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

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.

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References

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

  2. 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

  3. 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

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

  5. 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

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

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

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

  9. 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

  10. 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

  11. 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

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

  13. 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

  14. 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

  15. 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

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

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

  18. 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

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

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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. 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

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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.

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Correspondence to Satoshi Harashima.

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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 (2008). https://doi.org/10.1007/s00253-008-1609-9

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Keywords

  • Deletion Strain
  • Chromosomal Deletion
  • Nonessential Gene
  • Autonomously Replicate Sequence
  • Specific Chromosomal Region