Transposon mutagenesis of Trypanosoma brucei identifies glycosylation mutants resistant to concanavalin A.
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: Man(7)GlcNAc(2)-PP-dolichyl-alpha6-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.
Leal
Simone
S
Laboratory of Molecular Parasitology, The Rockefeller University, New York, New York 10021, USA.
Acosta-Serrano
Alvaro
A
Morris
James
J
Cross
George A M
GA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003208:Concanavalin A / Q000494:pharmacology*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005785:Gene Conversion; D016678:Genome; D006031:Glycosylation; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D010641:Phenotype; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019895:Transposases; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; C111652:mariner transposases; C053277:procyclic acidic repetitive protein, Trypanosoma; D003208:Concanavalin A; D008364:Mannosyltransferases; D019895:Transposases