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

  • Minetaka Sugiyama
  • Toshimasa Nakazawa
  • Kiriko Murakami
  • Takahiro Sumiya
  • Atsushi Nakamura
  • Yoshinobu Kaneko
  • Masafumi Nishizawa
  • Satoshi Harashima
Methods
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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.

Keywords

Deletion Strain Chromosomal Deletion Nonessential Gene Autonomously Replicate Sequence Specific Chromosomal Region 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Notes

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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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Minetaka Sugiyama
    • 1
  • Toshimasa Nakazawa
    • 1
  • Kiriko Murakami
    • 3
  • Takahiro Sumiya
    • 2
  • Atsushi Nakamura
    • 2
  • Yoshinobu Kaneko
    • 1
  • Masafumi Nishizawa
    • 3
  • Satoshi Harashima
    • 1
  1. 1.Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuita-shiJapan
  2. 2.Research Institute for Information Science and EducationHiroshima UniversityHigashi-hiroshima-shiJapan
  3. 3.Department of Microbiology and ImmunologyKeio University School of MedicineShinjukuJapan

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