The transposable element Ac has been used in maize to identify and clone specific genes (Fedoroff et al., PNAS 81:3825, 1984). The presence of the element within a mutated gene provides a tag by which the gene may be recovered for molecular analysis. Baker et al. (PNAS 83:4844, 1986) have shown that Ac will transpose in tobacco callus, thus extending the range of species in which Ac may be useful as a gene tag. Since many qf the mutations which may be obtained by this method require whole plant screening, we have analyzed the behavior of Ac in tobacco plants regenerated directly after transformation with Ac.
The Ac element from wx-m9 (Fedoroff et al., Cell 35:325, 1983) was inserted into the binary Agrobacterium vector pGA482 (An. Plant Physiol. 81:86) and transferred by leaf disk transformation (Horsch et al., Science 227:1229, 1985) into Wisconsin 38 tobacco. Thirty-five kanamycin resistant plants were obtained, 23 of which exhibited Southern hybridization patterns consistent with at least one unrearranged copy of Ac. This was assessed by the presence of a 7.5kb BglII fragment consisting of the 4.5kb Ac in a 0.25kb PstI wx gene fragment (Fedoroff et al., 1983) and pUC8, a 2.7kb cloning vector (Vieira and Messing, Gene 19:259, 1982).
When Ac transposes from the initial site of insertion, a 3.0kb BglII fragment is created. This fragment was detected at a copy number of 0.5 to 1.0 copies per cell in 9 plants, and was just detectable (< 0.1 copy) in a further 11 plants. Three plants showed no evidence of Ac transposition.
In order to determine whether the frequency of Ac transposition is related to the level of Ac transcription, we isolated RNA from 19 transformants with different Ac transposition frequencies and determined the level of Ac transcription by S1 analysis (Weaver and Weissman, Nucl. Acids Res. 7:1175, 1979). All plants which showed transposition also showed transcription; however, no correlation could be made between the level of transcription and the relative intensity of the 3.0kb band. Ac transcripts were detected in 2 plants which were negative for Ac transposition, while one plant which was negative for transposition had no detectable transcription.
We are currently analyzing DNA from shoots which have undergone sequential regenerations from the same transformed root stock to determine whether the frequency of transposition is constant with time or if it changes during extended residence of the element within a genome.
B.H. Taylor, E.J. Finnegan and E.S. Dennis
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