Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae.

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MATa and MATalpha strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation.

Creating a Saccharomyces cerevisiae haploid strain having 21 chromosomes.

Chromosome engineering techniques that can manipulate a large segment of chromosomal DNA are useful not only for studying the organization of eukaryotic genomes but also for the improvement of industrially important strains. Toward the development of techniques that can efficiently manipulate a large segment of chromosome, we have previously reported a one-step chromosome splitting technique in a haploid Saccharomyces cerevisiae cell, with which we could successfully split yeast chromosome 11, XIII, or XI into two halves to create a haploid strain having 17 chromosomes. We have now constructed chromosome splitting vectors bearing ADE2, HIS3, LEU2, or TRP1 marker, and by using these vectors, we could successively split yeast chromosomes to create a novel yeast haploid strain having up to 21 chromosomes.