The maize controlling element Ac was previously shown to transpose in tobacco cells (Baker et al., Proc. Natl. Acad. Sci. USA. 83:4484, 1986). We report here a phenotypic assay designed to detect excision of Ac from a selectable marker gene, neomycin phosphotransferase II (NPTII). An NPTII gene which expresses Km resistance in tobacco cells, and contains a unique restriction site in the transcriptional leader, was constructed in plasmid pKU2. Ac, or an internally deleted defective Ac element, was inserted into the leader of this gene in plasmids pKU3 and pKU4, respectively. These insertions inactivate the NPTII gene. These three plasmids were inserted into the T-DNA of the Agrobacterium tumefaciens Ti-plasmid pGV3850, and then transferred to regenerating tobacco protoplasts. The transformed cells were selected with 100 or 200 mg/l kanamycin (Km). Protoplasts transformed with pGV3850::pKU3 formed approximately 22% (100 mg/l Km) or 30% (200 mg/l Km) as many Kmr calli as protoplasts transformed with pGV3850::pKU2. Protoplasts transformed with pGV3850::pKU4 formed approximately 4% (100 mg/l Km) or 0% (200 mg/l Km) of the number of KMr calli obtained after transformation with pGV3850::pKU2 (see Table 1). Southern blot analysis of five of the rare KMr calli transformed with pGV3850::pKU4 and selected on 100 mg/l Km showed no evidence of excision of the transposon sequences, and were produced by an unknown mechanism. However, similar analysis of seven KMr calli formed after transformation with pGV3850::pKU3 revealed that in all cases Ac had excised, restoring the structure of the NPTII gene.
This assay is being used to perform functional analysis of Ac (see Coupland et al., this issue). Several features of the phenotypic assay can be employed in the design of appropriate plasmid vectors using the AclDs family of elements as mutagens and gene tags.
Barbara Baker1, George Coupland2, Nina Fedoroff3, Peter Starlinger2 and Jeff Schell2
1Max-Planck-Institut fur Zuchtungsforschung
2Institut fur Genetik
3Carnegie Institution of Washington,
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