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Synthesis vs. Abundance, Bradyzoite Development

ABSTRACT: Parasites grown as either tachyzoites or bradyzoites for 40 hours were pulsed with 2,4-dithiouracil (DTU) for 1 hour. Following the pulse, mRNA was extracted and either used directly in microarray experiments (Abundance Arrays) or biotinylated and purified to select DTU labeled RNAs that were then used in microarray experiments (Synthesis Arrays). Two independent tachyzoite and bradyzoite preparations were made and duplicate microarrays were performed for each sample. A development or differentiation experiment design type assays events associated with development or differentiation or moving through a life cycle. Development applies to organism(s) acquiring a mature state, and differentiation applies to cells acquiring specialized functions. Keywords: development_or_differentiation_design

ORGANISM(S): Toxoplasma gondii

PROVIDER: GSE2946 | GEO | 2005/07/16

SECONDARY ACCESSION(S): PRJNA104763

REPOSITORIES: GEO

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Synthesis vs. Abundance, Bradyzoite Development

Project description:Parasites grown as either tachyzoites or bradyzoites for 40 hours were pulsed with 2,4-dithiouracil (DTU) for 1 hour. Following the pulse, mRNA was extracted and either used directly in microarray experiments (Abundance Arrays) or biotinylated and purified to select DTU labeled RNAs that were then used in microarray experiments (Synthesis Arrays). Two independent tachyzoite and bradyzoite preparations were made and duplicate microarrays were performed for each sample. A development or differentiation experiment design type assays events associated with development or differentiation or moving through a life cycle. Development applies to organism(s) acquiring a mature state, and differentiation applies to cells acquiring specialized functions. User Defined

2005-07-16 | E-GEOD-2946 | biostudies-arrayexpress

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Project description:5 arrays per condition (covering 3 biological replicate experiments) were performed on Human Foreskin Fibroblasts (HFFs) at 44 hours post infection. The four experimental conditions include uninfected "standard", uninfected "stress", tachyzoite-infected "standard+TZ", and bradyzoite-infected "stress+BZ". Bradyzoite conversion is induced at 4 hours post infection with replacement of standard DMEM+10% FCS with RPMI+1%FCS buffered with 50mM Hepes to pH 8.15. Approximately 20% of cells are infected and bradyzoite conversion is >90% in these experiments. These data are normalized using 2-D loess (span factor .4). The calculations in the manuscript are based on are log2 ratios of normalized channel 2/channel 1 medians. A development or differentiation experiment design type assays events associated with development or differentiation or moving through a life cycle. Development applies to organism(s) acquiring a mature state, and differentiation applies to cells acquiring specialized functions. Developmental Stage: Uninfected (standard or stress medium), or Tachyzoite-infected (standard medium) or Bradyzoite-infected (stress medium) Keywords: development_or_differentiation_design

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Transcription profiling of parasites grown as either tachyzoites or bradyzoites for 40 hours pulsed with 2,4-dithiouracil (DTU) for 1 hour

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Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase. An RNA stablity experiment design type examines stability and/or decay of RNA transcripts. Keywords: RNA_stability_design

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The protein phosphatase 2A holoenzyme is a key regulator of starch metabolism and bradyzoite differentiation in Toxoplasma gondii

Project description:Phenotypic switching from tachyzoite to bradyzoite and vice versa is the fundamental mechanism underpinning the pathogenicity and adaptability of the protozoan parasite Toxoplasma gondii. Accumulation of cytoplasmic starch granules is a hallmark of the quiescent bradyzoite stage. The regulatory factors and mechanisms that contribute to amylopectin storage in bradyzoites remain incompletely known. Here, we show that T. gondii protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit (PP2A-C), a structural subunit (PP2A-A) and a regulatory subunit (PP2A-B). Disruption of any of these subunits increased starch accumulation and disrupted the parasite differentiation. The putative PP2A holoenzyme substrates were identified by phosphoproteomics. PP2A contributes to the regulation of amylopectin metabolism via dephosphorylation of calcium-dependent protein kinase 2 at S679. Several putative PP2A substrates were found to play important roles in bradyzoite differentiation. Our findings establish PP2A as an integral component of the regulatory network mediating amylopectin metabolism and tachyzoite-bradyzoite transformation in T. gondii.

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Thiouracil Pulse-chase, Tachyzoites and Bradyzoites mRNA stability data

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Project description:Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set

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