A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2,000 μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.
Keywords: 3′ UTR; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 6-methylpurine sensitive; 90% inhibitory concentration; BTU2; Ciliate; Gene manipulation; HHF1; HHO1; Hepes; IC90; MTT1; Puromycin-N-acetyltransferase; SEH1; Selection marker; Tetrahymena thermophila; Transformation; beta-tubulin gene; blasticidin S-resistant gene; bsr; chx1-1; cy-s; cycloheximide sensitive; gene mutation expressing 6-methylpurine sensitive; gene mutation expressing cycloheximide sensitive; histone H1 gene; histone H4 gene; metallothionein gene; mp-s; mpr1-1; neo; neomycin-resistant gene; nucleoporin Seh1 gene; pVGF1; pac; plasmid vector for the expression of GFP fusion protein in vegetative Tetrahymena cells; puromycin-N-acetyltransferase gene; three prime untranslated region.
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