Suppressor activity of Ac transposase in transfected Petunia cells
-- Lutz Essers and Reinhard Kunze

The non-autonomous 0.4 kb Ds1 element has, in contrast to most other Ds elements, only few AAACGG-like TPase binding motifs. Therefore Ds1 seems to be an ideal subject for investigating their influence on the excision rate. The element was cloned in both directions between the 1'-promotor and the GUS-gene. Although Ds1 contains several closed reading frames in both orientations, GUS-expression was obtained in transfected Petunia protoplasts. In further experiments promotor activities of Ds1 were found in both orientations. However, these activities are too weak to explain the GUS-activity. We consider it more likely that the element may be read-through transcribed and spliced out, as was described for Ds1 in maize.

We determined that the above-mentioned GUS expression was reduced to about 50% when an Ac transposase (TPase) expression plasmid was cotransfected into the cells. A possible explanation for this phenomenon could be a suppression effect of TPase binding to its target DNA, similar to the action of the En/Spm-encoded TnpA-protein (Grant et al., EMBO J. 9:2029-2035, 1990). Consistently, if the Ds1-element was substituted by a 16mer of the AAACGG-motif, GUS expression was reduced to about 30%. A transpositionally defective, but DNA binding-positive TPase-derivative has retained the suppressor activity, whereas mutations in the DNA-binding region abolish the suppressor effect. These findings suggest that the TPase recognizes in vivo the same DNA sequences as the renatured TPase of bacterial origin in vitro, and that the binding of the TPase to the DNA is strong enough to interfere with read-through transcription. 


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