Skip to main content
SpringerLink
Log in

PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae

  • Methods
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Chromosome rearrangements, especially chromosomal deletions, have been exploited as important resources for functional analysis of genomes. To facilitate this analysis, we applied a previously developed method for chromosome splitting for the direct deletion of a designed internal or terminal chromosomal region carrying many nonessential genes in haploid Saccharomyces cerevisiae. The method, polymerase chain reaction (PCR)-mediated chromosomal deletion (PCD), consists of a two-step PCR and one transformation per deletion event. In this paper, we show that the PCD method efficiently deletes internal regions in a single transformation. Of the six chromosomal regions targeted for deletion by this method, five regions (16 to 38 kb in length) containing 10 to 19 nonessential genes were successfully eliminated at high efficiency. The one targeted region on chromosome XIII that was not deleted was subsequently found to contain sequences essential for yeast growth. While 14 individual genes in this region have been reported to be nonessential, synthetic lethal interactions may occur among these nonessential genes. Phenotypic analysis showed that four deletion strains still exhibited normal growth while possible synthetic growth defects were observed in another strain harboring a 19-gene deletion on chromosome XV. These results demonstrate that the PCD method is a useful tool for deleting genes and for analyzing their functions in defined chromosomal regions.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Amberg DC, Burke DJ, Strathern JN (2005) Methods in yeast genetics. Cold Harbor Spring Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132

    Article  CAS  PubMed  Google Scholar 

  • Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dean N (1995) Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci USA 92:1287–1291

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Giaever G, Chu AM, Ni L, Connelly C, Riles L, Veronneau S, Dow S, Lucau-Danila A, Anderson K, Andre B, Arkin AP, Astromoff A, Bakkoury ME, Bangham R, Benito R, Brachat S, Campanaro S, Curtiss M, Davis K, Deutschbauer A, Entian KD, Flaherty P, Foury F, Garfinkel DJ, Gerstein M, Gotte D, Guldener U, Hegemann JH, Hempel S, Herman Z, Jaramillo DF, Kelly DE, Kelly SL, Kotter P, LaBonte D, Lamb DC, Lan N, Liang H, Liao H, Liu L, Luo C, Lussier M, Mao R, Menard P, Ooi SL, Revuelta JL, Roberts CJ, Rose M, Ross-Macdonald P, Scherens B, Schimmack G, Shafer B, Shoemaker DD, Sookhai-Mahadeo S, Storms RK, Strathern JN, Valle G, Voet M, Volckaert G, Wang CY, Ward TR, Wilhelmy J, Winzeler EA, Yang Y, Yen G, Youngman E, Yu K, Bussey H, Boeke JD, Snyder M, Philippsen P, Davis RW, Johnston M (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391

    Article  CAS  PubMed  Google Scholar 

  • Gietz RD, Schiestl RH (1995) Transforming yeast with DNA. Methods Mol Cell Biol 5:255–269

    Google Scholar 

  • Giga-Hama Y, Tohda H, Takegawa K, Kumagai H (2007) Schizosaccharomyces pombe minimum genome factory. Biotechnol Appl Biochem 46:147–155

    Article  CAS  PubMed  Google Scholar 

  • Gong M, Rong YS (2003) Targeting multi-cellular organisms. Curr Opin Genet Dev 13:215–220

    Article  CAS  PubMed  Google Scholar 

  • Gueldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassete for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524

    Article  CAS  Google Scholar 

  • Hirano Y, Sugimoto K (2007) Cdc13 telomere capping decreases mec1 association but does not affect tel1 association with DNA ends. Mol Biol Cell 18:2026–2036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hirashima K, Iwaki T, Takegawa K, Giga-Hama Y, Tohda H (2006) A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast. Nucleic Acids Res 34:e11

    PubMed  PubMed Central  Google Scholar 

  • Kawasaki H, Ouchi K (1994) A DNA construct useful for specific chromosome loss in Saccharomyces cerevisiae. J Ferment Bioeng 77:125–130

