Genomic hybridizations for the parents and progeny of the Toxoplasma gondii I X III genetic cross
Ontology highlight
ABSTRACT:
ORGANISM(S): Toxoplasma gondii
SUBMITTER: Michael Behnke
PROVIDER: E-GEOD-26607 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:This SuperSeries is composed of the following subset Series: GSE26558: Expression Quantitative Trait Locus (eQTL) Mapping of Stage-specific Gene Expression in Progeny from a type I X III Genetic Cross of Toxoplasma gondii GSE26607: Genomic hybridizations for the parents and progeny of the Toxoplasma gondii I X III genetic cross Refer to individual Series
2012-01-11 | E-GEOD-26608 | biostudies-arrayexpress
Project description:The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages, types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between types I vs. III and II vs. III. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on a new genetic cross and linkage mapping. Genome-wide association revealed a single quantitative trait locus controls the > 4 log difference in lethality between these strains. Neither ROP16 nor ROP18, previously implicated in virulence differences in T. gondii, were found to contribute to differences between types I and II. Instead, the major virulence locus contained a cluster of pseudokinases denoted as rhoptry protein 5 (ROP5); this locus contains a tandem cluster of polymorphic alleles that differed in expression levels between strains. ROP5 alleles contained only part of the catalytic triad of canonical S/T kinases, and consistent with this they lack demonstrable kinase activity in vitro. Genetic disruption of the rop5 locus in the type I lineage lead to a > 5 log increase in the lethal dose, and surviving mice developed lasting immunity and were protected from an otherwise lethal challenge. These findings reveal that amplification of a polymorphic cluster of pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model. We used a new genotyping protocol based on hybridization of parental and recombinant progeny genomic DNA (gDNA) to Affymetrix gene arrays constructed for T. gondii (http://ancillary.toxodb.org/docs/Array-Tutorial.html). We identified 1,603 single feature polymorphisms (SFP) based on probes that successfully discriminated perfect match (type II) from mismatch (type I) hybridization across all clones and provided reliable SFP markers for this cross.
2011-05-03 | E-GEOD-24905 | biostudies-arrayexpress
Project description:PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. We generated T. gondii mutants lacking PRMT1 (∆prmt1) by deletion of the PRMT1 gene. ∆prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the complemented strain ∆prmt::PRMT1mRFP. PRMT1 localizes primarily in the cytoplasm with enrichment at the centrosome, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ∆prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole genome expression profiling demonstrated differences in expression of cell cycle regulated genes in ∆prmt1 relative to the complemented ∆prmt1::PRMT1mRFP and parental wild-type strains, but these changes did not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our genetic studies suggest that the most critical function of PRMT1 is within the centrosome as a regulator of daughter cell counting to assure the proper replication of the parasite. RNA samples were isolated in triplicates from RH-hxgprt parent strain (W), PRMT1 knockout (K) strain and PRMT1 knockout strain complemented with RFP-tagged PRMT1 protein (C). Parasites were grown for 32h at 37C. Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies).
2015-09-18 | E-GEOD-73177 | biostudies-arrayexpress
Project description:The parents and progeny of the I X III genetic cross were genotyped using the ToxoGeneChip in order to generate a more detailed genetic map. We also used the genomic hybridization data to look for copy number variations (CNV) and segmental duplications.
