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New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe
 
Mariana C. Gadaleta1, Osamu Iwasaki2, Chiaki Noguchi1, Ken-ichi Noma2, and Eishi Noguchi1
1Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA
2Department of Gene Expression and Regulation, The Wistar Institute, Philadelphia, PA
BioTechniques, Vol. 55, No. 5, November 2013, pp. 257–263
Full Text (PDF)
Abstract

We describe a series of new vectors for PCR-based epitope tagging and gene disruption in the fission yeast Schizosaccharomyces pombe, an exceptional model organism for the study of cellular processes. The vectors are designed for amplification of gene-targeting DNA cassettes and integration into specific genetic loci, allowing expression of proteins fused to 12 tandem copies of the Pk (V5) epitope or 5 tandem copies of the FLAG epitope with a glycine linker. These vectors are available with various antibiotic or nutritional markers and are useful for protein studies using biochemical and cell biological methods. We also describe new vectors for fluorescent protein-tagging and gene disruption using ura4MX6, LEU2MX6, and his3MX6 selection markers, allowing researchers in the S. pombe community to disrupt genes and manipulate genomic loci using primer sets already available for the widely used pFA6a-MX6 system. Our new vectors may also be useful for gene manipulation in Saccharomyces cerevisiae.

The fission yeast Schizosaccharomyces pombe is an excellent model organism for studying a variety of biological processes (1). The pFA6a-MX6 plasmid is a commonly used backbone plasmid for generating epitope tagging vectors (2, 3). To facilitate protein detection, many vectors are designed to express proteins with tandem copies of epitopes from their endogenous genomic loci. For example, 13xMyc (13Myc), 5xFLAG (5FLAG), and 3xHA (3HA) epitope tagging vectors are available (2, 4). However, a system of tandem Pk epitope tagging vectors for genomic integration has not been reported, although episomal 3xPk (3Pk)-tagging vectors are available (5).

Method Summary

Our 12Pk-tagging vectors for genomic integration significantly improve the sensitivity of protein detection by Western blotting. We also describe genomic 5FLAG-tagging vectors with a glycine linker, which allows flexibility between the epitope and the protein, enhancing immunoprecipitation efficiency. This report also describes vectors for fluorescent-tagging and gene deletion useful in S. pombe.

The Pk epitope, which is also called V5, is a short amino acid epitope with the sequence GKPIPNPLLGLDST from the P and V proteins of the paramyxovirus SV5 (6). Antibodies against the Pk epitope are readily available from commercial sources, and Pk has been widely used for protein purification and detection. In addition, Pk-tagged proteins have successfully been used for chromatin precipitation (ChIP) assay and the related ChIP-seq approach (7). Although some vectors for genomic tagging with the Pk epitope are available in the budding yeast Saccharomyces cerevisiae, they contain only one copy of the epitope (8). Therefore, in order to facilitate Pk-epitope detection, we constructed a series of vectors for genomic tagging with 12 tandem copies of the Pk epitope (12Pk).

To construct Pk-tagging vectors using the pFA6a-MX6 system, the PacI-AscI 3HA fragment in the pFA6a-3HA-kanMX6 plasmid (2) was replaced with an XhoI adaptor sequence. Six copies of a DNA fragment containing two copies of the Pk sequence were introduced into the XhoI site, resulting in the pFA6a-12Pk-kanMX6 plasmid (Figure 1A). The kanamycin-resistant gene (kanMX6) (3) in this plasmid was then replaced with various selection marker genes, including hphMX6 (hygromycin-resistance gene) (9, 10), natMX6 (nourseothricin-resistantce gene) (9, 10), LEU2MX6 (S. cerevisiae LEU2 gene complementing S. pombe leu1 mutations), and ura4MX6 (S. pombe ura4+ gene) (Figure 1A).





These vectors have structures similar to those of pFA6a-MX6-related epitope tagging vectors previously developed by Bähler et al. (2). Therefore, the same PCR primers described in their article can be used to perform the one-step PCR to generate gene-targeting DNA cassettes for 12Pk epitope tagging at the C terminus of a protein. The two-step PCR primers described by Krawchuk and Wahls (11) are also compatible with our constructs. Because our vectors are designed to introduce epitope sequences to the 3′ end of an open reading frame at its own genomic locus, tagged proteins are expressed from their endogenous promoter, allowing us to investigate protein function under physiological conditions.

To confirm expression of 12Pk-tagged proteins, we generated a rad52(3′)-12Pk-kanMX6 cassette using the two-step PCR method (11). In this cassette, the 250 bp genomic DNA upstream of the rad52 stop codon is fused to 12Pk-KanMX6. This gene-targeting DNA cassette was integrated into the rad52 locus of wild-type S. pombe cells, and Rad52–12Pk expression was confirmed (Figure 2A).





Rad52 (previously called Rad22 in S. pombe) is a DNA repair protein and is recruited to DNA damage sites (12). To validate the functionality of the Rad52–12Pk protein, we performed a ChIP assay, showing that Rad52–12Pk associates with the programmed DNA damage site at the mating-type locus in wild-type S. pombe cells (Figure 2B). These results indicate that our vectors are useful for studying the molecular functions of various proteins expressed under physiological conditions.

