Transfection of Leishmania and Trypanosoma brucei by Electroporation

Beverley, Stephen M.; Clayton, Christine

doi:10.1385/0-89603-239-6:333

Cited by 85 publications

(66 citation statements)

References 10 publications

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“…Inducible ectopic expression of Tb-SepSecS was done by using pLew-100 carrying the phleomycin resistance gene (25). Transfection, selection of transformants, and production of clonal cell lines were done by using standard procedures as described (12).…”

Section: Methodsmentioning

confidence: 99%

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The canonical pathway for selenocysteine insertion is dispensable in Trypanosomes

Aeby

Palioura²,

Pusnik

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Section: Methodsmentioning

confidence: 99%

“…The study was done in the insect form of the parasitic protozoon Trypanosoma brucei as double allelic KO cell lines can easily be produced in this system by homologous recombination-directed gene replacements (12). Because T. brucei is diploid, double KO cell lines are null mutants in both alleles.…”

mentioning

confidence: 99%

The canonical pathway for selenocysteine insertion is dispensable in Trypanosomes

Aeby

Palioura²,

Pusnik

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…2 The plasmids were linearized with NotI and transfected into the procyclic T. brucei strain 29 -13, which expresses T7 RNA polymerase and the tetracycline repressor. Selection with phleomycin, cloning, and induction with tetracycline were done as described previously (24). For the IF2/MTF double RNAi cell line (see Fig.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

Charrière

Tan

Schneider

2005

Journal of Biological Chemistry

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The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA Met , all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA Met present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA Met carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA Met . In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA Met . Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA Met , suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knockdown of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyltRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.

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“…The resulting RNAi plasmids were linearized with NotI and transfected into the procyclic T. brucei strain 29-13, which expresses T7 RNA polymerase and the tetracycline repressor (7,8). Selection with phleomycine, cloning, and induction with tetracycline were done as described previously (9).…”

Section: Methodsmentioning

confidence: 99%

Non-canonical Eukaryotic Glutaminyl- and Glutamyl-tRNA Synthetases Form Mitochondrial Aminoacyl-tRNA in Trypanosoma brucei

Rinehart

Horn

Wei

et al. 2004

Journal of Biological Chemistry

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Glutaminyl-tRNA synthetase is thought to be absent from organelles. Instead, Gln-tRNA is formed via the transamidation pathway, the other route to this essential compound in protein biosynthesis. However, it was previously shown that glutaminyl-tRNA synthetase activity is present in Leishmania mitochondria. This work identifies genes encoding glutaminyl-and glutamyltRNA synthetase in the closely related organism Trypanosoma brucei. Down-regulation of their respective gene products by RNA interference showed that (i) they are essential for the growth of insect stage T. brucei and (ii) they are responsible for essentially all of the glutaminyl-and glutamyl-tRNA synthetase activity detected in both the cytosol and the mitochondria. In vitro aminoacylation experiments with the recombinant T. brucei enzymes and total tRNA confirmed the identity of the two aminoacyl-tRNA synthetases. Interestingly, T. brucei uses the same eukaryotic-type glutaminyl-tRNA synthetase to form mitochondrial and cytosolic Gln-tRNA. The formation of Glu-tRNA in mitochondria and the cytoplasm is catalyzed by a single eukaryotic-type discriminating glutamyl-tRNA synthetase. T. brucei, similar to Leishmania, imports all of its mitochondrial tRNAs from the cytosol. The use of these two eukaryotictype enzymes in mitochondria may therefore reflect an adaptation to the situation in which the cytosol and mitochondria use the same set of tRNAs.

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Transfection of Leishmania and Trypanosoma brucei by Electroporation

Cited by 85 publications

References 10 publications

The canonical pathway for selenocysteine insertion is dispensable in Trypanosomes

The canonical pathway for selenocysteine insertion is dispensable in Trypanosomes

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

Non-canonical Eukaryotic Glutaminyl- and Glutamyl-tRNA Synthetases Form Mitochondrial Aminoacyl-tRNA in Trypanosoma brucei

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