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.

• PMID: 16018552
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Sugiyama M, Ikushima S, Nakazawa T, Kaneko Y, Harashima S

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