    Article  CAS  Google Scholar 

  • Kolisnychenko V, Plunkett G III, Herring CD, Feher T, Posfai J, Blattner FR, Posfai G (2002) Engineering a reduced Escherichia coli genome. Genome Res 12:640–647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar A, Harrison PM, Cheung KH, Lan N, Echols N, Bertone P, Miller P, Gerstein MB, Snyder M (2002) An integrated approach for finding overlooked genes in yeast. Nat Biotechnol 20:58–63

    Article  CAS  PubMed  Google Scholar 

  • Murakami K, Tao E, Ito Y, Sugiyama M, Kaneko Y, Harashima S, Sumiya T, Nakamura A, Nishizawa M (2006) Large scale deletions in the Saccharomyces cerevisiae genome create strains with altered regulation of carbon metabolism. Appl Microbiol Biotechnol 75:589–597

    Article  Google Scholar 

  • Murray AW, Claus TB, Szostak JW (1988) Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae. Mol Cell Biol 8:4642–4650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pennock E, Buckley K, Lundblad V (2001) Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104:387–396

    Article  CAS  PubMed  Google Scholar 

  • Silberstein S, Schlenstedt G, Silver PA, Gilmore R (1998) A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum. J Cell Biol 143:921–933

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Su H, Wang X, Bradley A (2000) Nested chromosomal deletions induced with retroviral vectors in mice. Nature 24:92–95

    CAS  Google Scholar 

  • Sugiyama M, Ikushima S, Nakazawa T, Kaneko Y, Harashima S (2005) PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae. BioTechniques 38:909–914

    Article  CAS  PubMed  Google Scholar 

  • Taneja V, Paul S, Ganesan K (2004) Directional ligation of long-flanking homology regions to selection cassettes for efficient targeted gene-disruption in Candida albicans. FEMS Yeast Research 4:841–847

    Article  CAS  PubMed  Google Scholar 

  • Vega LR, Mateyak MK, Zakian VA (2003) Getting to the end: telomerase access in yeast and humans. Nat Rev Mol Cell Biol 4:948–959

    Article  CAS  PubMed  Google Scholar 

  • Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808

    Article  CAS  PubMed  Google Scholar 

  • Winston F, Dollard C, Ricupero-Hovasse S (1995) Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11:53–55

    Article  CAS  PubMed  Google Scholar 

  • Yamagishi K, Sugiyama M, Kaneko Y, Harashima S (2008) Conditional chromosome splitting in Saccharomyces cerevisiae using the homing endonuclease PI-SceI. Appl Microbiol Biotechnol 79:699–706 doi:https://doi.org/10.1007/s00253-008-1465-7

    Article  CAS  PubMed  Google Scholar 

  • Yu BJ, Sung BH, Koob MD, Lee CH, Lee JH, Lee WS, Kim MS, Kim SC (2002) Minimization of the Escherichia coli genemo using a Tn5-targeted Cre/loxP excision system. Nat Biotechnol 20:1018–1023

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by the Senri Life Science Foundation and was carried out as a part of the Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of METI and NEDO.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka, 565-0871, Japan

    Minetaka Sugiyama, Toshimasa Nakazawa, Yoshinobu Kaneko & Satoshi Harashima

  2. Research Institute for Information Science and Education, Hiroshima University, 1-4-2 Kagamiyama, Higashi-hiroshima-shi, Hiroshima, 739-8511, Japan

    Takahiro Sumiya & Atsushi Nakamura

  3. Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan

    Kiriko Murakami & Masafumi Nishizawa

Authors
  1. Minetaka Sugiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toshimasa Nakazawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kiriko Murakami
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takahiro Sumiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Atsushi Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yoshinobu Kaneko
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Masafumi Nishizawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Satoshi Harashima
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Satoshi Harashima.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sugiyama, M., Nakazawa, T., Murakami, K. et al. PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae . Appl Microbiol Biotechnol 80, 545–553 (2008). https://doi.org/10.1007/s00253-008-1609-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1609-9

Keywords

Search

Navigation