A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome

Abstract

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).

Figure 1

Genetic map position on chromosome 5 of the three start lines used in this study. The nearest RFLP marker and genetic distance are shown. CEN, Centromere. This map is derived from Smith et al. (1996).

Figure 2

Distribution of Ds insertions on the Arabidopsis genome. Inserted sites are plotted at 500-kb intervals. Red, blue, and green dots indicate transpositions from Ds391-20, Ds392-13, and Ds389-13, respectively. Red, blue, and green arrowheads represent the respective start positions. The circle represents the centromere.

Figure 3

Distribution of Ds insertions in hot spots near NORs on chromosomes 2 and 4. Arrows on the chromosomes show insertions. Positions of the sequenced BACs are also indicated.

Figure 4

Transposition rate within a given distance from a start site. Circles, triangles, and rectangles represent transposition rates from Ds391-20, Ds392-13, and Ds389-13, respectively.

Figure 5

Nucleotide sequences around Ds insertion sites. A, Examples of irregularly inserted patterns. B, Examples of authentic patterns. Arrows indicate IR sequences of a Ds element necessary for transposition. Ds elements in the insertion lines are surrounded by a rectangle. Sequences duplicated by transposition are underlined. Sequences shown as red and blue indicate those of the original Ds and the insertion line, respectively. The sequence shown as black is the Nossen wild-type sequence. Hyphens and dots show deleted nucleotides in the insertion lines compared with original Ds element (--) and wild-type genomic DNA (·). Additional nucleotides in the insertion lines are indicated in lowercase.