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GrainGenes Reference Report: PLC-11-1841

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Reference	PLC-11-1841
Title	Multiple independent defective Suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics
Journal	Plant Cell
Year	1999
Volume	11
Pages	1841-1852
Author	Tissier A [ Show all 7 ]
Abstract	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 approximately 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.
Keyword	[ Hide all but 1 of 18 ] arabidopsis thaliana genetic transformation genome-analysis glufosinate herbicide-resistance in-vitro-selection insertional-mutagenesis molecular genetics mutation nucleotide sequence polymerase chain reaction reporter genes screening t-dna transgenic-plants transposable-elements transposition zea mays

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