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Molecular and Cellular Biology, August 1999, p. 5466-5473, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

In Vitro Analysis of alpha -Amanitin-Resistant Transcription from the rRNA, Procyclic Acidic Repetitive Protein, and Variant Surface Glycoprotein Gene Promoters in Trypanosoma brucei

Gabriele Laufer, Gabriel Schaaf, Sigrid Bollgönn, and Arthur Günzl*

Abteilung Zellbiologie, Zoologisches Institut der Universität Tübingen, D-72076 Tübingen, Germany

Received 3 March 1999/Returned for modification 19 April 1999/Accepted 25 May 1999

In Trypanosoma brucei, transcription resistant to the mushroom toxin alpha -amanitin is not restricted to the rRNA genes (rDNA), as in higher eukaryotes, but extends to genes encoding the major cell surface proteins variant surface glycoprotein (VSG) and procyclin or procyclic acidic repetitive protein (PARP). Here, we report the development of a homologous cell extract from procyclic T. brucei cells in which rDNA and PARP A and VSG gene promoters drive efficient, accurate, and alpha -amanitin-resistant transcription. A comparative analysis revealed that transcription from the three promoters generally required identical reaction conditions for maximal efficiency. Nevertheless, PARP promoter transcription proved to be exceptional by its high efficiency, its lag phase, a high template DNA concentration optimum, and its tolerance to increasing concentrations of Mn2+. Mutational analysis for both the PARP and rDNA promoters showed that the proximal and distal core elements were essential for efficient transcription in vitro. Deletion of the upstream control regions (UCRs), however, had a different effect. Whereas PARP UCR deletion reduced transcription efficiency almost 10-fold, deletion of the rDNA UCR had only a minor effect on transcription efficiency.

* Corresponding author. Mailing address: Abteilung Zellbiologie, Zoologisches Institut der Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Phone: 49-7071-2978862. Fax: 49-7071-294634. E-mail: arthur.guenzl{at}uni-tuebingen.de.

Molecular and Cellular Biology, August 1999, p. 5466-5473, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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