The number of the Pk epitope-sequence tagged to a protein should influence the sensitivity of protein detection by Western blotting. Indeed, when we constructed cut14–5Pk and cut14–12Pk strains using the method described above, the level of Cut14–12Pk was much higher than that of Cut14–5Pk (Figure 2C), indicating that our 12Pk-tagging vectors improve protein detection sensitivity when compared with previous versions, such as 1Pk and 3Pk-tagging vectors (5, 8).

We previously described a system of vectors for C-terminal 5FLAG tagging (4). Others have found that introducing a linker sequence allows flexibility between the epitope and the protein, enhancing antibody-epitope interaction (13). Therefore, to improve our 5FLAG-tagging system, we introduced 11 glycine sequences (G11) immediately before 5FLAG in the pFA6a-5FLAG-kanMX6 plasmid (4), resulting in pFA6a-G11–5FLAG-kanMX6 (Figure 1B). A shorter version with 9 glycine sequences (G9) is also available (pFA6a- G9–5FLAG-kanMX6) (Figure 1B). The reliability of this plasmid was also confirmed by introducing the G9–5FLAG or G11–5FLAG tag into the C terminus of Trt1, the catalytic subunit of the S. pombe telomerase. We also introduced the 5FLAG tag (without a linker) into the Trt1 C terminus as a control.

As shown in Figure 2D, Trt1–5FLAG (no linker), Trt1-G9–5FLAG, and Trt1-G11–5FLAG expressed from the trt1 locus were detected at similar levels in S. pombe whole cell extract (Figure 2D). This is to be expected because all vectors are designed to express proteins with five tandem copies of the FLAG epitope. It is known that Trt1 immunoprecipitation is inefficient when tagged with Myc immediately after the Trt1 C terminus. However, eight glycine sequences (G8) introduced between the Trt1 and Myc epitopes allow for efficient immunoprecipitation (13). Importantly, Trt1-G9– 5FLAG was precipitated much more efficiently than Trt1–5FLAG (Figure 2D). In addition, the level of immunoprecipitated Trt1 further increased when the G11 linker was used (Figure 2D). Therefore, our pFA6a-G9–5FLAG-kanMX6 and pFA6a-G11–5FLAG-kanMX6 vectors are suitable for protein studies when direct epitope tagging compromises protein conformation and/or function.

To facilitate imaging analyses of proteins in S. pombe, we also constructed vectors for green fluorescent protein (GFP(S65T)) and monomeric red fluorescent protein (mRFP) genomic tagging. For both fluorescent tags, only antibiotic markers (kan, hph, nat) were available to the S. pombe research community. Therefore, we first replaced the PmeI-BglII kanMX6 fragment of pFA6a-GFP-kanMX6 (2) with ura4MX, resulting in pFA6A-GFP-ura4MX6 (Figure 1C). Then, the PacI-AscI GFP fragment of this vector was replaced with a PCR-amplified mRFP sequence, resulting in pFA6a-mRFP-ura4MX6 (Figure 1C). We also constructed pFA6A-GFP-bleMX6 (Figure 1C), providing a more flexible marker selection.

Finally, we describe new vectors for gene deletion in S. pombe based on the commonly used pFA6a-MX6-based plasmids, which contain antibiotic-resistance markers (2, 9, 10). However, these pFA6a-MX6 plasmids do not include nutritional markers. For this reason, the PmeI-BglII kanMX6 fragment of pFA6a-kanMX6 (2) was replaced with PCR-amplified S. cerevisiae LEU2, S. pombe his3+, and ura4+ fragments, generating pFA6a-LEU2MX6, pFA6a-his3MX6, and pFA6a-ura4MX6, respectively (Figure 1D). All of these vectors have structures similar to that of pFA6a-kanMX6, allowing us to conveniently and systematically manipulate genomic loci using primer sets already available for the pFA6a-kanMX system (2, 11).

The kan, hph, and nat genes have also been used in S. cerevisiae (14), and similar pFA6a-MX vectors are available for S. cerevisiae research (3, 8). Therefore many of the vectors described in this report may also be applicable to gene manipulation in S. cerevisiae. The plasmids constructed in this report (Table 1) and their maps will be available through the Addgene web site (http://www.addgene.org).

Table 1. 





Author contributions

Conceived and designed the experiments: MCG, OI, KN, EN. Performed the experiments: MCG, OI, CN. Analyzed the data: MCG, OI, KN, EN. Contributed reagents/materials/analysis tools: MCG, OI, CN, KN, EN. Wrote the paper: MCG, EN.

Acknowledgments

We thank Jürg Bähler for generously donating plasmids, and Jordan Asam, Mukund Das, and Rochelle Vollmerding for technical assistance. We also express special thanks to Roger Y. Tsien for generous permission to use the mRFP containing plasmid. This study was supported by NIH (R01GM077604 to E.N. and DP2-OD004348 to K.N.) and the G. Harold & Leila Y. Mathers Foundation (to K.N). This paper is subject to the NIH public Access Policy.

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Eishi Noguchi, Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102-1192. E-mail: [email protected]">[email protected]

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