SITE-SPECIFIC CLEAVAGE OF CHROMOSOMES IN-VITRO THROUGH CRE-LOX RECOMBINATION

Site-specific recombination systems are useful tools for chromosome en gineering in vivo and site-specific DNA cleavage methods have applicat ions in genome analysis and gene isolation. Here, we report a new meth od to fragment chromosomes in vitro using the Cre-lox site-specific re combination system. Two lox sites were targeted into the 5.7 Mb chromo some I of Schizosaccharomyces pombe. In vitro recombination between ch romosomal lox sites and exogenously provided lox oligonucleotides 'cle aved' the chromosome at the defined lox sequences. Site-specific cleav age of lox sites in the tobacco genome was also demonstrated. This rec ombination-based cleavage method provides a novel approach for structu ral and functional analyses of eukaryotic chromosomes as it allows dir ect isolation of chromosome regions that correspond to phenotypes reve aled through Cre-lox mediated chromosome rearrangements in vivo. Moreo ver, recombination with end-labeled lox oligonucleotides would permit the specific end-labeling of chromosome segments to facilitate the lon g range mapping of chromosomes.