Skip to main content
SpringerLink
Log in

Generation of expression vectors for high-throughput functional analysis of target genes in Schizosaccharomyces pombe

  • Articles
  • Published:
The Journal of Microbiology Aims and scope Submit manuscript

Abstract

An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, urw4 + and Saccharomyces cerevisiae LEU2 complementing leul. These vectors contain 3 different strengths of the inducible promoter nmtl, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adams, C., D. Haldar, and R.T. Kamakaka. 2005. Construction and characterization of a series of vectors for Schizosaccharomyces pombe. Yeast 22, 1307–1314.

    Article  CAS  PubMed  Google Scholar 

  • Alberti, S., A.D. Gitler, and S. Lindquist. 2007. A suite of Gateway® cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913–919.

    Article  CAS  PubMed  Google Scholar 

  • Alting-Mees, M.A., E.P. Risseeuw, E. Liu, M. Desautels, W.A. Crosby, and S.M. Hemmingsen. 2006. Intracellular expression of recombinant antibody fluorescent protein fusions for localization of target antigens in Schizosaccharomyces pombe. Methods Mol. Biol. 313, 97–105.

    CAS  PubMed  Google Scholar 

  • Apolinario, E., M. Nocero, M. Jin, and C.S. Hoffman. 1993. Cloning and manipulation of the Schizosaccharomyces pombe his7 + gene as a new selectable marker for molecular genetic studies. Curr. Genet. 24, 491–495.

    Article  CAS  PubMed  Google Scholar 

  • Basi, G., E. Schmid, and K. Maundrell. 1993. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123, 131–136.

    Article  CAS  PubMed  Google Scholar 

  • Bischoff, J.R., D. Casso, and D. Beach. 1992. Human p53 inhibits growth in Schizosaccharomyces pombe. Mol. Cell. Biol. 12, 1405–1411.

    CAS  PubMed  Google Scholar 

  • Campbell, R.E., O. Tour, A.E. Palmer, P.A. Steinbach, G.S. Baird, D.A. Zacharias, and R.Y. Tsien. 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882.

    Article  CAS  PubMed  Google Scholar 

  • Chung, K.S., Y.J. Jang, N.S. Kim, S.Y. Park, S.J. Choi, J.Y. Kim, J.H. Ahn, H.J. Lee, J.H. Lim, J.H. Song, J.H. Ji, J.H. Oh, K.B. Song, H.S. Yoo, and M. Won. 2007. Rapid screen of human genes for relevance to cancer using fission yeast. J. Biomol. Screen 12, 568–577.

    Article  CAS  PubMed  Google Scholar 

  • Chung, K.S., M. Won, S.B. Lee, Y.J. Jang, K.L. Hoe, D.U. Kim, J.W. Lee, K.W. Kim, and H.S. Yoo. 2001. Isolation of a novel gene from Schizosaccharomyces pombe: stm1 + encoding a seven-transmembrane loop protein that may couple with the heterotrimeric Gα2 protein, Gpa2. J. Biol. Chem. 276, 40190–40201.

    CAS  PubMed  Google Scholar 

  • Das, S., J.E. Dixon, and W. Cho. 2003. Membrane-binding and activation mechanism of PTEN. Proc. Natl. Acad. Sci. USA 100, 7491–7496.

    Article  CAS  PubMed  Google Scholar 

  • Forsburg, S.L. 1993. Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res. 21, 2955–2956.

    Article  CAS  PubMed  Google Scholar 

  • Forsburg, S.L. and D.A. Sherman. 1997. General purpose tagging vectors for fission yeast. Gene 191, 191–195.

    Article  CAS  PubMed  Google Scholar 

  • Grimm, C. and J. Kohli. 1988. Observations on integrative transformation in Schizosaccharomyces pombe. Mol. Gen. Genet. 215, 87–93.

    Article  CAS  PubMed  Google Scholar 

  • Ink, B., M. Zornig, B. Baum, N. Hajibagheri, C. James, T. Chittenden, and G. Evan. 1997. Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells. Mol. Cell. Biol. 17, 2468–2474.

    CAS  PubMed  Google Scholar 

  • Jang, Y.J., M. Won, K.S. Chung, D.U. Kim, K.L. Hoe, C. Park, and H.S. Yoo. 1997. A novel protein, Psp1, essential for cell cycle progression of Schizosaccharomyces pombe is phosphorylated by Cdc2-Cdc13 upon entry into G0-like stationary phase of cell growth. J. Biol. Chem. 272, 19993–20002.

    Article  CAS  PubMed  Google Scholar 

  • Lander, E.S., L.M. Linton, B. Birren, C. Nusbaum, M.C. Zody, J. Baldwin, K. Devon, K. Dewar, M. Doyle, W. FitzHugh, R. Funke, D. Gage, K. Harris, A. Heaford, J. Howland, L. Kann, J. Lehoczky, R. LeVine, P. McEwan, K. McKernan, et al. 2001. Initial sequencing and analysis of the human genome. Nature 409, 860–921.

