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Keywords:

  • expression vector;
  • minimal media;
  • nourseothricin-resistance marker;
  • Gateway system;
  • Schizosacchromyces pombe

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In the post-genomic era, an immediate challenge is to assign biological functions to novel proteins encoded by the genome. This challenge requires the use of a simple organism as a genetic tool and a range of new high-throughput techniques. Schizosacchromyces pombe is a powerful model organism used to investigate disease-related genes and provides useful tools for the functional analysis of heterologous genes. To expand the current array of experimental tools, we constructed two series of Sz. pombe expression vectors, i.e. general and Gateway vectors, containing nourseothricin-resistance markers. Vectors carrying nourseothricin-resistance markers possess advantages in that they do not limit the parental strains with auxotrophic mutations with respect to availability for use in clone selection and can be used together with vectors carrying nutrient markers in minimal media. We modified the pSLF173, pSLF273 and pSLF373 vectors carrying a triple haemagglutinin epitope (3×HA) and an Ura4 marker. The vectors described here contain the nmt1 promoter with three different episomal expression strengths for proteins fused with 3×HA, EGFP or DsRed at the N-terminus. These vectors represent an important contribution to the genome-wide investigation of multiple heterologous genes and for functional and genetic analysis of novel human genes. Copyright © 2013 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The fission yeast Schizosaccharomyces pombe is well suited for the study of interspecies gene functions because key cellular pathways of Sz. pombe have been highly conserved in mammalian cells. Overexpression of a heterologous gene in Sz. pombe may cause growth defects or morphological changes by interfering with the cell cycle or basic metabolic pathways. This behaviour can be used as a simple indication of a gene's role in the dysregulation of basic cellular processes (Bharathi et al., 1997; Grallert and Nurse, 1997; Gachet et al., 2005; Chung et al., 2007).

Expression vectors are crucial tools to facilitate functional analysis of heterologous genes in both model organisms and humans. Numerous expression vectors are available to study the functions of ectopic genes, such as the effect of mutation and overexpression in Sz. pombe (Siam et al., 2004; Adams et al., 2005; Van Driessche et al., 2005). Vectors containing auxotrophic markers, including Ura4, His3, Ade6 and LEU2, are available for episomic expression in Sz. pombe (Grimm and Kohli, 1988; Apolinario et al., 1993; Toh-e, 1995; Waddell and Jenkins, 1995; Adams et al., 2005; Ahn et al., 2009). However, plasmids with an auxotrophic marker have the disadvantage of functioning only in strains with the corresponding auxotrophic mutation. In contrast, vectors carrying a dominant drug-resistant marker do not require corresponding auxotrophic mutations and thus can be used for novel experiments in Sz. pombe. While overexpression of a heterologous gene is an effective method for investigating its function, cloning of the gene of interest is the laborious and time-consuming first step. This step is especially challenging in the functional analysis of genes in a genome-wide study, which requires a large number of genes cloned in multiple vectors.

As described in this report, we constructed a series of autonomously replicating vectors containing a dominant drug-resistance gene, nourseothricin acetyltransferase (NAT), and/or a Gateway conversion cassette for easy manipulation of gene cloning. Although the antibiotics geneticin and hygromycin B do not work in minimal defined Edinburgh minimal medium (EMM) containing ammonium chloride as a nitrogen source, the antibiotic nourseothricin works effectively in this medium (Hentges et al., 2005). Thus, vectors carrying a NAT gene possess an advantage in that they can be used together with nutrient marker-carrying vectors in minimal media. For functional studies of ectopic gene expression and facilitated protein purification in Sz. pombe, vectors were constructed to produce proteins fused to different N-terminal tag sequences in Sz. pombe. These vectors could be exploited for high-throughput functional analysis of multiple heterologous genes in Sz. pombe.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Strains and media

The fission yeast Sz. pombe 972 (h, wild-type) and ED665 (h, ade6-M210 leu1-32 ura4-D18) were used for vector validation. Yeast cells were grown in rich medium (YES; 0.5% yeast extract and 3% glucose with supplements), Edinburgh minimal medium (EMM) supplemented with adenine, uracil and/or leucine, and EMMG, which replaced NH4Cl in EMM with 1 g/l sodium glutamate. Media preparation and basic manipulation methods of Sz. pombe were carried out as described by Moreno et al. (1991). Escherichia coli DH5α and DB3.1 strains were used to subclone PCR products and destination cassettes and to amplify the constructed plasmids.

