Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites
| Autor: | Makkuni Jayaram, Jay H. Konieczka, Yuri Voziyanov, Andrew L. Paek, Chien-Hui Ma, Swetha Bolusani |
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| Rok vydání: | 2006 |
| Předmět: |
Recombination
Genetic Genetics Binding Sites Base Sequence FLP-FRT recombination Cre recombinase DNA Genomics Biology Genome Interleukin-10 Yeasts DNA Nucleotidyltransferases Escherichia coli Recombinase Humans Site-specific recombinase technology Cre-Lox recombination Site-specific recombination Directed Molecular Evolution Molecular Biology Floxing Plasmids |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkl548 |
| Popis: | As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites. |
| Databáze: | OpenAIRE |
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