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Originally published In Press as doi:10.1074/jbc.M011725200 on January 30, 2001

J. Biol. Chem., Vol. 276, Issue 19, 16257-16264, May 11, 2001
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New Insights into Host Factor Requirements for Prokaryotic beta -Recombinase-mediated Reactions in Mammalian Cells*

Vicente DíazDagger , Pilar ServertDagger , Ignacio PrietoDagger , Manuel A. GonzalezDagger , Carlos Martínez-ADagger , Juan C. Alonso§, and Antonio BernadDagger

From the Dagger  Departamento de Inmunología y Oncología and § Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain

The prokaryotic beta -recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in mammalian cells, both on episomic and chromatin-integrated substrates. Using a specific recombination activated gene expression system, we report the site-specific recombination activity of an enhanced green fluorescent protein (EGFP) fused version of beta -recombinase (beta -EGFP). This allows expression of active beta -recombinase detectable in vivo and in fixed cells by fluorescence microscopy. In addition, cellular viability is compatible with a substantial level of expression of the beta -EGFP protein. Using fluorescence-activated cell sorting, we have been able to enrich cell populations expressing this fusion protein. Application of this strategy has allowed us to study in more depth the host factor requirements for this system. Previous work showed that eukaryotic HMG1 protein was necessary and sufficient to help beta -recombinase activity in vitro. The influence of ectopic expression of HMG1 protein in the recombination process has been analyzed, indicating that HMG1 overexpression does not lead to a significant increase on the efficiency of beta -recombinase-mediated recombination both on episomal substrates and chromatin-associated targets. In addition, beta -recombinase-mediated recombination has been demonstrated in HMG1 deficient cells at the same levels as in wild type cells. These data demonstrate the existence of cellular factors different from HMG-1 that can act as helpers for beta -recombinase activity in the eukaryotic environment.

* This work was partially supported by Grants 07/057/96 and 08.6/0021/1997 from the Consejería de Cultura de la Comunidad Autónoma de Madrid, Grant SAF98-0008-CO4-O3 del Plan Nacional de Salud y Farmacia, Comisión Interministerial de Ciencia y Tecnología, and Grant BIO4-CT95-0274 from the Biotechnology Program, EU (to A. B.), and Grant PB96-0817 from Dirección General de Investigación, Ciencia y Tecnología, Grants 2FD97-1692-C02-02 and 08.3/0028/98 from Consejería de Cultura de la Comunidad Autónoma de Madrid (to J. C. A.). The Department of Immunology and Oncology was founded and is supported by the Spanish Research Council (Consejo Superior de Investigaciones Científicas) and the Pharmacia Corporation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 34-91-585-4562; Fax: 34-91-372-0493; E-mail: abernad@cnb.uam.es.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
P. Servert, J. Garcia-Castro, V. Diaz, D. Lucas, M. A. Gonzalez, C. Martinez-A, and A. Bernad
Inducible model for {beta}-six-mediated site-specific recombination in mammalian cells
Nucleic Acids Res., January 3, 2006; 34(1): e1 - e1.
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