Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A nucleolar AAA-NTPase is required for Toxoplasma gondii division

ABSTRACT: 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).

ORGANISM(S): Toxoplasma gondii

SUBMITTER: Elena Suvorova

PROVIDER: E-GEOD-43784 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling of FV-P6 mutant parasites at 35C and 40C, as well as FV-P6 complemented with a wild-type Cactin encoding cosmid (TOXO93 or comp) and as a pseudo-diploid wild-type line encoding both the wild-type and mutant Cactin locus (S4.2), also at both temperatures. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. Since TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development. The role of Cactin in G1 progression of Toxoplasma gondii tachyzoites was assessed by expression profiling a temperature senstive mutant of Cactin at both the permissive (35C) as well as the restrictive (40C) temperature. Controls under the same temperature conditions inlcude the mutant line complemented with a wild-type Cactin allele encoded on a cosmid and a wild-type parasite line expressing an additional, mutant allele of Cactin (pseudo-diploid line).

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A nucleolar AAA-NTPase is required for Toxoplasma gondii division

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