Figure 3

T. gondii Cdk7, CyclinH and Mat1 interact with each other.

(A) Interactions between TgCdk7, TgCyclinH and TgMat1 were detected by yeast two hybrid analyses. T. gondii CyclinH and Mat1 proteins fused to the GAL4 DNA-binding domain (BD) were expressed in combination with either Cdk7 or CyclinH or Cdk2 fused to the GAL4 activation domain (AD) in yeast strain Y2HGold. Each transformant was spotted in serial dilutions from 10¹ to 10⁻⁴ on SD plates lacking Leu and Trp (DDO); His, Leu and Trp (TDO) and Ade, His, Leu and Trp (QDO). Transformants TgCyclinH-BD/TgCdk7-AD, TgMat1-BD/TgCdk7-AD, TgMat1-BD/TgCyclinH-AD, TgCyclinH-BD/TgCdk2-AD and not the TgMat1-BD/TgCdk2-AD were capable of growth in the absence of Ade and His. The p53-BD/T antigen-AD and Lamin-BD/T antigen-AD were used as a positive and negative control interaction respectively. (B) GST pull down assays were performed using GST beads bound TgCyclinH and GST alone proteins in the presence of His-Cdk7. Western blot analysis using anti-Cdk7 antibody showed the specific binding of TgCyclinH with TgCdk7. The bottom panel showed the Coomassie-stained gel following protein transfer as loading control. (C) Similar interaction experiment using GST beads bound TgCyclinH, TgCdk7 and GST alone proteins in the presence of His-TgMat1 showed the specific interactions of TgCdk7 and TgCyclinH with TgMat1. (D) TgCdk7, TgCyclinH and TgMat1 proteins form a complex in vitro. IP with anti-MBP antibody could pull down His-TgCdk7 and GST-TgCyclinH along with MBP-TgMat1. Proteins eluted from anti-MBP antibody crosslinked agarose beads were subjected to SDS-PAGE and silver staining analysis. (E) Immunofluorescence assay (IFA) in RH strain T. gondii. IFA using anti-Cdk7 and anti-CyclinH antibodies showed colocalization in the parasite nucleus. (F) Similarly, IFA using anti-Cdk7 and anti-Mat1 antibodies showed nuclear colocalization. (G) IF using anti-CyclinH and anti-Mat1 antibodies showed colocalization mostly in the parasite nucleus. DAPI was used to stain the parasite nucleus. Scale bar 5 μm. (H) IP using anti-GFP antibody or control IgG followed by immunoblotting (IB) with anti-Mat1 antibody. TgMat1 co-immunoprecipitated with TgCdk7-YFP using specific anti-GFP antibody, but not with control IgG.