The prokaryotic beta-recombinase catalyzes site-specific recombination in mammalian cells.
The development of new strategies for the in vivo modification of eukaryotic genomes has become an important objective of current research. Site-specific recombination has proven useful, as it allows controlled manipulation of murine, plant, and yeast genomes. Here we provide the first evidence that the prokaryotic site-specific recombinase (beta-recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments. beta-Recombinase, encoded by the beta gene of the Gram-positive broad host range plasmid pSM19035, has been functionally expressed in eukaryotic cell lines, demonstrating high avidity for the nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence. In simian COS-1 cells, transient beta-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA substrate. The same result was obtained in a recombination-dependent lacZ activation system tested in a cell line that stably expresses the beta-recombinase protein. In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient beta-recombinase expression also promoted deletion of the intervening DNA, independently of the insertion position of the target sequences. The utility of this new recombination tool for the manipulation of eukaryotic genomes, used either alone or in combination with the other recombination systems currently in use, is discussed.
Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.
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V
Díaz
F
F
Rojo
C
C
Martínez-A
J C
JC
Alonso
A
A
Bernad
D000818:Animals; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D016678:Genome; D016680:Genome, Bacterial; D016681:Genome, Fungal; D019426:Integrases; D045522:Recombinases; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004271:DNA, Fungal; D045522:Recombinases; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1