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Eukaryotic Cell, September 2006, p. 1539-1549, Vol. 5, No. 9
1535-9778/06/$08.00+0     doi:10.1128/EC.00141-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Chromosome-Wide Analysis of Gene Function by RNA Interference in the African Trypanosome

Chandra Subramaniam ,1,3,§,{ddagger} Paul Veazey,2,4,{ddagger} Seth Redmond,2,{ddagger} Jamie Hayes-Sinclair,1 Emma Chambers,1,5 Mark Carrington,6 Keith Gull,1,3 Keith Matthews,1,7* David Horn,4* and Mark C. Field2,8*

School of Biological Sciences, University of Manchester, Oxford Road, Manchester, United Kingdom,1 Department of Biochemistry, Imperial College of Science Technology and Medicine, London, United Kingdom,2 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, United Kingdom,3 London School of Hygiene and Tropical Medicine, Keppel Street, London, United Kingdom,4 Department of Biological Sciences, Lancaster University, Lancaster, United Kingdom,5 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom,6 Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, United Kingdom,7 Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom8

Received 15 May 2006/ Accepted 5 June 2006

Trypanosomatids of the order Kinetoplastida are major contributors to global disease and morbidity, and understanding their basic biology coupled with the development of new drug targets represents a critical need. Additionally, trypanosomes are among the more accessible divergent eukaryote experimental systems. The genome of Trypanosoma brucei contains 8,131 predicted open reading frames (ORFs), of which over half have no known homologues beyond the Kinetoplastida and a substantial number of others are poorly defined by in silico analysis. Thus, a major challenge following completion of the T. brucei genome sequence is to obtain functional data for all trypanosome ORFs. As T. brucei is more experimentally tractable than the related Trypanosoma cruzi and Leishmania spp. and shares >75% of their genes, functional analysis of T. brucei has the potential to inform a range of parasite biology. Here, we report methods for systematic mRNA ablation by RNA interference (RNAi) and for phenotypic analysis, together with online data dissemination. This represents the first systematic analysis of gene function in a parasitic organism. In total, 210 genes have been targeted in the bloodstream form parasite, representing an essentially complete phenotypic catalogue of chromosome I together with a validation set. Over 30% of the chromosome I genes generated a phenotype when targeted by RNAi; most commonly, this affected cell growth, viability, and/or cell cycle progression. RNAi against approximately 12% of ORFs was lethal, and an additional 11% had growth defects but retained short-term viability in culture. Although we found no evidence for clustering or a bias towards widely evolutionarily conserved genes within the essential ORF cohort, the putative chromosome I centromere is adjacent to a domain containing genes with no associated phenotype. Involvement of such a large proportion of genes in robust growth in vitro indicates that a high proportion of the expressed trypanosome genome is required for efficient propagation; many of these gene products represent potential drug targets.

* Corresponding author. Mailing address for M. Field (data and website questions): University of Cambridge, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom. Phone: 44 1223 333734. Fax: 44 1223 333346. E-mail: mcf34{at}cam.ac.uk. Mailing address for D. Horn (RNAi technology questions): London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom. Phone: 44 20 7927 2352. Fax: 44 20 7636 8739. E-mail: david.horn{at}lshtm.ac.uk. Mailing address for K. Matthews (transfection and phenotyping questions): Institute of Immunology and Infection Research, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, United Kingdom. Phone: 44 131 651 3639. Fax: 44 131 650 6564. E-mail: keith.matthews{at}ed.ac.uk.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

§ Present address: Institute for Animal Health, Compton, Newbury RG20 YNN, United Kingdom.

{ddagger} C.S., P.V., and S.R. contributed equally to this work.

Eukaryotic Cell, September 2006, p. 1539-1549, Vol. 5, No. 9
1535-9778/06/$08.00+0     doi:10.1128/EC.00141-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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