title: 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.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita-shi, Osaka, Japan.
authors.forename: Minetaka
authors.initials: M
authors.lastname: Sugiyama
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Nakazawa
authors.forename: Kiriko
authors.initials: K
authors.lastname: Murakami
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sumiya
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakamura
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Kaneko
authors.forename: Masafumi
authors.initials: M
authors.lastname: Nishizawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Harashima
mesh_terms: D002872:Chromosome Deletion; D015825:Chromosomes, Fungal / Q000235:genetics*; D006238:Haploidy; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D029701:Saccharomyces cerevisiae Proteins