Materials

Antibiotics, hygromycin B (HmB) and nourseothricin (clonNAT) were purchased from GibcoBRL and Werner BioAgents, respectively. The Gateway conversion cassette system and LR clonase enzyme were purchased from Invitrogen (Carlsbad, CA, USA). Restriction endonucleases and Klenow fragments of DNA polymerase I were purchased from Roche (Mannheim, Germany) and NEB (MA, UK). Anti-G3BP1, anti-HA (12CA5), anti-GFP (G6539) and anti-RFP (sc-33353) antibodies were purchased from Roche (Mannheim, Germany), Sigma-Aldrich (St. Louis, MO, USA), and Santa Cruz Biotech (CA, USA), respectively.

Development of expression plasmids with dominant drug-resistant markers

We modified the pSLF173, pSLF273 and pSLF373 vectors, which are widely used for functional genetic analysis in Sz. pombe. The plasmids pSLF173, pSLF273 and pSLF373 were purchased from a collection of recombinant DNA vectors at American type culture collection (ATCC). The hygromycin B-resistant marker (hptMX4) and nourseothricin-resistant marker (natMX4) were amplified by PCR, using the following primers: 5′-GCATGCATTGTTTAGCTTGCCTCGTCCC-3′ and 5′-GCGCATGCCGTTTTCGACACTGGATGGC-3′ from pAG32 (Goldstein and McCusker, 1999); and 5′-GCGCAAGCTTTGTTTAGCTTGCCTCGTCCC-3′ and 5′-GCGCCTGCAGCGTTTTCGACACTGGATGGC-3′ from pAG25 (Goldstein and McCusker, 1999). To construct the pSLF174 vector (natMX4), the PCR-amplified natMX4 fragment was digested with HindIII/PstI to generate two fragments, both of which were recovered. The pSLF173 vector, was also digested with HindIII/PstI into three fragments; two fragments were recovered, while the DNA fragment carrying the Ura4 gene was not. Next, all four fragments transferred from natMX4 and pSLF173 were quadruple-ligated to generate pSLF174 vectors, as shown in Figure 1. The PCR-amplified hptMX4 fragment was inserted into the NsiI/SphI site of the pSLF173 vector (Ura4), yielding the pSLF184 vector (hptMX4).

image

Figure 1. Construction of the expression plasmids carrying dominant drug-resistance markers for Sz. pombe. (A) pSLF174 vector with N-terminal 3×HA and nourseothricin-resistant marker natMX4. (B) pSLF184 vector with N-terminal 3×HA and hygromycin B-resistant marker hptMX4

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To construct vectors with fluorescence tags for translational fusion with a target gene, the DNA fragments of coding sequences were cloned into Sz. pombe ARS-based vectors. Enhanced green fluorescent protein (EGFP) and Discosoma red fluorescent protein (DsRed) were amplified by PCR, using the following primers: 5′-GCTCTCGAGATGGTGAGCAAGGGCGAGGAG-3′ and 5′-TCAGGATCCCTTGTACAGCTCGTCCATGCC-3′ from pEGFP-1 (Clontech, CA, USA); and 5′-GATCTCGAGAT GGCCTCCTCCGAGGACGTC-3′ and 5′-ATCGTCGACGGCGCCGGTGGAGTGGCGGCC-3′ from pDsRed-C1 (Clontech). PCR-generated GFP fragments were inserted into the XhoI/BamHI sites of pSLF174, pSLF274 and pSLF374 vectors to yield the EGFP-translational fusion vectors pSLF176, pSLF276 and pSLF376, respectively. The PCR products of RFP were inserted into the XhoI/SalI site of pSLF174, pSLF274 and pSLF374 vectors to make pREP6X, pREP46X and pREP86X vectors (DsRed). To construct vectors without a tag sequence, the 3×HA tag sequences in pSLF174, pSLF274 and pSLF374 vectors were removed by digestion with XhoI/NotI, replaced with Klenow enzyme and self-ligated, resulting in pREP4X, pREP44X and pREP84X, respectively.