    Article  CAS  PubMed  Google Scholar 

  • Matsuyama, A., A. Shirai, and M. Yoshida. 2008. A series of promoters for constitutive expression of heterologous genes in fission yeast. Yeast 25, 371–376.

    Article  CAS  PubMed  Google Scholar 

  • Moon, D., J.A. Bae, H.J. Cho, and J.H. Yoon. 2008. The fission yeast homologue of Gle1 is essential for growth and involved in mRNA export. J. Microbiol. 46, 422–428.

    Article  CAS  PubMed  Google Scholar 

  • Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823.

    Article  CAS  PubMed  Google Scholar 

  • Ozeki, S., J. Cheng, K. Tauchi-Sato, N. Hatano, H. Taniguchi, and T. Fujimoto. 2005. Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane. J. Cell Sci. 118, 2601–2611.

    Article  CAS  PubMed  Google Scholar 

  • Park, S.H. and R.T. Raines. 2004. Fluorescence polarization assay to quantify protein-protein interactions. Methods Mol. Biol. 261, 161–166.

    CAS  PubMed  Google Scholar 

  • Prasher, D.C. 1995. Using GFP to see the light. Trends Genet. 11, 320–323.

    Article  CAS  PubMed  Google Scholar 

  • Rodrigues, F., M. van Hemert, H.Y. Steensma, M. Corte-Real, and C. Leao. 2001. Red fluorescent protein (DsRed) as a reporter in Saccharomyces cerevisiae. J. Bacteriol. 183, 3791–3794.

    Article  CAS  PubMed  Google Scholar 

  • Sheff, M.A. and K.S. Thorn. 2004. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 21, 661–670.

    Article  CAS  PubMed  Google Scholar 

  • Siam, R., W.P. Dolan, and S.L. Forsburg. 2004. Choosing and using Schizosaccharomyces pombe plasmids. Methods 33, 189–198.

    Article  CAS  PubMed  Google Scholar 

  • Toh-e, A. 1995. Construction of a marker gene cassette which is repeatedly usable for gene disruption in yeast. Curr. Genet. 27, 293–297.

    Article  CAS  PubMed  Google Scholar 

  • Van Driessche, B., L. Tafforeau, P. Hentges, A.M. Carr, and J. Vandenhaute. 2005. Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance. Yeast 22, 1061–1068.

    Article  PubMed  Google Scholar 

  • Venter, J.C., M.D. Adams, E.W. Myers, P.W. Li, R.J. Mural, G.G. Sutton, H.O. Smith, M. Yandell, C.A. Evans, R.A. Holt, J.D. Gocayne, P. Amanatides, R.M. Ballew, D.H. Huson, J.R. Wortman, Q. Zhang, C.D. Kodira, X.H. Zheng, L. Chen, M. Skupski, et al. 2001. The sequence of the human genome. Science 291, 1304–1351.

    Article  CAS  PubMed  Google Scholar 

  • Waddell, S. and J.R. Jenkins. 1995. arg3 +, a new selection marker system for Schizosaccharomyces pombe: Application of ura4 + as a removable integration marker. Nucleic Acids Res. 23, 1836–1837.

    Article  CAS  PubMed  Google Scholar 

  • Yan, Y. and G. Marriott. 2003. Analysis of protein interactions using fluorescence technologies. Curr. Opin. Chem. Biol. 7, 635–640.

    Article  CAS  PubMed  Google Scholar 

  • Zheng, M.Z., L.M. Zheng, and Y.X. Zeng. 2008. SCC-112 gene is involved in tumor progression and promotes the cell proliferation in G2/M phase. J. Cancer Res. Clin. Oncol. 134, 453–462.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Medical Genomics Research Center, KRIBB, Daejeon, 305-806, Republic of Korea

    Jiwon Ahn, Chung-Hae Choi, Kwang-Lae Hoe, Misun Won & Kyung-Sook Chung

  2. Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul, 139-706, Republic of Korea

    Chang-Mo Kang & Chun-Ho Kim

  3. Department of Microbiology, Chungnam National University, Daejeon, 305-764, Republic of Korea

    Jiwon Ahn & Hee-Moon Park

  4. Department of Food Science and Technology, Chungnam National University, Daejeon, 305-764, Republic of Korea

    Kyung-Bin Song

Authors
  1. Jiwon Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chung-Hae Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chang-Mo Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chun-Ho Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hee-Moon Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kyung-Bin Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kwang-Lae Hoe
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Misun Won
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kyung-Sook Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Misun Won or Kyung-Sook Chung.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ahn, J., Choi, CH., Kang, CM. et al. Generation of expression vectors for high-throughput functional analysis of target genes in Schizosaccharomyces pombe . J Microbiol. 47, 789–795 (2009). https://doi.org/10.1007/s12275-009-0010-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-009-0010-4

Keywords

Search

Navigation