Conditional chromosome splitting in Saccharomyces cerevisiae using the homing endonuclease PI-SceI.
A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes.
Yamagishi
Kazuo
K
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.
Sugiyama
Minetaka
M
Kaneko
Yoshinobu
Y
Harashima
Satoshi
S
D020029:Base Pairing; D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D044783:F-Box Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D016615:Telomere / Q000235:genetics; D014170:Transformation, Genetic; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics
D017931:DNA Primers; D044783:F-Box Proteins; C470285:MET30 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific