Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes
Section snippets
Strains, media, and transformation
S. cerevisiae strain FY834 (17) (MATα his3Δ200 ura3-52 leu2Δ1 lys2Δ202 trp1Δ63) was the principal strain used in the study; other strains that were used are listed in Table 1. Yeast cells were grown on YPDA medium containing 1% yeast extract, 2% peptone, 2% glucose, and 0.04% adenine (Sigma-Aldrich, St. Louis, MO, USA) or SD medium containing 2% glucose and 0.67% Difco™ yeast nitrogen base without amino acids (YNB; BD Biosciences, Sparks, MD, USA). SG medium was also used in this study and
Splitting chromosome IV into 11 new chromosomes
We derived the yeast stock ZN1 (Fig. 1A) in our laboratory from FY834, and after splitting chromosome IV into 2 derivative chromosomes at the LYS4 and LYS14 loci (12; data not shown), we subsequently derived strain ZN3. Using ZN3 as the starting strain, we sought to further split the derivatives of chromosome IV into 11 mini-chromosomes with the PCS method (8). For this purpose, the target sequence was extended to 650 bp using overlap-extension PCR as follows: a 30 base overlap sequence was
Acknowledgments
This work was supported by Grant-in-Aid for Scientific Research (B) 12460044 (to S. H.), (B) 15380064 (to S. H.), Grant-in-Aid for challenging Exploratory Research 19658132 (to S. H.), and Grant-in-Aid for Young Scientists (B) 23780080 (to M. S.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. This work was also carried out as a part of the project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of METI and NEDO.
References (23)
- et al.
A yeast artificial chromosome-splitting vector designed for precise manipulation of specific plant chromosome region
J. Biosci. Bioeng.
(2005) - et al.
Repeated chromosome splitting targeted to delta sequences in Saccharomyces cerevisiae
J. Biosci. Bioeng.
(2003) - et al.
Creating a Saccharomyces cerevisiae haploid strain having 21 chromosomes
J. Biosci. Bioeng.
(2003) - et al.
Construction and characterization of single-gene chromosomes in Saccharomyces cerevisiae
J. Biosci. Bioeng.
(2008) - et al.
A versatile and general splitting technology for generating targeted YAC subclones
Appl. Microbiol. Biotechnol.
(2005) - et al.
Chromosome XII context is important for rDNA function in yeast
Nucleic Acids Res.
(2006) - et al.
A polymerase chain reaction-mediated yeast artificial chromosome-splitting technology for generating targeted yeast artificial chromosomes subclones
Methods Mol. Biol.
(2006) - et al.
Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA
Biotechniques
(2003) - et al.
Large scale deletions in the Saccharomyces cerevisiae genome create strains with altered regulation of carbon metabolism
Appl. Microbiol. Biotechnol.
(2007) - et al.
PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae
Biotechniques
(2005)
Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae
Appl. Microbiol. Biotechnol.
Cited by (18)
Single-chromosome fission yeast models reveal the configuration robustness of a functional genome
2022, Cell ReportsCitation Excerpt :The single-chromosome and two-chromosome budding yeast Saccharomyces cerevisiae strains artificially constructed displayed drastic changes in global chromosome structures, but show little change in gene expression profiles and exhibit no apparent defects in various phenotypes (Luo et al., 2018; Shao et al., 2018). Conversely, increasing chromosome numbers from 16 to 21 or 30 by splitting native chromosomes in Saccharomyces cerevisiae also seems phenotypically inert (Ueda et al., 2012; Widianto et al., 2003). Whether strong tolerance to chromosome configuration changes is a ubiquitous attribute of eukaryotic genomes is unknown.
Design, building, and challenges in synthetic genomics
2022, New Frontiers and Applications of Synthetic BiologyCRISPR-PCD and CRISPR-PCRep: Two novel technologies for simultaneous multiple segmental chromosomal deletion/replacement in Saccharomyces cerevisiae
2020, Journal of Bioscience and BioengineeringLarge fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae
2016, Analytical BiochemistryCitation Excerpt :After several repeated chromosome operations, a high number of foreign sequences that remain may result in chromosome instability. The PCR-mediated approach [3] combines two-step PCR and one transformation per splitting event with the Cre/loxP system for marker rescue, and using this method, chromosomes have been successfully split collectively into minichromosomes that were occasionally lost during mitotic growth in various combinations. This innovative method is simple and efficient, but the minichromosomes have uncertainty.
Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering inSaccharomyces cerevisiae
2015, Journal of Bioscience and Bioengineering
- †
The first two authors contributed equally to this work.
- §
Present address: Central Laboratories for Frontier Technology, KIRIN Holdings Co., Ltd., Yokohama, Kanagawa, Japan.