To disrupt the MEC-17 gene in Tetrahymena, we used homologous DNA recombination with a fragment carrying the neo4 marker that replaced the coding region. MEC-17 was overexpressed in Tetrahymena using the MTT1 cadmium-dependent promoter. In C. elegans, MEC-12-K40, MEC-12-Q40, and MEC-12-R40 transgenes were introduced into a single site on chromosome II in the EG4322 strain. Animals homozygous for a MEC-12 transgene and homozygous for the mec-12(e1607) allele were obtained by standard crosses. All touch sensation assays in C. elegans were done using blind scoring. To deplete human MEC-17 (C6Orf134) mRNA in Hela cells, we introduced MEC-17-specific siRNAs (ON-TARGETplus pool, Dharmacon) using Oligofectamine (Invitrogen). To knockdown mec17 expression in zebrafish, MOs designed to target the MEC-17 mRNA (Open Biosystems) were injected into early embryos. ATG-MEC17 MO targets the translation initiation site of mec17 mRNA. SP-MEC17 MO targets the exon3/intron3–4 splice junction, and is expected to result in an aberrant splicing isoform of exon2 to exon 4, producing a frameshift mutation and associated protein truncation. As a negative control, we injected MO with a random sequence (oligo-25N, Gene Tools) or a 5bp mismatch to the ATG-MEC17 MO.Live embryos were scored for phenotypes at 48 hpf. To produce a recombinant MEC-17 protein, the cDNA sequence of the murine MEC-17 (BF135007, Open Biosystems) was subcloned into pGEX-3X plasmid (GE Healthcare), expressed in BL21 E. coli cells as a GST fusion and purified using GST-Bind kit (Novagen). The in vitro acetylation assays were performed in 50 mM Tris-HCl pH 8.0, 10 mM glycerol, 0.1 mM EDTA, with purified Tetrahymena MEC-17-KO axonemes or tubulin (purified using DEAE chromatography), recombinant GST-MmMEC-17 enzyme and 10 µM acetyl-CoA. The reaction was detected by western blotting using anti-acetyl–K antibodies.