2012-01-11 | GSE26607 | GEO
Project description:The control of gene expression in Toxoplasma is not well understood. As we and others have shown, this organism does contain bipartite promoters that are sufficient to induce gene expression. This information can be used in downstream experiments to identify proteins that bind to experimentally confirmed cis elements. One of these methods, yeast one-hybrid is currently being employed to identify proteins which bind to the BAG1 and B-NTPase promoter elements. Another powerful method that can be used to identify genomic regions containing controllers of gene expression takes advantage of genetic linkage mapping provided by genetic crosses, called expression quantitative trait locus (eQTL) mapping. In the eQTL approach, one uses gene expression as quantitative traits, or phenotypes, in QTL mapping. QTL mapping is a proven method in Toxoplasma that has recently identified apicomplexan-specific virulence factors in both the Type II X III and Type I X III crosses (Saeij et al., 2006, Taylor et al., 2006, Su et al., 2002). With the recent design of the Affymetrix Toxoplasma GeneChip one can gather gene expression profiles for all predicted genes in the Toxoplasma genome in a rapid and reproducible way. Combining the power of Affymetrix gene expression technology and QTL mapping can lead to the identification of genes with differential expression in the parents and progeny of a genetic cross and then to the identification of the genomic regions that contain mechanisms that have an effect on expression. The first description of combining global gene expression profiles with QTL mapping was published by Brem et al. using the model organism Saccharomyces cerevisiae (Brem et al., 2002). Since that time, eQTL projects have been described for several other organisms including human, mouse, rat, maize, Arabidopsis, barley, eucalyptus, and wheat (Gibson & Weir, 2005). There are two mapping location types expected when using gene expression profiles as phenotypes. Mapping of gene expression profiles can lead to linkage of a locus (region) containing the gene being mapped, thus cis to the gene. Although the boundaries of the locus will contain many genes, it is presumed that for most cis QTLs sequence polymorphisms in the regulatory region(s) of the gene being mapped are the cause of the observed expression differences. The other mapping location type expected is one where the expression profile maps to a locus not containing the gene being mapped, or trans to the gene (Ronald et al., 2005). The most interesting trans loci are those that have multiple genes mapping to the same locus in trans, or to a trans hotspot. These hotspots likely contain factor(s) that regulate many genes, such as transcription factors or factors involved in upstream signal transduction pathways that can eventually lead to a number of transcriptional changes (Yvert et al., 2003). Here we describe the results of an eQTL project using the gene expression profiles for the parents and 34 informative progeny of the type I-GT1-Fudr X type III-CTGara cross for both the tachyzoite and bradyzoite stages. This is the first time global gene expression profiles have been used to determine linkage to regions presumed to be controllers of transcription using the Affymetrix Toxoplasma GeneChip. The dominant genomic associations found were those mapping cis and there were several regions, or hotspots, where multiple genes map in trans. In addition, we observed an unexpected finding suggesting there are several duplication events in the parasites of the Type I X III cross which may also have consequences on gene expression levels. The ToxoGeneChip microarray (http://ancillary.toxodb.org/docs/Array-Tutorial.html) was used to measure both tachyzoite and bradyzoite mRNA expression in the parents and progeny of the I X III cross. For each of the two developmental expression sets, genome-wide eQTL scans were run for each gene specific probeset on the microarray. The parents of the cross are CTGara and GT1-Fudr. The rest comprise the informative progeny.
2012-01-11 | E-GEOD-26558 | biostudies-arrayexpress
Project description:Toxoplasma gondii temperature sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. We describe transcriptome of the temperature sensitive mutant 12-109C6. Transcriptome studies demonstrated that gene expression is largely downregulated in the mutant at the restrictive temperature 40oC and is accompanied by a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. RNA samples were isolated in duplicate from mutant 12-109C6 parasites grown for 24h at permissive (34oC) and 6h and 24h at non-permissive temperatures (40oC). Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies, Santa Clara CA).
