Elizabeth Wirtz

A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei

First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.

Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.

Trypanosoma brucei variant surface glycoprotein regulation involves coupled activation/inactivation and chromatin remodeling of expression sites.

Trypanosoma brucei is an extracellular protozoan parasite that cycles between mammalian hosts and the tsetse vector. In bloodstream‐form trypanosomes, only one variant surface glycoprotein gene (VSG) expression site (ES) is active at any time. Transcriptional switching between ESs results in antigenic variation. No VSG is transcribed in the insect procyclic stage. We have used bacteriophage T7 RNA polymerase (T7RNAP) to study the transcriptional accessibility of ES chromatin in vivo. We show that T7RNAP‐mediated transcription from chromosomally integrated T7 promoters is repressed along the entire length of the ES in the procyclic form, but not in the bloodstream form, suggesting that the accessible chromatin of inactive bloodstream‐form ESs is remodeled upon differentiation to yield a structure that is no longer permissive for T7RNAP‐mediated transcription. In the bloodstream form, replacing the active ES promoter with a T7 promoter, which is incapable of sustaining high‐level transcription of the entire ES, prompts an ES switch. These data suggest two distinct mechanisms for ES regulation: a chromatin‐mediated developmental silencing of the ES in the procyclic form and a rapid coupled mechanism for ES activation and inactivation in the bloodstream form.

Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair

Trypanosome RNA editing utilizes a seven polypeptide complex that includes two RNA ligases, band IV and band V. We now find that band IV protein contributes to the structural stability of the editing complex, so its lethal genetic knock‐out could reflect structural or catalytic requirements. To assess the catalytic role in editing, we generated cell lines which inducibly replaced band IV protein with an enzymatically inactive but structurally conserved version. This induction halts cell growth, showing that catalytic activity is essential. These induced cells have impaired in vivo editing, specifically of RNAs requiring uridylate (U) deletion; unligated RNAs cleaved at U‐deletion sites accumulated. Additionally, mitochondrial extracts of cells with reduced band IV activity were deficient in catalyzing U‐deletion, specifically at its ligation step, but were not deficient in U‐insertion. Thus band IV ligase is needed to seal RNAs in U‐deletion. U‐insertion does not appear to require band IV, so it might use the other ligase of the editing complex. Furthermore, band IV ligase was also found to serve an RNA repair function, both in vitro and in vivo.

Conditional expression of glycosylphosphatidylinositol phospholipase C in Trypanosoma brucei.

Trypanosoma brucei glycosylphosphatidylinositol phospholipase C (GPIPLC) is expressed in the bloodstream stage of the life cycle, but not in the procyclic form. It is capable of hydrolyzing GPI-anchored proteins and phosphatidylinositol (PI) in vitro. Several roles have been proposed for GPIPLC in vivo, in the release of variant surface glycoprotein during differentiation or in the regulation of GPI and PI levels, but none has been substantiated. To explore GPIPLC function in vivo, tetracycline-inducible GPIPLC gene (GPIPLC) conditional knock-out bloodstream form and tetracycline-inducible GPIPLC-expressing procyclic cell lines were constructed. We were unable to generate GPIPLC null mutants. Cleavage of GPI-anchored proteins was abolished in extracts from uninduced conditional knock-outs and was restored upon induction. Despite the barely detectable level of GPIPLC activity in uninduced conditional knock-out bloodstream forms, their growth was not affected. GPI-protein cleavage activity could be induced in procyclic cell extracts, up to wild-type bloodstream levels. Myo-[3H]inositol incorporation into [3H]inositol monophosphate was about 14-fold lower in GPIPLC conditional knock-out bloodstream forms than in the wild type. Procyclic cells expressing GPIPLC showed a 28-fold increase in myo-[3H]inositol incorporation into [3H]inositol monophosphate and a 1.5-fold increase in [3H]inositol trisphosphate levels, suggesting that GPIPLC may regulate levels of inositol phosphates, by cleavage of PI and phosphatidylinositol 4,5-bisphosphate.