Biomedical subjects

O Dhellin

Publications and source records attributed to O Dhellin.

3 recordsLinked to original sources

PubMed1997

Functional differences between the human LINE retrotransposon and retroviral reverse transcriptases for in vivo mRNA reverse transcription.

We have analysed the reverse transcriptase (RT) activity of the human LINE retrotransposon and that of two retroviruses, using an in vivo assay within mammalian (murine and human) cells. The assay relies on transfection of the cells with expression vectors for the RT of the corresponding elements and PCR analysis of the DNA extracted 2-4 days post-transfection using primers bracketing the intronic domains of co-transfected reporter genes or of cellular genes. This assay revealed high levels of reverse-transcribed cDNA molecules, with the intron spliced out, with expression vectors for the LINE. Generation of cDNA molecules requires LINE ORF2, whereas ORF1 is dispensable. Deletion derivatives within the 3.8 kb LINE ORF2 allowed further delineation of the RT domain: > 0.7 kb at the 5'-end of the LINE ORF2 is dispensable for reverse transcription, consistent with this domain being an endonuclease-like domain, as well as 1 kb at the 3'-end, a putative RNase H domain. Conversely, the RT of the two retroviruses tested, Moloney murine leukemia virus and human immunodeficiency virus, failed to produce similar reverse transcripts. These experiments demonstrate a specific and high efficiency reverse transcription activity for the LINE RT, which applies to RNA with no sequence specificity, including those from cellular genes, and which might therefore be responsible for the endogenous activity that we previously detected within mammalian cells through the formation of pseudogene-like structures.

3T3 Cells↗

PubMed1995

mRNA retroposition in human cells: processed pseudogene formation.

Using a sensitive assay for detection of reverse transcription events, we demonstrate that human HeLa cells can 'retropose', i.e. reverse transcribe and integrate, the mRNA of a naive reporter gene, at a low but detectable frequency. Furthermore, we show that the retroposed copies have all the hallmarks of the processed pseudogenes naturally found in the mammalian genome: they lack intron and 5' promoter sequence, they have acquired a 3' poly(A) tail, and they are flanked by short repeats (< 15 bp) of target DNA sequence. These results demonstrate that human cells possess an endogenous reverse transcription activity, which is not restricted to transcripts of transposable elements, and which is likely to be involved in the formation, still ongoing, of a large fraction of the eukaryotic genome.

Base Sequence↗

PubMed1994

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.

Animals↗