SAS4 genes in Tetrahymena. (A) Endogenous TTHERM_00194700:HaloTag (grayscale) localizes to oral apparatus and oral primordium cilia, cortical cilia, and to cortical rows. Scale bar, 5 µm. Enlarged panel is rotated 90° counter-clockwise and shows weak cortical row and cilia localization. Scale bar, 1 µm. (B) Phylogenetic analysis of Sas4 proteins, known and predicted (indicated by “pred.”). The SAS4 gene described in this paper (TTHERM_00382220) is closely related to the three previously studied Paramecium SAS4 paralogs, whereas TTHERM_00194700 is distantly related. Phylogenetic tree was created using phylogeny.fr in one-click mode. BLAST analysis of the TTHERM_00194700 protein sequence in the Paramecium genome revealed two additional Paramecium genes, which we identify as Pt_00194700-like(L). The phylogenetic tree shows that the TTHERM_00194700 and Pt_00194700-like proteins cluster together and are distantly related to the Paramecium Sas4 proteins. This supports the hypothesis that TTHERM_00382220 is a Sas4 homologue and that TTHERM_00194700 is not likely a true Sas4 homologue or a paralog to TTHERM_00382220. Dm, Drosophila melanogaster; Mm, Mus musculus; Dr, Danio rerio; Xl, Xenopus laevis; Hs, Homo sapiens; Sp, Strongylocentrotus purpuratus; Tt, T. thermophila; Im, Ichthyophthirius multifiliis; Pt, Paramecium tetraurelia; Cr, Chlamydomonas reinhardtii. (C) Table showing the E-values and percent identities of TTHERM_00382220 and TTHERM_00194700 aligned to the human CPAP protein sequence. (D) Gene expression levels for TTHERM_00382220 and TTHERM_00194700 in different stages of Tetrahymena growth, starvation, and mating (adapted from the Tetrahymena Functional Genomics Database, http://tfgd.ihb.ac.cn). Average of two replicate datasets is shown for each gene. (E) Alignment of human CPAP, TTHERM_00382220, and TTHERM_00194700 amino acid sequence in the C-terminal T complex protein 10 (TCP)/G-box region.