Development of an efficient two-element transposon tagging system in Arabidopsis thaliana

Abstract

Modified Ac and Ds elements, in combination with dominant markers (to facilitate monitoring of excision, reinsertion and segregation of the elements) were introduced into Arabidopsis thaliana ecotype Landsberg erecta. The frequencies of somatic and germinal transactivation of the Ds elements were monitored using a streptomycin resistance assay. Transactivation was significantly higher from a stable Ac (sAc) carrying a 537 bp deletion of the CpG-rich 5' untranslated leader of the transposase mRNA than from a wild-type sAc. However, substitution of the central 1.77 kb of the transposase open reading frame (ORF) with a hygromycin resistance marker did not alter the excision frequency of a Ds element. beta-Glucuronidase (GUS) or iaaH markers were linked to the transposase source to allow the identification of plants in which the transposase source had segregated away from the transposed Ds element, eliminating the possibility of somatic or germinal re-activation. Segregation of the excision marker, Ds and sAc was monitored in the progeny of plants showing germinal excision of Ds. 29% of the plants inheriting the excision marker carried a transposed Ds element.

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon‐induced allelic variation and meiosis‐associated expression

“…One promising potential application of the AtDMCl gene is using its promoter for improving transposon tagging efficiency. Different heterologous transposon tagging systems have been employed in Arabidopsis (Aarts et aL, 1995;Bancroft et al, 1992;Dean et aL, 1992;Lucas et aL, 1995). The main factors determining the efficiency of each system in the tagging of host genes are the frequencies of transposon excision and reinsertion, and the independence of transposition events.…”

Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

“…This does not, therefore, represent a significant increase in efficiency over the use of one-component and two-component (Bancroft et al, 1992) systems in wild-type Arabidopsis, where the equivalent frequencies of useful plants were found to be 0.6% and 1.1%, respectively. Dean et al (1992) analysed the transposition of Ac in the wild-type Landsberg erecta background and found that, whereas the frequency of germinal excision was relatively low (1.1%), the number of revertants inheriting a transposed element was very high (58%).…”

“…Dean et al (1992) analysed the transposition of Ac in the wild-type Landsberg erecta background and found that, whereas the frequency of germinal excision was relatively low (1.1%), the number of revertants inheriting a transposed element was very high (58%). Using a stabilized Ac element with a deleted untranslated leader sequence (the Nael deletion) to drive the excision of a Ds element, Bancroft et al (1992) found that higher germinal excision frequencies (3.8%) were accompanied by a reduction in the proportion of revertants carrying transposed elements (29%). Whereas higher germinal excision frequencies can be achieved by using the CaMV 35S promoter to drive Ac TPase expression (Swinburne et al, 1992), Ds re-insertion frequencies under these circumstances are very much reduced [approximately 10% of germinal revertants were found to carry new Ds insertions (Long et aL, 1993)].…”

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast‐derived cells

“…Ac activity has been monitored using artificial Ds elements produced by deletion of the autonomous Ac element; the Ds elements transpose in a heterologous host provided the Ac transposase encoded by ORFa is also present (Altmann et al, 1992;Bancroft et aL, 1992;Grevelding et aL, 1992;Lassner et al, 1989;Swinburne eta/., 1992).…”