|Thomason, L - UC BERKELEY|
|Calendar, Richard - UC BERKELEY|
Submitted to: Molecular Genetics and Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 16, 2001
Publication Date: July 16, 2001
Interpretive Summary: This article describes testing an irreversible type of recombination system derived from phage íC31 for site-specific integration into a simple eukaryotic cell. Insertional and DNA exchange reactions were demonstrated. The DNA exchange reactions were particular effective in placing DNA fragments into the target locus, reaching efficiencies close to that of autonomously replicating plasmid vectors.
Technical Abstract: The Streptomyces bacteriophage íC31 site-specific recombination system was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the íC31 integrase gene. High efficiency transformation to Ura+ occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attB x attP recombination was precise. In another strategy, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe bearing an attP duplication. The íC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision, the reverse reaction, was observed from maintenance of the integrase gene in the integrant lines. The irreversibility of the íC31 site-specific recombination system sets it apart from other systems currently used for eukaryotic cells, which reverse readily. Deployment of the íC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eucaryotic systems.