In the Gateway system, target genes in the donor vector are transferred to the destination vector by recombination in the conversion cassettes of both vectors, a process called the LR reaction. To construct destination vectors containing nourseothricin-resistant marker natMX4, we inserted the chloramphenicol/ccdB resistance Gateway cassette B or C in-frame to the GFP or DsRed tags (Figure 3). The ‘B’ conversion cassette was introduced into the SmaI site of the pSLF174 series and the BamHI site of the pSLF176 series, constructing the pDES174 (3×HA) and pDES176 series (EGFP), respectively. The conversion cassette ‘C’ was ligated into SalI-digested pREP6X series vectors to make destination vectors pDES6X series (DsRed). To construct the Gateway vectors without a tag sequence, the pSLF174 series were digested with XhoI/NdeI to remove the 3×HA tag sequence. Fragment ends were filled in with Klenow enzyme and ligated to make the pDES4X series. Proper integration and orientation of the Gateway cassette were confirmed by DNA sequencing.

Target gene cloning in destination vectors and their ectopic expression in Sz. pombe

For easy generation of recombined proteins using the Gateway system in a model organism, the full-length cDNA of human genes was amplified by PCR and cloned into the entry vector pENTR3C. To transfer target genes from the entry to the destination vector for recombined protein expression, 200 ng of each entry and destination vector were mixed with 2 µl of the LR clonase enzyme (Invitrogen, CA, USA) and incubated for 3 h at room temperature. Then, 1 µl protease K was added to the mixture, which was incubated for 10 min at 37°C to terminate the clonase reaction.

The destination vectors containing cloned target genes were introduced into the ED665h auxotroph haploid strain by the lithium acetate transformation method (Moreno et al., 1991). The colonies carrying the recombined vectors were first grown in EMM containing thiamine, which repressed the expression of the ectopic genes, and then were washed three times with distilled water. Cultured cells were transferred to thiamine-free EMM and grown for an additional 12 h to deplete residual thiamine in the cells. For ectopic expression of the target genes, the cells were transferred again to fresh thiamine-free EMM (Forsburg and Sherman, 1997; Chung et al., 2001). The overexpression of cloned genes was confirmed by immunoblot analysis, using G3BP1, HA, GFP and RFP antibodies.

Immunoblot analysis

Cells were lysed by the glass bead method in a lysis buffer (20 mm Tris–HCl, pH 7.5, 10 mm EGTA, 2 mm EDTA, 0.25 m sucrose, 1% NP-40) containing a protease inhibitor cocktail (Complete TM-mini, Roche, Mannheim, Germany) and separated by SDS–PAGE. Immunodetections were performed using monoclonal antibodies against the HA, GFP and DsRed tags, a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotech, CA, USA) and the ECL detection kit (Millipore, MA, USA).

Microscopy

The morphology and fluorescence of the cells were examined using a fluorescence microscope and differential interference contrast microscopy (Carl Zeiss, Oberköchen, Germany). Cells were harvested and resuspended in a small volume of 50 mm citrate/phosphate buffer and immobilized for microscopic image analysis.

Request for plasmids

Send plasmid requests for non-commercial purposes to Kyung-Sook Chung (fax: 82-42-860-4144; e-mail: kschung@kribb.re.kr). Plasmid sequence information is provided in the supporting information on the internet.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Selection of the nourseothricin-resistant marker natMX4 as a dominant drug resistance marker

Simultaneous expression of several exogenous genes using plasmids is an important tool for molecular genetic studies in Sz. pombe. Therefore, the development of various vectors for Sz. pombe is required. Vectors carrying antibiotic resistance markers are a desirable alternative to typical vectors carrying auxotrophic markers. It has been reported that three antibiotic resistance cassettes were used in PCR-based targeted gene disruption and tagging by homologous recombination of chromosomes in Sz. pombe: kanMX encodes the geneticin-resistant marker, natMX4 encodes the nourseothricin-resistant marker and hptMX4 encodes the hygromycin B-resistant marker (Hentges et al., 2005). Other vectors containing drug resistance markers, such as the blasticidin S deaminase gene (BSD) and the bleomycin resistance gene (bleMX6) have also been reported in Sz. pombe (Kimura et al., 1994; Benko and Zhao, 2011). However, only a limited number of episomal vectors containing drug-resistance markers are available in Sz. pombe (Matsuyama and Yoshida, 2012). In the combined use of auxotrophic and antibiotic resistance markers, it would be advantageous to use vectors carrying the antibiotic resistance marker effective in EMM. Therefore, we chose the nourseothricin resistance marker, which functions effectively in EMM (Hentges et al., 2005), and the backbone pSLF173 containing three copies of the influenza virus haemagglutinin (HA) epitope (Field et al., 1988) for the construction of new episomal vectors.

