Evidence for a reverse transcription intermediate for a marked line transposon in tumoral rat cells

We have marked a "reconstituted" LINE element from rat with an intron-containing indicator gene, to test for its RNA-mediated transposition in tumoral rat cells in culture. Three cloned LINE promoter-containing fragments were tested by a transient transfection assay using a LacZ reporter gene, and the promoter with maximum expression was substituted--in an homologous manner--to the 5' domain of a close to full-length genomic LINE. The resulting marked LINE was stably introduced by transfection into tumoral rat cells. PCR amplification of the DNA from several transfected clones, using primers bracketting the intronic domain of the indicator gene, yielded fragments with a reduced size: their DNA sequencing, in four cases out of four, demonstrated splicing out of the intron as expected for the passage of the marked LINE through an RNA intermediate and its reverse transcription. Fractionation of cellular DNA by the Hirt procedure indicated that reverse transcribed copies are present in the "extrachromosomal" fraction. Their abundance is close to 1 copy per 10(4) cells. These results strongly suggest that rat LINEs transpose through an RNA intermediate and its reverse transcription, as previously demonstrated for the Drosophila LINE I element and, further, that reverse transcription might take place prior to integration, resulting in extrachromosomal DNA molecules as preintegrative transposition intermediates.