Retrotransposition of a marked Drosophila line-like I element in cells in culture.
We have marked a Drosophila transposable element--the LINE-like I element--with an intron-containing indicator gene inserted in place of a large deletion in the I element second ORF encompassing the reverse transcriptase domain, and this marked element was placed downstream to a potent actin promoter. An expression vector for the I element ORFs was also constructed, under the same heterologous promoter. The indicator gene contains a lacZ reporter gene the expression of which is conditioned by retrotransposition of the marked element, thus allowing detection of transposition events by testing for either beta-galactosidase expression or occurrence of spliced DNA molecules. The marked I element was introduced into Drosophila melanogaster cells in culture by transfection. Spliced DNA copies of the marked element and specifically stained beta-galactosidase-expressing cells were detected only upon co-transfection with the I expression vector, thus indicating that an ORF2-deleted element can be complemented in trans for transposition. This simple assay for retrotransposition in Drosophila cells in culture provides a tool for the rapid analysis of the mechanism of I transposition in its cis and trans sequence requirements.
Jensen
S
S
Unités de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS, Villejuif, France.
Cavarec
L
L
Dhellin
O
O
Heidmann
T
T
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA; D004251:DNA Transposable Elements; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014162:Transfection
D004251:DNA Transposable Elements; D004247:DNA