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J. Biol. Chem., Vol. 279, Issue 28, 28979-28988, July 9, 2004

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Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A^*

Simone Leal, Alvaro Acosta-Serrano¶, James Morris||**, and George A. M. Cross

From the Laboratory of Molecular Parasitology, The Rockefeller University, New York, New York 10021, the Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom, and the ^||Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, Maryland 21205

We have engineered Trypanosoma brucei with a novel mariner transposition system that allows large populations of mutant cells to be generated and screened. As a proof of principle, we isolated and characterized two independent clones that were resistant to the cytotoxic action of concanavalin A. In both clones, the transposon had integrated into the locus encoding a homologue of human ALG12, which encodes a dolichyl-P-Man: Man₇GlcNAc₂-PP-dolichyl-6-mannosyltransferase. Conventional knock-out of ALG12 in a wild-type background gave an identical phenotype to the mariner mutants, and biochemical analysis confirmed that they have the same defect in the N-linked oligosaccharide synthesis pathway. To our surprise, both mariner mutants were homozygous; the second allele appeared to have undergone gene conversion by the mariner-targeted allele. Subsequent experiments showed that the frequency of gene conversion at the ALG12 locus, in the absence of selection, was 0.25%. As we approach the completion of the trypanosome genome project, transposon mutagenesis provides an important addition to the repertoire of genetic tools for T. brucei.

Received for publication, March 29, 2004

^* This work was supported by National Institutes of Health Grant AI21729 (to G. A. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a Wellcome Trust Traveling Research Fellowship. Present address: Wellcome Centre for Molecular Parasitology, Anderson College, University of Glasgow, Glasgow G11 6NU, Scotland, UK.

^** Supported by National Institutes of Health Grant AI21334. Present address: Dept. of Genetics and Biochemistry, Clemson University, Clemson, SC 29634-0324.

To whom correspondence should be addressed: Lab. of Molecular Parasitology, The Rockefeller University, 1230 York Ave., New York, NY 10021-6399. Tel.: 212-327-7571; Fax: 212-327-7845; E-mail: george.cross{at}rockefeller.edu.

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