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

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)545-553
Number of pages9
JournalApplied Microbiology and Biotechnology
Volume80
Issue number3
DOIs
Publication statusPublished - 2008 Sep

Fingerprint

Haploidy
Polymerase chain reaction
Yeast
Saccharomyces cerevisiae
Genes
Polymerase Chain Reaction
Chromosomes
Growth
Gene Deletion
Functional analysis
Yeasts
Genome
Defects

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering
  • Microbiology (medical)

Cite this

Sugiyama, M., Nakazawa, T., Murakami, K., Sumiya, T., Nakamura, A., Kaneko, Y., ... Harashima, S. (2008). PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 80(3), 545-553. https://doi.org/10.1007/s00253-008-1609-9

PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae. / Sugiyama, Minetaka; Nakazawa, Toshimasa; Murakami, Kiriko; Sumiya, Takahiro; Nakamura, Atsushi; Kaneko, Yoshinobu; Nishizawa, Masafumi; Harashima, Satoshi.

In: Applied Microbiology and Biotechnology, Vol. 80, No. 3, 09.2008, p. 545-553.

Research output: Contribution to journalArticle

Sugiyama, M, Nakazawa, T, Murakami, K, Sumiya, T, Nakamura, A, Kaneko, Y, Nishizawa, M & Harashima, S 2008, 'PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae', Applied Microbiology and Biotechnology, vol. 80, no. 3, pp. 545-553. https://doi.org/10.1007/s00253-008-1609-9
Sugiyama, Minetaka ; Nakazawa, Toshimasa ; Murakami, Kiriko ; Sumiya, Takahiro ; Nakamura, Atsushi ; Kaneko, Yoshinobu ; Nishizawa, Masafumi ; Harashima, Satoshi. / PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae. In: Applied Microbiology and Biotechnology. 2008 ; Vol. 80, No. 3. pp. 545-553.
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