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Eukaryotic Cell, February 2006, p. 422-425, Vol. 5, No. 2
1535-9778/06/$08.00+0     doi:10.1128/EC.5.2.422-425.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Metallothionein Gene from Tetrahymena thermophila with a Copper-Inducible-Repressible Promoter

Francesco Boldrin,^1* Gianfranco Santovito,¹ Jacek Gaertig,² Dorota Wloga,² Donna Cassidy-Hanley,³ Theodore G. Clark,³ and Ester Piccinni¹

Department of Biology, University of Padova, Padova, Italy 35100,¹ Department of Cellular Biology, University of Georgia, Athens, Georgia 30602,² Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853³

Received 28 July 2005/ Accepted 25 October 2005

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We describe a novel metallothionein gene from Tetrahymena thermophila that has a strong copper-inducible promoter. This promoter can be turned on and off rapidly, making it a useful system for induction of ectopic gene expression in Tetrahymena and enhancing its applications in cell and molecular biology, as well as biotechnology.

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The fresh-water ciliate Tetrahymena thermophila is a highly evolved cell with a functional complexity that rivals that of metazoa. Moreover, it combines ease of growth with the availability of advanced genetic tools, which has made T. thermophila a key model for studies of cell and molecular biology (13). More recently, Tetrahymena has shown considerable promise within the biotechnology arena, in particular as a system for the expression of heterologous membrane proteins (4, 5). In 2002, the first inducible-repressible promoter for driving high-level expression of heterologous genes was introduced (11). This promoter belongs to a Cd-inducible metallothionein gene (MTT1) from T. thermophila, and it could be rapidly activated or deactivated by simply adding or depleting cadmium in the growth medium. Metallothioneins (MTs) are ubiquitous, low-molecular-weight, cysteine-rich metal-binding proteins that can be transcriptionally induced by a plethora of stimuli, the most effective being heavy metals, in particular cadmium, zinc, and copper (6). Usually multiple MT isoforms are present in one organism. Indeed, in other Tetrahymena species (T. pyriformis and T. pigmentosa) MT-1 and MT-2 isoforms, induced by Cd and Cu, respectively, have been identified (2, 3, 7, 8, 9, 10).

Based on the coding region of the Tetrahymena pigmentosa MT-2 gene (GenBank accession no. AF479586 [GenBank] ), degenerate PCR was used to amplify the MTT2 coding region of T. thermophila.

MTT2 gene expression was examined by Northern blotting analysis of total RNA extracted from wild-type T. thermophila strain CU428.1 grown in the presence or absence of copper. MTT2 transcript levels were extremely low in the absence of Cu but increased rapidly within 30 min of treatment (Fig. 1A). This increase was followed by a transient down regulation and then a second increase over the next hour. Transcript levels then slowly declined, reaching a plateau at roughly 4 h (Fig. 1A). A rapid decline occurred when copper was withdrawn from the medium. Following overnight growth in 630 µM Cu, no MTT2 transcripts were detectable in cells 30 min after transfer to medium without Cu (Fig. 1B).

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FIG. 1. Northern blot analyses of MTT2 and NRK2-GFP mRNAs in T. thermophila. T. thermophila wild-type strain CU428.1 was grown in SPP (1% proteose peptone, 0.8% glucose, 0.1% yeast extract, 0.003% EDTA ferric sodium salt) medium to log phase (3 x 105 to 5 x 105 cells per ml), and cells were exposed to 630 µM Cu. (A) Time course of induction of MTT2 mRNA. Total RNA (20 µg) from control cells (harvested at 24 h without Cu) and cells treated with Cu for various periods of time was hybridized with a [-32P]dATP-labeled, randomly primed MTT2-specific cDNA probe. The nylon membranes were then stripped and rehybridized with a [-32P]dATP end-labeled oligonucleotide probe against T. thermophila 17S rRNA (12). (B) Cells were treated with 630 µM Cu overnight, washed twice with 10 mM Tris-HCl (pH 7.5), resuspended in SPP medium without Cu, and harvested at the times indicated. (C) Northern blot analysis of NRK2-GFP mRNAs. The plasmid replacement vector pMTT2-NRK2-GFP was biolistically introduced into strain CU522, and macronuclear transformants were selected based on paclitaxel resistance resulting from loss of function at the BTU1 locus. The MTT2-NRK2-GFP strain was grown in SPP medium and exposed to 630 µM Cu. Samples of total RNA (20 µg) were hybridized with a [-32P]dATP-labeled, randomly primed NRK2-GFP probe and rehybridized with a [-32P]dATP end-labeled T. thermophila 17S rRNA oligonucleotide probe. Controls were harvested at 24 h; treated cells were harvested after the indicated times. Northern blots were scanned, and densitometer readings for MTT2 and NRK2-GFP mRNAs, expressed as arbitrary units (a.u.), were normalized against those for T. thermophila 17S rRNA.

