FUNCTIONAL DIFFERENCES BETWEEN THE HUMAN LINE RETROTRANSPOSON AND RETROVIRAL REVERSE TRANSCRIPTASES FOR IN-VIVO MESSENGER-RNA REVERSE TRANSCRIPTION

We have analysed the reverse transcriptase (RT) activity of the human LINE retrotransposon and that of two retroviruses, using an in vivo as say within mammalian (murine and human) cells, The assay relies on tra nsfection of the cells with expression vectors for the RT of the corre sponding elements and PCR analysis of the DNA extracted 2-4 days post- transfection using primers bracketing the intronic domains of co-trans fected reporter genes or of cellular genes, This assay revealed high l evels of reverse-transcribed cDNA molecules, with the intron spliced o ut, 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 dom ain: >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, Con versely, the RT of the two retroviruses tested, Moloney murine leukemi a virus and human immunodeficiency virus, failed to produce similar re verse transcripts, These experiments demonstrate a specific and high e fficiency reverse transcription activity for the LINE RT, which applie s to RNA with no sequence specificity, including those from cellular g enes, and which might therefore be responsible for the endogenous acti vity that we previously detected within mammalian cells through the fo rmation of pseudogene-like structures.