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Nucleic Acids Research, 1992, Vol. 20, No. 21 5625-5631
© 1992

GENOME STRUCTURE AND MAPPING

Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes

Agnès Thierry and Bernard Dujon

Unité de Génétique Moléculaire des Levures, Département de Biologie Moléculaire (URA 1149 du CNRS), Institut Pasteur 25 rue du Dr Roux, F-75724 Paris-Cedex 15, France

Received August 21, 1992. Revised October 12, 1992. Accepted October 12, 1992.

We have developed a new method for the physical mapping of genomes and the rapid sorting of genomic libraries which is based on chromosome fragmentation by the meganuclease I-Sce I, the first available member of a new class of endonucleases with very long recognition sequences. I-Sce I allows complete cleavage at a single artificially inserted site in an entire genome. Sites can be inserted by homologous recombination using specific cassettes containing selectable markers or, at random, using transposons. This method has been applied to the physical mapping of chromosome XI (620 kb) of Saccharomyces cerevisi and to the sorting of a cosmid library. Our strategy has potential applications to various genome mapping projects. A set of transgenic yeast strains carrying the I-Sce I sites at various locations along a chromosome defines physical intervals against which new genes, DNA fragments or clones can be mapped directly by simple hybridizations.


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