To assess whether the 5' region of MTT2 could act as a promoter to drive the expression of other genes, a region 1,456 bp upstream of the start codon in MTT2 was amplified using a genome-walking technique (Clontech). This region was juxtaposed with the coding sequence of a chimeric gene specifying the endogenous NIMA (never in mitosis in Aspergillus nidulans) kinase, NRK2, linked to green fluorescent protein (designated NRK2-GFP). MTT2^P-NRK2-GFP was biolistically introduced into the ß-tubulin 1 (BTU1) locus of T. thermophila strain CU522 by homologous recombination followed by growth in the microtubule-stabilizing drug paclitaxel (5). Expression of chimeric NRK2-GFP gene products in transformed cell lines was investigated by Northern blotting and fluorescence microscopy. As shown by Northern blotting using an NRK2-specific probe, transcripts from the chimeric gene accumulated rapidly, reaching maximal levels 1 h after the addition of Cu to the growth medium and then gradually declining (Fig. 1C). At the protein level, GFP fluorescence was readily visible within transformed cells 2 h after their exposure to Cu (Fig. 2). NRK2-GFP fluorescence was present in the basal bodies and at the tips of cilia (mainly in the anterior region), with the label appearing more evident in dividing cells (Fig. 2A and B). Furthermore, the affected cilia underwent shortening, presumably due to kinase activity associated with the NRK-GFP fusion protein (D. Wloga, unpublished data). Control cells showed only a weak autofluorescence of cytoplasmic granules and vesicles (Fig. 2C). Taken together, these results argue that the region upstream of the start codon of MTT2 can act as a functional promoter when introduced at an ectopic site in the Tetrahymena genome.

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FIG. 2. Fluorescence localization of the chimeric protein NRK2-GFP in transgenic cells. Living MTT2-NRK2-GFP cells were observed by fluorescence (A to C) and Nomarsky contrast (D to F) microscopy. (A, D) Single cells grown in SPP medium and exposed to 630 µM Cu for 2 h; (B, E) treated cells in division phase; (C, F) single untreated cells. Bars, 10 µm.

The overall kinetics of MTT2 gene expression, including the transient decrease following induction by Cu (Fig. 1A), is similar to that described for many organisms, including other Tetrahymena species (10). However, it should be noted that a second Cu-inducible MT isoform encoded within the T. thermophila genome has recently been described (MTT4; GenBank accession no. AY660008 [GenBank] ) (http://www.tigr.org/tdb/e2k1/ttg/). Because the cDNA sequences of MTT2 and MTT4 are nearly identical and because the probe used in Northern blots would not be expected to distinguish them, it is entirely possible that the transcript levels shown in Fig. 1A represent the combined mRNAs of both genes.

From a practical standpoint, use of the MTT2 promoter to drive inducible, high-level expression of heterologous genes in T. thermophila is quite significant. In the case of potentially toxic gene products such as the endogenous NIMA-related kinase NRK2, controlled expression is often a prerequisite for functional assays, as well as for localization studies of expressed proteins. At the same time, overexpression of foreign genes has considerable importance from a biotechnological perspective. We are currently focusing on two broad categories of proteins for overexpression in the Tetrahymena system: (i) vaccine antigens and potential drug targets against protozoan pathogens of humans and animals (in particular, apicomplexans) and (ii) eukaryotic membrane and secretory proteins that are difficult to express in more conventional systems. To date, these studies have utilized the Cd-sensitive MTT1 promoter for gene expression (11). While this promoter is a powerful tool in basic research, it requires the addition of a highly toxic metal to the growth medium, which must be disposed of during the production process (1). Clearly, the availability of alternative promoters for driving inducible expression of foreign genes in T. thermophila would be useful. To our knowledge, only two such promoters exist. The first is a temperature-sensitive promoter from the hsp70 gene of T. thermophila (C. Miceli, personal communication), whose relative strength has yet to be determined. The second is the Cu-dependent promoter from MTT2 reported here.

In conclusion, we have described a new MT gene from Tetrahymena thermophila that has a strong promoter induced by an essential metal, Cu. This promoter can be turned on and off rapidly, making it a good system for induction of ectopic gene expression in Tetrahymena and enhancing its applications in cell and molecular biology, as well as biotechnology. Because MTT2 gene expression requires Cu rather than Cd, its promoter may offer an attractive alternative to the previously described MTT1 promoter for driving foreign gene expression in T. thermophila. Although this will depend to some extent on its relative strength as a promoter, preliminary studies (not shown) clearly suggest that the upstream region of MTT2 can direct high-level expression of heterologous genes.

 
We thank Shay Soker (Wake Forest University School of Medicine, Winston-Salem, NC) for his help with degenerate PCR. We are also indebted to Paola Irato for her essential help with radiochemicals.

This work was partially supported by Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR).

 
* Corresponding author. Mailing address: Department of Biology, Via Ugo Bassi 58/B, 35100 Padova, Italy. Phone: 39-049-827-6310. Fax: 39-049-827-6300. E-mail: francesco.boldrin{at}unipd.it.

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Eukaryotic Cell, February 2006, p. 422-425, Vol. 5, No. 2
1535-9778/06/$08.00+0     doi:10.1128/EC.5.2.422-425.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Boldrin, F. Articles by Piccinni, E. Search for Related Content PubMed PubMed Citation Articles by Boldrin, F. Articles by Piccinni, E.