Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phi C31 site-specific recombination system

The site-specific recombination system used by the Streptomyces bacteriopha ge phi C31 was tested in the fission yeast Schizosaccharomyces pombe. A tar get strain with the phage attachment site attP inserted at the leu1 locus w as co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4(+) marker, and a second plasmid expressing the phi C 31 integrase gene. High-efficiency transformation to the Ura(+) phenotype o ccurred when the integrase gene was expressed. Southern analysis revealed t hat the attB-ura4(+) plasmid integrated into the chromosomal attP site. Seq uence analysis showed that the attBxattP recombination was precise. In anot her approach, DNA with a ura4(+) marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplicati on. The phi C31 integrase catalyzed two reciprocal cross-overs, resulting i n a precise gene replacement. The site-specific insertions are stable, as n o excision (the reverse reaction) was observed on maintenance of the integr ase gene in the integrant lines. The irreversibility of the phi C31 site-sp ecific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phi C31 reco mbination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.