To compare natMX4 with hptMX4, two drug-resistance markers encoding natMX4 and hptMX4 were amplified by PCR (Figure 1). The PCR-amplified fragments were cloned into the pSLF173 vector (Figure 1), resulting in pSLF174 (natMX4) and pSLF184 (hptMX4). It was previously reported that Sz. pombe did not grow in both rich and minimal media at concentrations of 100 µg/ml of nourseothricin (clonNAT). Hygromycin B inhibits the growth of Sz. pombe at a concentration of 100 µg/ml in rich medium, but not in minimal medium. To measure the drug resistance offered from multi-copy plasmid, the growth of cells transformed with pSLF174 (natMX4) and pSLF184 (hptMX4) were tested in a concentration-dependent manner. In rich medium, Sz. pombe cells carrying pSLF174 and pSLF184 grew well with 100–300 µg/ml clonNAT and hygromycin B compared to cells carrying pSF173 (Ura4) (Figure S1). In minimal medium, clonNAT showed a growth-inhibitory effect at concentrations > 100 µg/ml (Figure 2A, C). However, when the plates were incubated for a further 2 weeks at 4°C, only a partial inhibitory effect was seen on cell growth in plates containing 100 µg/ml clonNAT, and the cells did grow, albeit slowly (Figure 2C). On the other hand, Sz. pombe cells carrying pSLF184 (hptMX4) did not show selective growth because hygromycin B was ineffective (lost its activity) in EMM (Figure 2B). We further demonstrated that hygromycin B kept its activity for growth inhibition of Sz. pombe cells in EMMG that replaced NH4Cl in EMM with sodium glutamate (Figure 2D). Thus, we chose the natMX4 marker to construct a series of autonomously replicating vectors with a dominant drug resistance marker for Sz. pombe. Additionally, we confirmed whether the vectors carrying the NAT gene can be used together with the vectors carrying nutrient markers such as Ura4 and/or LEU2 in minimal medium. Auxotrophic mutant ED665 cells were transformed with pSLF173 (Ura4), pSLF175 (LEU2) and/or pSLF174 (natMX4) and spotted onto EMM – UL and EMM + UL plates in the presence or absence of clonNAT. Each gene, NAT, Ura4 and LEU2, is independently worked in minimal medium, implying that NAT and nutrient marker can be used concurrently in minimal medium (Figure 2E).

image

Figure 2. Sensitivity and resistance of Sz. pombe cells to nourseothricin (clonNAT) and hygromycin B (HmB) in minimal medium. (A, B) Wild-type cells (972 h) were transformed with pSLF174 (natMX4) or pSLF184 (hptMX4) plasmids and spread onto EMM or YES in the absence or presence of antibiotics (50, 100 or 200 µg/ml clonNAT or 200 µg/ml HmB). The plates were incubated at 30°C for 2–3 days. (C) Long-term activity of clonNAT; five-fold dilution series of wild-type cells or cells transformed pSLF174 plasmid were spotted onto EMM plates containing either 100 or 200 µg/ml clonNAT. The plates were incubated at 30°C for 3 days (upper panel) or incubated for a further 2 weeks at 4°C (lower panel). (D) Activity of hygromycin B in EMMG medium. Wild-type cells or cells transformed with pSLF184 plasmid were spotted onto EMM or EMMG containing 200 µg/ml HmB. (E) Concurrent use of auxotrophic marker (Ura4 and/or LEU2) and drug resistance marker (natMX4): ED665 cells transformed with pSLF173 (Ura4), pSLF175 (LEU2) and/or pSLF174 (natMX4) and spotted onto EMM-UL or EMM + UL plates in the presence or absence of clonNAT (200 µg/ml)

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Construction of general episomal expression vectors with tag sequences

Analysis of protein expression level and its effect is important in functional genetic analysis. Because the preparation of antibodies against proteins of interest can be lengthy and difficult, epitope tagging of target proteins has become a popular substitute for protein-specific antibodies in facilitating detection, purification and other functional studies. Furthermore, fluorescent tags provide additional evidence of function by revealing subcellular protein localization. EGFP and DsRed tags are both commonly used to determine protein localization and live-cell fluorescence imaging (Rodrigues et al., 2001; Campbell et al., 2002; Sheff and Thorn, 2004). We used both GFP and DsRed to construct new vectors for Sz. pombe.

