Transcription of integrated MMTV LTR DNA in fibroblast clones is not associated with DNA methylation but with the integration site.
By transfecting 3T3 TK- fibroblasts with a plasmid DNA containing the MMTV LTR promotor and the thymidine kinase gene, we have isolated TK+ clones that contain only one copy of the recombinant DNA. The newly acquired DNA is integrated at different sites in each TK+ clones and can be amplified as a tandem repeat in some cases. The basal level of expression of the newly acquired LTR sequence is variable among the TK+ clones studied. Among those producing detectable amounts of MMTV RNA, we found that transcription is initiated from the LTR promotor and that the synthesis is stimulated by a short dexamethasone treatment. In addition, the presence of tandem repeated LTR DNA does not amplify the MMTV RNA synthesis. Endogenous LTR sequences have been found to be methylated, whereas we show that the newly integrated sequences, both as single copy and tandem repeats, remain unmethylated. Thus, non-methylation of MMTV LTR DNA is necessary but not significant to allow its transcription. Our results suggest that the level of LTR promotor activity could be modulated by the nature of its integration site within the cellular genome.
D000818:Animals; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002999:