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

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

Content Provider	PubMed Central
Author	Baker, B. Coupland, G. Fedoroff, N. Starlinger, P. Schell, J.
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.
Starting Page	1547
File Format	PDF
ISSN	14602075
e-ISSN	14602075
Journal	The EMBO Journal
Issue Number	6
Volume Number	6
Language	English
Publisher Date	1987-06-01
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Neuroscience Immunology and Microbiology Medicine Molecular Biology

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