Fig. 3

Tetrahymena Cac2 and Hir1 knockout analysis. A Left, Schematic representation of homologous recombination-mediated gene replacement strategy. The gene targeting vector carries a NEO drug marker which is flanked by 1 kb of DNA that shares sequence identity to upstream and downstream regions of the gene of interest. Right, RT-PCR analyses of ∆HIR1 and ∆CAC2 strains in comparison with wildtype Tetrahymena cells. The positions of the primers encompassing exon–exon junctions are indicated for both ∆HIR1 and ∆CAC2. Bands were observed at the expected sizes. Primers specific to unrelated genes were used as loading controls. B Indirect immunofluorescence analysis of Hir1^Tt-, Cac2^Tt-, and Asf1^Tt-FZZ in growing Tetrahymena. DAPI was used to stain the nuclei, and the positions of the MAC and MIC are indicated with arrows and arrowheads, respectively. C Indirect immunofluorescence analysis of RebL1-FZZ in dividing cells during Tetrahymena vegetative growth. DAPI was used to stain the nuclei, and the positions of the MAC and MIC are indicated with arrows and arrowheads, respectively. RebL1 localization at different cell cycle stages is also indicated as a cartoon in the left panel