PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae.
Chromosome engineering is playing an increasingly important role in the functional analysis of genomes. A simple and efficient technology for manipulating large chromosomal segments is key to advancing these analyses. Here we describe a simple but innovative method to split chromosomes in Saccharomyces cerevisiae, which we call PCR-mediated chromosome splitting (PCS). The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the CreAoxP system for marker rescue. Using this novel method, chromosomes I (230 kb) and XV (1091 kb) of a haploid cell were split collectively into 10 minichromosomes ranging in size from 29-631 kb with high efficiency (routinely 80%) that were occasionally lost during mitotic growth in various combinations. These observations indicate that the PCS method provides an efficient tool to engineer the yeast genome and may offer a possible approach to identify minimal genome constitutions as a function of culture conditions through further splitting, followed by combinatorial loss of minichromosomes.
Sugiyama
Minetaka
M
Osaka University, Osaka, Japan.
Ikushima
Shigehito
S
Nakazawa
Toshimasa
T
Kaneko
Yoshinobu
Y
Harashima
Satoshi
S
D015139:Blotting, Southern; D015825:Chromosomes, Fungal; D016521:Electrophoresis, Gel, Pulsed-Field; D016681:Genome, Fungal; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*