.3.1 and plant 14-68.1.4 to as2 and cer5 were determined by three-point test and Southern blot analysis as described in the text.