Results: 5

1.
FIG. 3.

FIG. 3. From: Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii .

Northern blot analysis indicates that the ZFP1 message is altered in TBD-6. A total of 10 μg of tachyzoite total RNA was used for Northern blot analysis, and the T. gondii tubulin gene was used as a loading control. In TBD-6, the signal from ZFP1 was more abundant with longer transcripts. ZFP1 transcript levels normalized to loading controls were 198% ± 47% (average ± standard error) in TBD-6 parasites compared to WT.

Padmini Vanchinathan, et al. Infect Immun. 2005 October;73(10):6680-6688.
2.
FIG. 5.

FIG. 5. From: Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii .

ZFP1 is decreased under bradyzoite conditions. (A) Western blot analyses under tachyzoite and bradyzoite (72 h at pH 8.1) conditions. ZFP1 was detected with antisera to recombinant protein, T. gondii anti-tubulin was used as a loading control, and anti-SRS9 was used as a bradyzoite marker. (B) Densitometric analyses of three independently generated tachyzoite and bradyzoite preparations revealed that the relative bradyzoite-to-tachyzoite abundance of ZFP1 was 39% ± 7% (average ± standard error).

Padmini Vanchinathan, et al. Infect Immun. 2005 October;73(10):6680-6688.
3.
FIG. 4.

FIG. 4. From: Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii .

ZFP1 is localized to the parasite nucleolus by the CCHC zinc finger motifs. YFP and HA fusion constructs with the full-length (1 to 444 aa) or short (1 to 141 aa) ZFP1 were transiently transfected into parasites and detected by autofluorescence (YFP) or an anti-HA antibody (HA). In rows 1 to 4, expression of ZFP1 is from a tubulin promoter; in row 5, expression of ZFP1 is from its endogenous promoter. Nuclear localization is with DAPI (row 1), or Hoechst (rows 2 to 4) staining, and by phase overlay (row 5). In rows 2 to 4 the host nucleus also stains with Hoechst.

Padmini Vanchinathan, et al. Infect Immun. 2005 October;73(10):6680-6688.
4.
FIG. 2.

FIG. 2. From: Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii .

Disruption of the ZFP1 upstream region in WT parasites recreates the TBD-6 phenotype. (A) Southern blot analysis shows the expected pattern in WT and ZFP1 upstream mutant parasites. Genomic DNA (10 μg) from each strain was digested with BamHI and probed with a 1.6-kb probe of the ZFP1, which contains an internal BamHI site. In WT parasites, two bands of the expected size were observed. In ZFP1 upstream mutant parasites, homologous insertion of the upstream disruption construct increases the size of the higher band. (B) Bradyzoite conversion of WT, TBD-6, ZFP1 upstream disruption mutant, and a control strain were quantitated after 72 h of bradyzoite induction. WT and the control strain had equivalent conversion efficiencies (>80%); both TBD-6 and ZFP1 upstream disruption mutant had statistically significantly decreased bradyzoite conversion phenotype. ✽, P < 0.05.

Padmini Vanchinathan, et al. Infect Immun. 2005 October;73(10):6680-6688.
5.
FIG. 1.

FIG. 1. From: Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii .

(A) Schematic of the genomic locus disrupted in TBD-6. The position of the mutagenesis vector (164 bp upstream of the transcription start site of ZFP1) and the location of ESTs for ZFP1 are diagrammed. The ESTs include TgEST 95052853 (labeled 1), TgESTzy28g11.r1 (labeled 2), TgEST_100109467 (labeled 3), TgESTzyd19h10.y1 (labeled 4), and TgESTzyj81d05.y2 (labeled 5). The ORFs as predicted by TgTwinscan predictions are listed. (B) The genomic locus of ZFP1 with the 5′ and 3′ untranslated regions, three introns, and the cDNA (as boxes) are diagrammed. The locations of the proteins used to generate recombinant antisera and CCHC motifs are labeled. (C) Protein sequence of ZFP1. The CCHC motifs are in italics, the NLS motifs are underlined, and a box indicates the position of aa 141, up to which was included in the “short” YFP and HA fusion constructs.

Padmini Vanchinathan, et al. Infect Immun. 2005 October;73(10):6680-6688.

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