High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay.
We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.
Tchenio
T
T
Unités de Biochimie-Enzymologie et de Physicochimie Macromoléculaire, Centre National de la Recherche Scientifique U147, Villejuif, France.
Heidmann
T
T
D000818:Animals; D002415:Cats; D002478:Cells, Cultured; D004251:DNA Transposable Elements; D003673:Defective Viruses / Q000235:genetics*; D015683:Gene Products, gag / Q000502:physiology; D015684:Gene Products, pol / Q000502:physiology; D015750:Genes, gag; D015751:Genes, pol; D005816:Genetic Complementation Test; D066298:In Vitro Techniques; D011533:Proviruses / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D014162:Transfection; D016662:Virus Integration; D001616:beta-Galactosidase / Q000235:genetics
D004251:DNA Transposable Elements; D015683:Gene Products, gag; D015684:Gene Products, pol; D012194:RNA-Directed DNA Polymerase; D001616:beta-Galactosidase