An insertional trap for conditional gene expression in Toxoplasma gondii

Identification of TAF250 as an essential gene

Lauren Jammallo, Keith Eidell, Paul H Davis, Fay J. Dufort, Courtney Cronin, Sivasakthivel Thirugnanam, Thomas C. Chiles, David S. Roos, Marc Jan Gubbels

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma.

Original languageEnglish (US)
Pages (from-to)133-143
Number of pages11
JournalMolecular and Biochemical Parasitology
Volume175
Issue number2
DOIs
StatePublished - Feb 1 2011

Fingerprint

Essential Genes
Toxoplasma
Tetracycline
Gene Expression
Cell Cycle
Chaperonins
Suppressor Genes
Cosmids
Trans-Activators
RNA Polymerase II
Toxoplasmosis
Protein Folding
Growth
DNA Repair
Genes
Plasmids
Phenotype
Proteins

Keywords

  • Cell cycle
  • Chaperonin
  • Genetic screen
  • Invasion
  • TAF250
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

Cite this

An insertional trap for conditional gene expression in Toxoplasma gondii : Identification of TAF250 as an essential gene. / Jammallo, Lauren; Eidell, Keith; Davis, Paul H; Dufort, Fay J.; Cronin, Courtney; Thirugnanam, Sivasakthivel; Chiles, Thomas C.; Roos, David S.; Gubbels, Marc Jan.

In: Molecular and Biochemical Parasitology, Vol. 175, No. 2, 01.02.2011, p. 133-143.

Research output: Contribution to journalArticle

Jammallo, Lauren ; Eidell, Keith ; Davis, Paul H ; Dufort, Fay J. ; Cronin, Courtney ; Thirugnanam, Sivasakthivel ; Chiles, Thomas C. ; Roos, David S. ; Gubbels, Marc Jan. / An insertional trap for conditional gene expression in Toxoplasma gondii : Identification of TAF250 as an essential gene. In: Molecular and Biochemical Parasitology. 2011 ; Vol. 175, No. 2. pp. 133-143.
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