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Chromosome- and genome-engineering technologies are becoming increasingly important in biotechnology as well as genome science. Since they have provided powerful approaches to analyze functions of large chromosomal regions or many genes simultaneously and are expected to contribute to breeding of novel microorganisms for a new phase of efficient production of valuable biomaterials, development of a simple and efficient technology for manipulating chromosome and genome is greatly demanded for promoting both basic and applied biosciences. As one of the key technology for manipulating chromosome and genome, we have developed a novel chromosome-engineering technology which we called chromosome-splitting technology. Chromosome-splitting technology is a procedure to cut a chromosome, at any chosen site, into two pieces, but to make them behave as functional chromosomes. Chromosomes must have three elements for their maintenance during mitotic growth and meiotic development, i.e., centromere, telomere, and replication origin. Therefore, if chromosomes are newly generated, whether derived from natural or artificially constructed, they must have these three elements for faithful transmission to daughter cells. Because the Saccharomyces cerevisiae has served as a valuable model eukaryote and also as an extremely useful industrial microorganism, we initiated work in this organism. We have developed the chromosome-splitting method that enables very simple, efficient, and repeatable splitting of chromosome1). This new
method was designated as PCS (PCR-mediated Chromosome Splitting) method. The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the Cre/loxP site-specific recombination system for marker rescue. The PCS method can be used for a wide variety of purposes such as: (1) chromosome shuffling to swap a particular chromosomal region from one strain with the corresponding region from another for exploring genotypephenotype relationships at the sub-chromosomal level, (2) genome reconstruction to create novel strains with a variety of genomic constitutions, (3) one-step deletion of a designed internal or terminal chromosomal region to survey functions of genes in defined chromosomal regions, and (4) application for the manipulation and functional analysis of chromosome fragments of higher eukaryotes cloned into yeast artificial chromosomes2-12). These advances to manipulate chromosome and genome in a large scale are expected to accelerate the breeding of novel strains for biotechnological purposes, and also to reveal functions of presently uncharacterized genes and chromosomal regions in S. cerevisiae.