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A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei

Nature volume 414, pages 759–763 (2001)Cite this article

Abstract

In the mammalian host, African trypanosomes generate consecutive waves of parasitaemia by changing their antigenic coat. Because this coat consists of a single type of variant surface glycoprotein (VSG), the question arises of how a trypanosome accomplishes the transcription of only one of a multi-allelic family of VSG expression site loci to display a single VSG type on the surface at any one time1. No major differences have been detected between the single active expression site and the cohort of inactive expression sites2. Here we identify an extranucleolar body containing RNA polymerase I (pol I) that is transcriptionally active and present only in the bloodstream form of the parasite. Visualization of the active expression site locus by tagging with green fluorescent protein3 shows that it is specifically located at this unique pol I transcriptional factory. The presence of this transcriptional body in postmitotic nuclei and its stability in the nucleus after DNA digestion provide evidence for a coherent structure. We propose that the recruitment of a single expression site and the concomitant exclusion of inactive loci from a discrete transcriptional body define the mechanism responsible for VSG mono-allelic expression.

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Figure 1: Identification of an extranucleolar body containing pol I in bloodstream-form nuclei of T. brucei.
Figure 2: The pol I extranucleolar body has a different function from that of the nucleolus.
Figure 3: Br-UTP labelling of nascent RNA in permeabilized bloodstream-form nuclei indicates that the extranucleolar pol I structure is transcriptionally active.
Figure 4: GFP–LacI tagging of the active expression site (ES) sequences in bloodstream-form nuclei reveals the association of this locus with α-amanitin-resistant transcription and with the extranucleolar pol I body.

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Acknowledgements

We thank A. F. Straight for the GFP–LacI tagging constructs; E. Wirtz and C. Ochatt for pLew100 and the SAT-derived construct; M. Hoek and G. A. M. Cross for the BAC clone; G. Pierron for the fibrillarin (P2G3) monoclonal antibody; and K. E. Sawin for the anti-GFP rabbit polyclonal antibody. We are grateful to K. Ersfeld for FISH protocols, and D. Robinson for technical advice. We thank A. Baines for technical assistance at the initial stage of this work, and all members of the Gull laboratory for discussions. This work was founded by the Wellcome Trust and the BBSRC.

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  1. Miguel Navarro

    Present address: Instituto de Parasitología y Biomedicina ‘López-Neyra’, CSIC, C/ Ventanilla 11, 18001, Granada, Spain

  2. Miguel Navarro: Correspondence and requests for materials should be addressed to M.N.

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  1. School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT, UK

    Miguel Navarro & Keith Gull

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41586_2001_BF414759a_MOESM1_ESM.mov

Movie (MOV 1.2 MB)

Immunofluorescence analysis of the bloodstream form of T. brucei using an anti-pol-I antibody (red) and DAPI staining (blue). The three-dimensional nature of the pol I body in the nucleoplasm is shown.

(Click here to download the Quicktime plugin: http://www.apple.com/quicktime/download/ )

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Navarro, M., Gull, K. A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei. Nature 414, 759–763 (2001). https://doi.org/10.1038/414759a

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