2013-01-08 | E-GEOD-43315 | biostudies-arrayexpress
Project description:Toxoplasma gondii cell cycle mutant 11-104A4 reversibly arrests in the G1 phase. The defect in this mutant was mapped by genetic complementation to a gene encoding a novel AAA-ATPase/CDC48 family member (TgNoAP1). A change in a tyrosine to a cysteine upstream of an AAA+ domain leads to protein instability and results in cell cycle arrest. This factor is cell cycle regulated and exclusively expressed in the nucleolus during the G1/S phases. At high temperature the mutant quickly arrests with a single nucleus and expresses transcripts enriched in the G1 subtranscriptome. This unique CDC48 ortholog operates in a parasite mechanism responsible for G1 progression and is the first G1-specific checkpoint factor described in Toxoplasma. We describe transcriptome of the temperature sensitive mutant 11-104A4. Transcriptome studies demonstrated that gene expression is reflective of the parasite arrest in G1 phase. mRNAs normally upregulated in S/M phase were largely downregulated in the mutant at the restrictive temperature 40oC. Genetic complementation with extra copy of the wild type TgNoAP1 rescued growth of the mutant 11-104A4 at 40oC and reversed changes in the transcriptome. RNA samples were isolated in duplicate from mutant 11-104A4 parasites grown for 32h at permissive (34oC) or non-permissive temperatures (40oC). In addition, a duplicate sample of RNA from mutant 11-104A4 complemented with cosmid TOXOV53 encoding wild type copy of TgNoAP1 similarly grown at 40oC was collected. Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies).
2013-01-27 | E-GEOD-43784 | biostudies-arrayexpress
Project description:We describe the cell cycle transcriptome of the Toxoplasma gondii that has emerged as a major genetic model for the study of Apicomplexa parasites. Two distinct transcriptional waves accompany the relatively simple binary replication of tachyzoite stage (endodyogeny) functionally separating conserved gene expression in the eukaryotic G1 phase from the lineage-specific expression that predominates in the parasite S phase and mitotic periods. This division of transcriptional focus closely mirrors the intimate relationship that has evolved between mitosis and building of the daughter parasites and invasion organelles. Promoter mechanisms appear to orchestrate the induction of gene expression in dividing tachyzoites with up to two dozen cell cycle AP2 factors likely acting within a transcriptional regulatory network to coordinate the parasite cell cycle transcriptome. To characterize the cell cycle transcriptome of Toxoplasma tachyzoites, we expanded a thymidine-synchrony model to isolate sufficient RNA for microarray expression studies. The ToxoGeneChip microarray (http://ancillary.toxodb.org/docs/Array-Tutorial.html) was used to measure mRNA expression in 13 duplicate samples (R0 to R12) covering 12 hours post-synchronization and nearly two tachyzoite replication cycles.
2010-08-27 | E-GEOD-19092 | biostudies-arrayexpress
Project description:Infection of RAW264.7 cells for 24 hours with 32 Toxoplasma Progeny from a Type II x Type III cross To measure changes in gene expression induced in macropahges upon Toxoplasma infection, we infected RAW 264.7 macrophages in cell culture with one of 32 Toxoplasma parasite progeny from a Type II x Type III cross. RNA was harvested 24 hours post infection. Cells were infected with Toxoplasma parasites, in vitro
2014-06-01 | E-GEOD-56534 | biostudies-arrayexpress
Project description:Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites (tachyzoites), replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. Whole genome expression profiling was carried out using the newly developed Affymetrix ToxoGeneChip (GeneChip Tgondiia520372) in order to analyze the ~8,000 predicted genes in the T. gondii genome of mutants and wild-type, allowing for full-scale expression profiling during bradyzoite differentiation in vitro. RNA from mutant and wild-type parasites was extracted and hybridized to the ToxoGeneChip. We harvested extracellular tachyzoites from freshly lysed fibroblasts (ET, 0h), intracellular tachyzoites (IT, 24h post-invasion) and parasites subjected to bradyzoite growth conditions for 72h (B72, 72h of induction). Extracellular parasites from freshly lysed fibroblasts were harvested for the seven mutant parasite lines (12K, 13P, B7, 11P, 11K, 7K and P11) and mutant parasites subjected to bradyzoite differentiation conditions for 72h. For a few samples we also harvested parasites 11h post egress of host cells. A time course was carried out with wild-type: parasites subjected to bradyzoite growth conditions for 24h, 36h and 48h.
2010-07-27 | E-GEOD-23174 | biostudies-arrayexpress