( Epitope-Tagging Vectors for Functi...)Epitope-Tagging Vectors for Functional Analysis in Yeast

Regulation of gene expression in yeast

Quinn Lu Tanya Hosfield
Stratagene

Stratagene has developed a series of epitope-tagging vectors to express and functionally analyze eukaryotic genes in the budding yeast Saccharomyces cerevisiae. Each of these pESC vectors contains one of four different yeast selective markers in the same vector backbone, which allows expression and epitope-tagging analysis of two different genes in a single yeast cell.

Characterizing the structure and function of every eukaryotic gene is a necessary task in the post genome era. Results of the yeast genome sequencing project indicate that S. cerevisiae shows homologous sequences for one-third of the human disease genes discovered to date.¹ Hence, the yeast makes an attractive model for studying these genes.

We constructed a series of epitope-tagging vectors to facilitate the in vivo study of eukaryotic genes in S. cerevisiae. With these vectors, any cloned gene can be introduced into yeast under the control of a repressible promoter, two different genes can be coexpressed in yeast, protein-protein interactions can be confirmed by immunoprecipitation analysis, and protein localization can be studied. Together with the use of other vectors, the pESC vectors can be used to characterize functional domains of an essential gene and genetically screen for suppressors and inhibitors of certain types of genes in yeast.

pESC Vectors

Figure 1

The pESC vectors (Figure 1) are specifically designed to express high levels of eukaryotic proteins in S. cerevisiae. They are derived from the pRS400 series vectors.² Each pESC vector contains the following sequences: a ColE1ori-amp^R fragment, which allows antibiotic selection and replication of the vector in E. coli for cloning; the 2-m sequence, which provides the origin of replication so that the vector can replicate autonomously in S. cerevisiae; an auxotrophic selectable marker gene (HIS, LEU2, TRP1, or URA3) to select and maintain the expression vector in yeast cells; GAL1 and GAL10 promoters in opposite orientation; a multiple cloning site (MCS); and a transcription termination sequence downstream of each promoter.

LEU2, TRP1, and HIS3 genes encode b-isopropylmalate dehydrogenase, N-(5-phosphoribosyl) anthranilate isomerase, and imidizoleglycerolphosphate dehydratase, respectively. These gene products are required for the biosynthesis of the amino acids leucine, tryptophan, and histidine, respectively. The URA3 gene encodes orotidine-5-phosphate decarboxylase, which is required for uracil biosynthesis. Plasmids bearing URA3 can be counter-selected by plating the cells on media containing 5-FOA (5-fluoroorotic acid). These features facilitate the functional analysis of genes by plasmid shuffling.³

Both the GAL1 and GAL10 promoters from S. cerevisiae are strictly regulated at the transcription level by the carbon source in the media. These promoters are tightly repressed when glucose is present in the media and are highly induced when galactose is the sole carbon source.⁴ In S. cerevisiae, the induction ratio of these promoters has been estimated to be greater than 1000 fold.^5,6 The presence of both the GAL1 and GAL10 promoters in opposite orientation allows two genes to coexpress in the same host cell.

The pESC vectors also contain DNA sequences coding for epitope peptides that can be specifically recognized by monoclonal antibodies. A sequence for the FLAG epitope (DYKDDDDK⁷) is located in the MCS downstream of the GAL1 promoter; a sequence for the c-myc epitope (EQKLISEEDL⁸) is located in the MCS downstream of the GAL10 promoter (Figure 1). The gene of interest can be inserted in front of the epitope sequence to generate C-terminal tagging or be inserted after the epitope sequence to generate N-terminal tagging.

Comparing GAL1 and GAL10 Promoter Strengths

We tested the pESC vectors by inserting the firefly luciferase gene into the MCS downstream of the GAL1 or GAL10 promoter. To compare the promoter activity under repressed and induced conditions, the luciferase gene was individually cloned in the BamH I site or the Not I site of pESC-TRP, so that the gene is under the control of the GAL1 promoter or the GAL10 promoter, respectively. Correct clones were identified by PCR using primers flanking the MCS downstream of the promoters.

The resulting constructs were designated pESC-TRP-Luc (GAL1) and pESC-TRP-Luc (GAL10). Note that in the two constructs, the luciferase gene was not fused to the epitope tags. These two constructs were transformed separately into S. cerevisiae, strain YPH499, and transformants were selected on media lacking the amino acid tryptophan (SD-TRP plates). The recovered transformants were inoculated into selective media with glucose or galactose as the carbon source (i.e., SD-TRP and SG-TRP, respectively). After incubating for 16 hours, at 30C, yeast cells were collected and lysed by vortexing with acid-washed glass beads. The cleared cell lysate was used to measure luciferase activity. In both cases, strong luciferase activity was detected when the promoters were induced (Figure 2, lanes 4 and 6). Only very low levels of luciferase activity were detected when the promoters were repressed (Figure 2, lanes 3 and 5).

