Manipulation of chromosomes in Saccharomyces cerevisiae : Construction of one-gene-chromosome and conditional chromosome splitting with homing endonuclease PI-SceI.
Kazuo Yamagishi, Minetaka Sugiyama, Yoshinobu Kaneko, Satoshi Harashima
Dept. Biotechnology, Osaka Univ., 2-1 Yamadaoka, Suita, 565-0871, Japan
We have improved the yeast chromosome splitting technology by adding H4 ARS sequence into transformed PCR fragment containing centromere and telomere-seed, and succeeded in constructing an extremely short (6 kb) chromosome having only one gene GLC7 (designated GLC7 one-gene-chromosome). This result suggested that the improved method makes it possible to construct mini-chromosome in any length from the yeast genome unless splitting chromosome destroys essential gene. However, since it is known that mini-chromosome with the length less than 100 kb are readily eliminated in mitotic division, the size contraction of mini-chromosome is an obstacle to construct cells harboring various one-gene-chromosomes. In fact, chromosome loss rate of various one-gene-chromosomes tested were 0.2 - 0.4 per cell division. Therefore, we further attempted to develop a conditional splitting method using a homing endonuclease, PI-SceI. Recognition sequence of PI-SceI, which is not present in the yeast genome, was introduced into downstream the GLC7 locus whose upstream site had been split. After induction of PI-SceI by MET3 promoter, about 50 % of clones were found to generate an expected GLC7 one-gene-chromosome. This conditional splitting followed by the loss of one-gene-chromosomes provides a novel tool to determine essential combination of one-gene-chromosomes in particular cultivation condition, which cannot be performed with sequential gene disruption.