We generated vectors for fluorescence tagging by eliminating 3×HA tag sequences at the N-termini of pSLF174, pSLF274 and pSLF374, which contain the nourseothricin antibiotic resistance marker, natMX4, and the nmt1 inducible promoter in three different strengths (see Figure 3 and Materials and methods). In place of these 3×HA tag sequences, the coding sequences of fluorescence proteins (EGFP and Ds-Red) were amplified by PCR and cloned into the N-termini of pSLF174, pSLF274 or pSLF374 vectors, which lack HA tags. Proper integration and orientation of these vectors was confirmed by DNA sequencing of the cloned gene. The resulting plasmids are listed in Table 1.

image

Figure 3. Structure of new episomal tagging plasmids containing a nourseothricin-resistant marker for Sz. pombe. (A) General-purpose plasmids with no tag and with tags such as 3×HA, EGFP or DsRed. The vectors contain natMX4 as a selection marker and the nmt1-inducible promoter in three different strengths. (B) Gateway vectors untagged and N-terminal-tagged with 3×HA, EGFP or DsRed

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Table 1. Summary of plasmids generated in this study
NameTagMarkerPromoter strengthCommentsReference
  • *

    HPT, hygromycin B phosphotransferase.

  • **

    These cassettes make the vectors Gateway-compatible by insertion to general purpose vectors.

General cloning vector
pSLF1733×HAUra4High Forsburg and Sherman (1997) (at ATCC)
pSLF273Medium
pSLF373Low
pSLF1843×HAHPT*HighDerived from pSLF173This study
pSLF1743×HANATHighDerived from pSLF173, 273, 373This study
pSLF274MediumThis study
pSLF374LowThis study
pREP4XNoNATHighDerived from pSLF173, 273, 373This study
pREP44XMediumThis study
pREP84XLowThis study
pSLF173EGFPNATHighDerived from pSLF173, 273, 373This study
pSLF173MediumThis study
pSLF173LowThis study
pREP4XDs-RedNATHighDerived from pSLF173, 273, 373This study
pREP4XMediumThis study
pREP4XLowThis study
Destination vectors
pDES1743×HANATHighDerived from pSLF174, 274, 374 /Cassette C**This study
pDES274MediumThis study
pDES374LowThis study
pDES4XNoNATHighDerived from pREP4X, 44X, 84X /Cassette B**This study
pDES44XMediumThis study
pDES84XLowThis study
pDES174EGFPNATHighDerived from pSLF176, 276, 376 /Cassette C**This study
pDES174MediumThis study
pDES174LowThis study
pDES174Ds-RedNATHighDerived from pSLF6X 46X, 86X /Cassette C**This study
pDES174MediumThis study
pDES174LowThis study

Constructions of Gateway expression vectors with tag sequences

A current challenge in the post-genomic era is to assign function to the increasing number of newly discovered genes. A comprehensive analysis including ectopic expression, subcellular localization and functional complementation of heterologous genes in a model organism can provide information to understand their biological functions. Although these approaches can efficiently identify function, the construction of expression vectors is labour-intensive and often complicated by limited restriction enzyme sites. Moreover, genome-wide functional analysis requires copious and time-consuming cloning work.

To address this problem and to develop a high-throughput cloning system for biological screening of target genes in Sz. pombe, we have constructed destination vectors compatible with the donor vector of the Gateway recombination-based cloning system. Destination vectors were constructed by inserting the chloramphenicol/ccdB resistance Gateway cassettes into expression vectors generated for tagging target proteins (see Figure 3 and Materials and methods). The resulting Gateway destination vectors are listed in Table 1.