Figure 2

This assay demonstrated the following: The carbon source in the media strictly regulated the GAL1 and GAL10 promoters in the pESC vectors, the galactose-induced levels achieved greater than a 1000-fold increase in luciferase activity, and the induced levels were comparable for both promotors.

Epitope Tagging of Luciferase Using pESC Vectors

Epitope tagging is a convenient technique in which a known peptide epitope that is recognized by an antibody is fused to the target protein of interest. With this technique, a tag-specific antibody can be used to detect the fusion protein within a cell, eliminating the need to make specific antibodies to each new protein under study.⁹

To test the FLAG and c-myc epitope tags in the pESC vectors, we cloned the luciferase gene downstream of the GAL promoters such that the gene is fused with c-myc at the C-terminus or with FLAG at the N-terminus. For C-terminal tagging with c-myc, the firefly luciferase gene was PCR amplified and cloned into the BamH I/Sal I sites of the pESC vectors, resulting in pESC-XXX-LucMC constructs. (XXX represents a plasmid backbone with a selectable marker, such as LEU2, HIS3, TRP1, or URA3.) For N-terminal tagging with FLAG, the luciferase gene was cloned in the Bgl II site of the pESC vectors, resulting in pESC-XXX-LucFN constructs. The constructs were transformed into S. cerevisiae, strain YPH499, and the transformants were selected on synthetic dropout plates specific for the particular vector used. For example, transformants with pESC-URA-LucMC or pESC-URA-LucFN were selected on SD-URA plates.

Figure 3

The transformants were grown in selective media with glucose or galactose as the carbon source, and the cleared cell lysate was assayed for luciferase activity. As shown in Figure 3, all eight constructs expressed active luciferase. When pESC-TRP-LucFN or pESC-TRP-LucMC was used, under induced conditions, the activity of the epitope-tagged luciferase showed a level similar to that of the untagged luciferase (Figure 2). The data indicate that the epitope-tagged luciferase retains its biological activity, and the tags do not interfere with the enzyme activity. We consistently observed that the luciferase activities derived from the pESC-URA vector are approximately two- to three-fold less, compared to those derived from other pESC vectors (Figure 3). Lower levels of luciferase expression were most likely due to the URA3 selectable marker contained in the vector. While the HIS3, LEU2, and TRP1 gene products are involved in amino acid biosynthesis, the URA3 gene product is involved in uracil biosynthesis. Marker gene activity has been shown to influence vector maintenance and levels of gene expression in yeast.¹⁰ To detect the epitope-tagged luciferase, we subjected the yeast lysates to Western blot analysis using antibodies specific to the epitope tag. A 64-kDa band, corresponding to the expected size of the epitope-tagged luciferase protein, was detected in samples derived from cells grown in galactose media (Figure 4, even lanes) but not in samples from cells grown in glucose media (Figure 4, odd lanes).

Figure 4

Conclusions

With Stratagenes new series of epitope-tagging vectors to express and functionally analyze eukaryotic genes in S. cerevisiae, high protein levels can be detected using available antibodies against the epitope tags.

Acknowledgments

The authors thank Edward Aranas, Alan Greener, Mary Buchanan, and John Bauer at Stratagene for discussions and suggestions.

REFERENCES

1. Foury, F. (1997) Gene 195: 1-10.

2. Christianson, T. W., et al. (1992) Gene 110: 119-122.

3. Sikorski, R.S. and Boeke, J. (1991) Methods Enzymol. 194: 302-318.

4. Johnston, M. (1977) Microbiol. Rev. 51: 458-476.

5. Marathe, S. V. and McEwen, J. E. (1995) Gene 154: 105-107.

6. Lohr, D., Venkov, P., and Zlatanova, J. (1995) FASEB J. 9: 777-787.

7. Hopp, H.P., et al. (1988) Bio/Technology 6: 1204-1210.

8. Evan, G. I., et al. (1985) Mol. Cell. Biol. 5: 3610-3616.

9. Kolodziej, P. A. and Young, R. A. (1991) Methods Enzymol. 194: 508-519.

10. Zealey, G. R., et al. (1988) Mol. Gen. Genet. 211: 155-159.

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