Transformation and strain engineering of Tetrahymena.
ABSTRACT Transformation of Tetrahymena by microinjection of DNA was established 25 years ago. This rather labor-intensive technique has since been shelved, replaced by less time consuming and more efficient methods, electroporation and biolistics. Conjugative electroporation is the method of choice for introducing autonomously replicating, rDNA-based vectors into Tetrahymena. These are maintained as high-copy linear mini-chromosomes. Versatile expression cassettes in these vectors facilitate expression of most genes. Transformation efficiencies are sufficiently high to permit screens using expression libraries. Biolistic transformation is primarily used to introduce DNA for integration into the genome by homologous recombination. This technique has greatly enhanced strain engineering of Tetrahymena through facilitating the disruption of genes (creating targeted knockout cell lines) or epitope-tagging coding regions, allowing researchers to take full advantage of the sequenced genome. The presence of both germline and somatic nuclei in these cells requires different strategies to target DNA to the desired compartment. This presents challenges, including the need to engineer the polygenic macronuclear genome, which has nearly 50 copies of each gene. However, separate manipulation of functionally distinct genomes provides experimental opportunities, especially for the analysis of essential genes, by modifying the silent micronucleus then subsequently examining phenotypes in the next sexual generation. The flexibility to engineer strains as needed makes Tetrahymena a facile system with which to answer many biological questions.
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ABSTRACT: The eukaryotic reverse transcriptase, telomerase, adds tandem telomeric repeats to chromosome ends to promote genome stability. The fully assembled telomerase holoenzyme contains a ribonucleoprotein (RNP) catalytic core and additional proteins that modulate the ability of the RNP catalytic core to elongate telomeres. Electron microscopy (EM) structures of Tetrahymena telomerase holoenzyme revealed a central location of the relatively uncharacterized p50 subunit. Here we have investigated the biochemical and structural basis for p50 function. We show that the p50-bound RNP catalytic core has a relatively slow rate of tandem repeat synthesis but high processivity of repeat addition, indicative of high stability of enzyme-product interaction. The rate of tandem repeat synthesis is enhanced by p50-dependent recruitment of the holoenzyme single-stranded DNA binding subunit, Teb1. An N-terminal p50 domain is sufficient to stimulate tandem repeat synthesis and bridge the RNP catalytic core, Teb1, and the p75 subunit of the holoenzyme subcomplex p75/p19/p45. In cells, the N-terminal p50 domain assembles a complete holoenzyme that is functional for telomere maintenance, albeit at shortened telomere lengths. Also, in EM structures of holoenzymes only the N-terminal domain of p50 is visible. Our findings provide new insights about subunit and domain interactions and functions within the Tetrahymena telomerase holoenzyme.Molecular and Cellular Biology 08/2013; · 5.04 Impact Factor
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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 2,000μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200μg/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.Gene 10/2013; · 2.20 Impact Factor
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ABSTRACT: Nucleoporin Nup98, an essential component of the nuclear pore complex, has multifunctional roles in nuclear functions including transcriptional regulation and nucleocytoplasmic transport. These functions mostly depend on a Gly-Leu-Phe-Gly (GLFG) sequence appearing repetitively in the N-terminal region of Nup98. As the GLFG sequence is well conserved among Nup98s from a wide variety of species including humans, yeasts, and ciliates such as Tetrahymena thermophila, a specific antibody that recognizes the GLFG sequence is expected to detect various Nup98s from a wide-range of species. To generate monoclonal antibodies specific to the GLFG repeat of Nup98, we used two synthetic polypeptides derived from the macronuclear Nup98 of T. thermophila as an antigen. We obtained two monoclonal antibodies (MAbs), 13C2 and 21A10, that recognize Nup98s in indirect immunofluorescence staining and Western blot analysis of T. thermophila. Peptide array analysis of these monoclonal antibodies located the position of their epitopes at or near GLFG residues: the epitope recognized by the 13C2 MAb is FGxxN (x being any amino acid), and the epitope recognized by the 21A10 MAb is GLF. As expected by their epitopes, these monoclonal antibodies also recognize Nup98 homologs expressed by human cells and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, indicating that 13C2 and 21A10 MAbs recognize Nup98 epitopes common to phylogenetically distinct organisms. Thus, these MAbs are useful in studying a wide variety of biological phenomena that involve Nup98, ranging from ciliate nuclear dimorphism to NUP98-related human leukemia.Monoclonal antibodies in immunodiagnosis and immunotherapy. 04/2013; 32(2):81-90.