Validation of the constructed Gateway plasmids

We tested the feasibility of these vectors using the human gene G3BP1, the binding protein 1 of GTPase activating protein (SH3 domain) gene. At first, G3BP1 was cloned in the entry vector to be transferred to various destination vectors of Sz. pombe. The LR reaction combined the entry vector with each of the destination vectors pDES4X (no tags), pDES174 (3×HA), pDES176 (EGFP) and pDES6X (DsRed) (Figure 4A). After introducing each vector into Sz. pombe, we used immunoblot analysis to detect the expression of G3BP1 protein and the expression of N-terminus tagged G3BP1 using antibodies against G3BP1, HA, EGFP and Ds-Red. In all constructs, G3BP1 and N-terminal tagged G3BP1 proteins were expressed successfully (Figure 4B). In addition, human cDNA clone SCC112, a novel nuclear cell cycle regulatory protein gene, was transferred via an entry vector to pDES178–SCC112 by the Gateway LR reaction (Figure 4A). The subcellular localizations of G3BP1 (Ozeki et al., 2005) and SCC112 (Zheng et al., 2008), which have been previously reported, were investigated in Sz. pombe. After 12 h induction of the cloned protein, localization was visualized under fluorescence microscopy (Figure 4C). Our results confirmed those previously described; the fusion protein EGFP–G3BP1 was localized to the cytoplasm, showing a spot-like pattern, whereas EGFP–SCC112 was localized to the nucleus. As a control, GFP fluorescence in cells expressing EGFP alone was distributed throughout both the nuclear and cytoplasmic compartments. For comparison with the auxotrophic marker vector, the Sz. pombe hsp9+ gene was cloned into the vectors pDES175N carrying the LEU2 marker (Ahn et al., 2009) and pDES176 carrying the NAT marker, and the localization of the GFP–hsp9 fusion protein expressed from these vectors, pDES175N–hsp9+ and pDES176–hsp9+, was compared. In agreement with our previous study (Ahn et al., 2012), we observed that GFP–hsp9+ fusion proteins expressed from pDES175N–hsp9+ and pDES176–hsp9+ were localized in the nucleus after heat shock, indicating that these vectors are valuable tools for the production of heterologous proteins, similar to auxotrophic marker vectors in Sz. pombe.

image

Figure 4. Validation of the vectors in Sz. pombe. (A) Practical application of Gateway vectors to test correct fusion. An entry clone containing human G3BP1 was used to generate expression constructs that were either untagged or tagged with HA, EGFP or DsRed. Additionally, an entry clone containing human SCC112 was used to generate GFP-tagged expression constructs to test the correct localization of the fusion protein. (B) Validation of protein tagging by immunoblot analysis, using 30 µg protein in SDS–PAGE. Immunoblot analysis detected appropriately sized fusion proteins with anti-G3BP1, anti-HA, anti-RFP and anti-GFP. (C) The Gateway-generated GFP-tagged proteins localized properly in accordance with previous reports: EGFP–G3BP1 in the cytoplasm and EGFP–SCC112 in the nucleus. As a control, EGFP alone was distributed throughout in both cytoplasm and nucleus. (D) The subcellular localization of GFP–hsp9+ fusion proteins expressed from pDES175N (LEU2 marker, GFP tag) and pDES176 (NAT marker, GFP tag) carrying the hsp9+ gene

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Here, we report the construction of episomal vectors carrying the drug-resistance marker, natMX4, with three different strengths of the nmt1 promoter in minimal medium. We also generated expression vectors containing a destination cassette, which is suitable for high-throughput cloning of target genes using the Gateway system. These vectors were also modified to express target proteins with various tags at the N-terminus, which can be used for affinity binding and functional analysis of target genes. The use of drug-resistance markers in vectors does not limit parental strains with auxotrophic mutations, and the strain can be available for use in clone selection, whereas vectors with auxotrophic markers are at a disadvantage because they function only in strains harbouring the corresponding auxotrophic mutation. Moreover, vectors containing the nourseothricin-resistant marker, natMX4, are particularly advantageous when used with other vectors with auxotrophic markers, because only nourseothricin is effective in minimal medium and can therefore be used for novel experiments involving Sz. pombe. Furthermore, owing to the fact that the entire set of protein-coding open reading frames (ORFeome) of Sz. pombe was generated using a recombination-based cloning system, the genes in these ORFeome clones can be transferred to the destination vector in this study by the Gateway LR reaction (Matsuyama et al., 2006). Therefore, vectors containing a dominant selection marker, natMX4, may contribute to genome-wide investigation of the cellular mechanisms underlying the functions of multiple heterologous genes and help exploit the experimental convenience of Sz. pombe as a model organism.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported in part by Basic Science Research (Grant No. RBM3301213) of the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology, and the Korea Research Council of Fundamental Science and Technology (Grant No. KGM2011211). Plasmids pAG25 and pAG32 were obtained from EUROSCARF.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The following supporting information may be found in the online version of this article:

FilenameFormatSizeDescription
yea_2955_sm_f1.tifTIFF image205KSensitivity and resistance of Sz. pombe cells to nourseothricin (clonNAT) and hygromycin B in rich media
yea_2955_supplementary material_Vector sequences.docxWord 2007 document41KVector sequences: general and destination vectors

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