Open AccessRetrotransposition of a markedDrosophilaline-like I element in cells in culture
Author(s) -
Silke Jensen,
Laurent Cavarec,
Olivier Dhellin,
Thiérry Heidmann
Publication year - 1994
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/22.8.1484
Subject(s) - biology , transposable element , p element , retrotransposon , transposition (logic) , orfs , microbiology and biotechnology , drosophila melanogaster , reporter gene , gene , transfection , drosophilidae , schneider 2 cells , genetics , gene expression , rna interference , open reading frame , rna , genome , peptide sequence , linguistics , philosophy
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.