PT  - JOURNAL ARTICLE
AU  - Tissier, Alain F.
AU  - Marillonnet, Sylvestre
AU  - Klimyuk, Victor
AU  - Patel, Kanu
AU  - Torres, Miguel Angel
AU  - Murphy, George
AU  - Jones, Jonathan D. G.
TI  - Multiple Independent Defective <em>Suppressor-mutator</em> Transposon Insertions in Arabidopsis: A Tool for Functional Genomics
AID  - 10.1105/tpc.11.10.1841
DP  - 1999 Oct 01
TA  - The Plant Cell
PG  - 1841--1852
VI  - 11
IP  - 10
4099  - http://www.plantcell.org/content/11/10/1841.short
4100  - http://www.plantcell.org/content/11/10/1841.full
SO  - Plant Cell1999 Oct 01; 11
AB  - A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which ∼80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.