New insights into host factor requirements for prokaryotic beta-recombinase-mediated reactions in mammalian cells.
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.
Diaz
V
V
Departamento de Inmunologia y Oncologia and Biotecnologia Microbiana, Centro Nacional de Biotecnologia, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.
Servert
P
P
Prieto
I
I
Gonzalez
M A
MA
Martinez-A
C
C
Alonso
J C
JC
Bernad
A
A
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006094:Gram-Positive Bacteria / Q000201:enzymology; D049452:Green Fluorescent Proteins; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D019426:Integrases; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D045522:Recombinases; D011995:Recombination, Genetic; D014162:Transfection
D006609:High Mobility Group Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D045522:Recombinases; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1