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.
-Amanitin-Resistant
Transcription from the rRNA, Procyclic Acidic Repetitive Protein,
and Variant Surface Glycoprotein Gene Promoters in
Trypanosoma brucei
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
-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
-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.
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