Apicomplexa
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Project description:Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although still little is known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. Here, we investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase (HDAC) inhibitor. We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi such as TSA and the clinically relevant compound, pyrimethamine. We identify TgHDAC3 as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (non replicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of ~370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDAC inhibitor of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and likely other Apicomplexa.
2009-03-19 | GSE15241 | GEO
Project description:Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although still little is known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. Here, we investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase (HDAC) inhibitor. We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi such as TSA and the clinically relevant compound, pyrimethamine. We identify TgHDAC3 as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (non replicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of ~370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDAC inhibitor of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and likely other Apicomplexa. Freshly released tachyzoites were needle-passed, and filtered using a 3-µm nucleopore membrane. Parasites were resuspended into fresh DMEM supplemented with 10% (v/v) FBS and 25 mM HEPES buffer pH7.2. Parasites were incubated in the presence of FR235222 (40 nM) or DMSO (0.1%) for 4 h at 37°C with 5% CO2. For ChIP-chip experiments freshly released tachyzoites (~5 x 109 at ~12 x 107 parasites/mL) were fixed for 15 min in 1% formaldehyde. Increase in AcH4 signals was verified by immunoblot to verify that FR235222 treatment was effective. To prepare chromatin samples, fixed parasites were lysed in MNase buffer (0.32 M Sucrose, 50 mM Tris-HCl pH7.8, 4 mM MgCl2, 3 mM CaCl2, 100 mM NaCl, 0.25% (v/v) NP40, 5% (v/v) glycerol, protease inhibitor EDTA-free cocktail (Roche)) and DNA was digested for 4 min at 37°C by MNase (2 units/mL). Digestion was stopped with 20 mM EDTA and chromatin was recovered in the soluble fraction after centrifugation at 10,000 g at 4°C; this constituted the S1 fractions. Pelleted materials were resuspended in dialysis buffer (1mM Tris-HCl pH7.8, 0.2 mM EDTA) containing 1 mM PMSF and protease inhibitor cocktail (Roche®) and dialyzed overnight at 4°C against the same solution. Then dialyzed materials were centrifuged and supernatant were harvested; this constitutes the S2 fractions. For chromatin immunoprecipitations, fractions S1 and S2 were pooled and DNA quality was verified by electrophoresis on 2% agarose gels; oligonucleosome ladder of 100-1000 bp were obtained. The histone-DNA complexes were immunoprecipitated with anti-acetyl histone H4 (Upstate®, catalog # 06-866) antibodies according to NimbleGenâs protocol (http://www.genomecenter.ucdavis.edu/expression_analysis/documents).
2010-06-21 | E-GEOD-15241 | biostudies-arrayexpress
Project description:Molecular analysis of Gregarinidae (Apicomplexa) parasites of Orthoptera Schistocerca gregaria and Locusta migratoria
| PRJEB38991 | ENA
Project description:BACKGROUND: In order to contribute to the establishment of a complete map of transcribed regions of the human genome, we constructed a testicular cDNA library for the cynomolgus monkey, and attempted to find novel transcripts for identification of their human homologues. RESULT: The full-insert sequences of 512 cDNA clones were determined. Ultimately we found 302 non-redundant cDNAs carrying open reading frames of 300 bp-length or longer. Among them, 89 cDNAs were found not to be annotated previously in the Ensembl human database. After searching against the Ensembl mouse database, we also found 69 putative coding sequences have no homologous cDNAs in the annotated human and mouse genome sequences in Ensembl.We subsequently designed a DNA microarray including 396 non-redundant cDNAs (with and without open reading frames) to examine the expression of the full-sequenced genes. With the testicular probe and a mixture of probes of 10 other tissues, 316 of 332 effective spots showed intense hybridized signals and 75 cDNAs were shown to be expressed very highly in the cynomolgus monkey testis, but not ubiquitously. CONCLUSIONS: In this report, we determined 302 full-insert sequences of cynomolgus monkey cDNAs with enough length of open reading frames to discover novel transcripts as human homologues. Among 302 cDNA sequences, human homologues of 89 cDNAs have not been predicted in the annotated human genome sequence in the Ensembl. Additionally, we identified 75 dominantly expressed genes in testis among the full-sequenced clones by using a DNA microarray. Our cDNA clones and analytical results will be valuable resources for future functional genomic studies. Keywords: other
2003-07-16 | GSE513 | GEO
Project description:Structural and functional analyses of full-length cDNAs
| PRJNA12621 | ENA
Project description:Project to identify and characterize full-length cDNAs
| PRJNA12757 | ENA
Project description:BACKGROUND: In order to contribute to the establishment of a complete map of transcribed regions of the human genome, we constructed a testicular cDNA library for the cynomolgus monkey, and attempted to find novel transcripts for identification of their human homologues. RESULT: The full-insert sequences of 512 cDNA clones were determined. Ultimately we found 302 non-redundant cDNAs carrying open reading frames of 300 bp-length or longer. Among them, 89 cDNAs were found not to be annotated previously in the Ensembl human database. After searching against the Ensembl mouse database, we also found 69 putative coding sequences have no homologous cDNAs in the annotated human and mouse genome sequences in Ensembl.We subsequently designed a DNA microarray including 396 non-redundant cDNAs (with and without open reading frames) to examine the expression of the full-sequenced genes. With the testicular probe and a mixture of probes of 10 other tissues, 316 of 332 effective spots showed intense hybridized signals and 75 cDNAs were shown to be expressed very highly in the cynomolgus monkey testis, but not ubiquitously. CONCLUSIONS: In this report, we determined 302 full-insert sequences of cynomolgus monkey cDNAs with enough length of open reading frames to discover novel transcripts as human homologues. Among 302 cDNA sequences, human homologues of 89 cDNAs have not been predicted in the annotated human genome sequence in the Ensembl. Additionally, we identified 75 dominantly expressed genes in testis among the full-sequenced clones by using a DNA microarray. Our cDNA clones and analytical results will be valuable resources for future functional genomic studies.
2010-06-05 | E-GEOD-513 | biostudies-arrayexpress
Project description:Large-scale sequencing of full-length cDNAs and clone distribution.
| PRJNA10873 | ENA