A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei

Alibu, Vincent P.; Storm, Lilian; Haile, Simon; Clayton, Christine; Horn, David

doi:10.1016/j.molbiopara.2004.10.002

Cited by 155 publications

(164 citation statements)

References 26 publications

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“…1A). Although the construct expresses dsRNA at a low but significant level in the absence of Tet, as previously reported (Alibu et al, 2005) the uninduced procyclic TbSPT2 RNAi cell line had only a slightly longer doubling time compared with the parental 29-13 cell line in the absence of Tet (Fig. 1B).…”

Section: Identification Of Genes Encoding the Subunits Of T Brucei Ssupporting

confidence: 80%

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Fridberg

Olson

Nakayasu

et al. 2008

Journal of Cell Science

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Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.

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Section: Identification Of Genes Encoding the Subunits Of T Brucei Ssupporting

confidence: 80%

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Fridberg

Olson

Nakayasu

et al. 2008

Journal of Cell Science

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“…The transfected cells were then selected in a medium containing hygromycin (2.5 g/ml) exactly following a published procedure (26). RNAi was induced by incubation with doxycycline (1 g/ml) for 48 h. To express GPIdeAc2 in the procyclic form, we cloned GPIdeAc2 into the HindIII-and BamHI-cut expression vector pPPMCS, which was constructed from pHD590 (28) by replacing its promoter and luciferase gene with the normal procyclic acidic repetitive protein promoter and the multiple cloning sites (10).…”

Section: Methodsmentioning

confidence: 99%

Removal or Maintenance of Inositol-linked Acyl Chain in Glycosylphosphatidylinositol Is Critical in Trypanosome Life Cycle

Hong¹,

Nagamune²,

Morita³

et al. 2006

Journal of Biological Chemistry

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The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase (GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.

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“…Trypanosomes expressing the Tet repressor [17,18] were cultured and transfected as previously described [17,19,20]. To induce tetracycline-regulatable promoters, tetracycline was added to 100 ng ml −1 .…”

Section: Trypanosome Culture and Transfectionmentioning

confidence: 99%

“…All nine Puf open reading frames (ORFs) were amplified from T. brucei Lister 427 genomic DNA, using Expand Polymerase (Roche), and cloned into the tetracycline-inducible vectors pHD615 and pHD617 [17]. Taq polymerase was used to amplify fragments for RNA interference, which were cloned into the p2T7 series vectors [18]. Relevant segments of the plasmids were commercially sequenced.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Briefly, for the genome-wide analysis of gene expression, 15 g of total RNA was incubated for 5 min at 65 • C with 500 ng of oligo d(T) [12][13][14][15][16][17][18] (Amersham), 30 mM dAGT-mix, 2 mM dCTP and 2 mM Cy3 (or Cy5) labelled dCTP (Amersham). The mixture was put on ice before addition of 8 l of 5× superscript buffer (Invitrogen), 2 l 0.1 M DTT, 1 l SuperScript III RT (Invitrogen).…”

Section: Microarraysmentioning

confidence: 99%

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Functional analysis of Trypanosoma brucei PUF1

Luu

Brems

Hoheisel

et al. 2006

Molecular and Biochemical Parasitology

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The genomes of Trypanosoma brucei, Leishmania major and Trypanosoma cruzi each encode 10 proteins with PUF domains. PUF domain proteins from yeast and metazoa have been shown to bind RNA and to regulate mRNA stability and translation. Phylogenetic analysis suggested that the PUF proteins were duplicated and diverged early in evolution, and that most PUF proteins were lost during the evolution of mammals. Depletion of any of the first nine T. brucei PUF protein mRNAs by RNA interference had no effect on cell growth; combined depletion of PUF1 and PUF3, PUF3 and PUF4, and PUF1 and PUF4 mRNAs also had no effect. In conflict with a previous report, procyclic trypanosomes lacking PUF1 genes grew normally and we could find no evidence that PUF1 is required for growth of trypanosomes in culture. Depletion or elimination of PUF1 mRNA did not affect the abundances of any other mRNAs, as detected in microarray analysis, and also had minimal effects on the proteome. (In control experiments, treatment of bloodstream and procyclic cells with 100 ng/ml tetracycline also had no detectable effects on the transcriptome and proteome.) PUF1 preferentially bound to retroposon RNAs and was not associated with polysomes. We suggest that, as in yeast, there may be functional redundancy among the Kinetoplastid PUF proteins, or they may be involved in fine-tuning gene expression together with other proteins. Alternatively, PUF proteins may be needed in differentiating trypanosomes or in non-culturable life-cycle stages.

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A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei

Cited by 155 publications

References 26 publications

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Removal or Maintenance of Inositol-linked Acyl Chain in Glycosylphosphatidylinositol Is Critical in Trypanosome Life Cycle

Functional analysis of Trypanosoma brucei PUF1

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