COLOGNE, WEST GERMANY

University of Cologne
 
 

Transcription of Ac and deleted derivatives of Ac in tobacco --Siegfried Feldmar, Reinhard Kunze and Peter Starlinger In order to study the influence of Ac deletions on transcription of Ac derivatives in transgenic tobacco, independently transformed tobacco plants were investigated by Northern blot analysis. The tobacco plants were transformed with Ti-plasmid vectors containing Ac or the derivatives integrated into the untranslated leader of thc neomycin phosphotransferase II (NPTII) gene of plasmid pKU2 as described by B. Baker (EMBO J. 6:1547, 1987). The insertions are located about 50bp downstream from the transcription initiation site of the 1' promoter.

The sequences deleted from the Ac derivatives are within the 5' untranscribed region of Ac (pKU19), the untranslated region of the Ac transcript (pKU33) or within the long open reading frame of the Ac sequence (pKU4). By Southern analysis the structural integrity of the Ac derivatives and the flanking T-DNA was confirmed. All Ac derivatives tested so far were integrated in the plant genome at their original position in the T-DNA.

Northern hybridisation with short Ac homologous probes was performed on ten independently transformed tobacco plants that had been selected for resistance to hygromycin. From previous experiments with kanamycin-resistant plants it is known that the intact Ac is transcribed after excision from the T-DNA in form of the 3.5kb mRNA also found in maize.

All Ac derivatives still integrated in the T-DNA are transcribed in a complex manner. In every case more than one Ac-homologous transcript spanning the whole transcription unit of Ac could be detected on the Northern blots. In some cases the autonomous Ac transcript starting at the Ac promoter and terminating at the polyadenylation signal near the 3' end of Ac could be detected (pKU19, pKU33), but most of the Ac homologous RNAs are readthrough transcripts of various sizes (2.0kb - 13kb) initiated outside of Ac, as was shown by hybridisation with a single stranded probe from the normally untranscribed 5' end of Ac. It is unclear whether readthrough transcripts starting at the 1' promoter or farther upstream on the T-DNA are able to produce the Ac transposase if the long open reading frame is intact and the element is inserted in sense orientation with respect to the 1' promoter sequence like in pKU19 or pKU33. B. Baker et al. showed that frequency of excision of the intact Ac is independent of the element's orientation. In this case readthrough transcription of Ac is not necessary for excision from the T-DNA.


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