CHROMOSOME ENGINEERING IN SACCHAROMYCES-CEREVISIAE BY USING A SITE-SPECIFIC RECOMBINATION SYSTEM OF A YEAST PLASMID

We have developed an effective method to delete or invert a chromosomal segment and to create reciprocal recombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specific recombination system of pSR1, a circular cryptic DNA plasmid resembling 2μm DNA of S. cerevisiae but originating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing the specific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or two different chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with a plasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placed downstream of the GAL1 promoter. When the transformants were cultivated in galactose medium, the recombination enzyme produced by expression of the R gene created the modified chromosome(s) by recombination between two specific recombination sites inserted on the chromosome(s).