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Eukaryotic Cell, December 2002, p. 1000-1009, Vol. 1, No. 6
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.6.1000-1009.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Ex Vivo and In Vitro Identification of a Consensus Promoter for VSG Genes Expressed by Metacyclic-Stage Trypanosomes in the Tsetse Fly

Michael L. Ginger,1,{dagger} Patricia A. Blundell,1 Alyson M. Lewis,1 Alison Browitt,1 Arthur Günzl,2 and J. David Barry1*

Wellcome Centre for Molecular Parasitology, Anderson College, University of Glasgow, Glasgow G11 6NU, United Kingdom,1 Abteilung Zellbiologie, Zoologisches Institut der Universität Tübingen, D-72076 Tübingen, Germany2

Received 19 June 2002/ Accepted 8 August 2002

The trypanosome variant surface glycoprotein (VSG) is first expressed during differentiation to the infective, metacyclic population in tsetse fly salivary glands. Unlike the VSG genes expressed by bloodstream form trypanosomes, metacyclic VSGs (MVSGs) have their own promoters. The scarcity of metacyclic cells has meant that only indirect approaches have been used to study these promoters, and not even their identities have been agreed on. Here, we isolated trypanosomes by dissection from salivary glands and used an approach involving 5' rapid amplification of cDNA ends to identify the transcription start site of three MVSGs. This shows that the authentic start site is that proposed for the MVAT series of MVSGs (K. S. Kim and J. E. Donelson, J. Biol. Chem. 272:24637-24645, 1997). In the more readily accessible procyclic trypanosome stage, where MVSGs are normally silent, we used reporter gene assays and linker scanning analysis to confirm that the 67 bp upstream of the start site is a promoter. This is confirmed further by accurate initiation in a homologous in vitro transcription system. We show also that MVSG promoters become derepressed when tested outwith their endogenous, subtelomeric loci. The MVSG promoters are only loosely conserved with bloodstream VSG promoters, and our detailed analysis of the 1.63 MVSG promoter reveals that its activity depends on the start site itself and sequences 26 to 49 bp and 56 to 60 bp upstream. These are longer than those necessary for the bloodstream promoter.

* Corresponding author. Mailing address: Wellcome Centre for Molecular Parasitology, Anderson College, University of Glasgow, 56 Dumbarton Rd., Glasgow G11 6NU, United Kingdom. Phone: 44 141 330 4875. Fax: 44 141 330 5422. E-mail: j.d.barry{at}bio.gla.ac.uk.

{dagger} Present address: University of Manchester, School of Biological Sciences, Manchester M13 9PT, United Kingdom.

Eukaryotic Cell, December 2002, p. 1000-1009, Vol. 1, No. 6
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.6.1000-1009.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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Copyright © 2002 by the American Society for Microbiology.