title: Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena.
abstract: 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.
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Iwamoto
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan.
authors.forename: Chie
authors.initials: C
authors.lastname: Mori
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan; Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan; Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan. Electronic address: tokuko@nict.go.jp.
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
mesh_terms: D000123:Acetyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D016798:Ciliophora / Q000235:genetics*; D003062:Codon; D004351:Drug Resistance; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D011691:Puromycin / Q000494:pharmacology*; D016808:Tetrahymena thermophila / Q000235:genetics*
chemical_list: D015415:Biomarkers; D003062:Codon; D011691:Puromycin; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase
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