Phenotypic assay for excision of the maize controlling element Ac in tobacco

Abstract

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac△), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 µg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed ˜25% as many calli resistant to 100 or 200 µg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac△ did not form any calli resistant to 200 µg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin‐resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.

A comparative study of Tam3 and Ac transposition in transgenic tobacco and petunia plants

“…The other explanation might be that the GUS reporter values represent the in vivo situation, because transposition of the element could be induced in cells of calli that were severly stressed by the dying, hygromycin-sensitive, cells surrounding it. An enrichment of resistant parts in calli under selection has already been proposed by Baker et al [2].…”

“…When the frequencies of marker gene restoration in pTT21820 and pTT224 (Tables 1 and 2) are normalized and corrected for the 65-70% expression of both markers in pTT218 and pBI121, the excision frequency can be estimated [2]. We estimated that the frequency of Tam3 excision lies between 20 and 66 ~o in tobacco and between 18 and 40 ~o in petunia, depending on the assay used ( Table 1).…”

“…Genetic analysis of transposition has been simplified by the use of a phenotypic assay allowing direct selection of excision events [2]. When Ac excises from the leader sequence of the neomycin phosphotransferase (NPTII) gene, the expression of the gene is restored and transformed calli become kanamycin-resistant.…”

“…When Ac excises from the leader sequence of the neomycin phosphotransferase (NPTII) gene, the expression of the gene is restored and transformed calli become kanamycin-resistant. No kanamycinresistant calli are found when a Ds, dissociator, element (an Ac element with an inactivated transposase function) is tested [2,21]. Subsequent molecular analysis of the resistant plants demonstrated the reintegration of the element.…”

Novel DNA structures resulting from dTam3 excision in tobacco

“…With the introduction of Ac into new hosts it became clear in subsequent analyses that the maize Ac and Ds elements retain most of their properties in heterologous hosts [ 1,11,13,14]. On the contrary, a Tam3 two-element system appears to be unable to perform the transposition process with the same efficiency and accuracy as the autonomous Tam3 element in both tobacco and Antirrhinum [20,21].…”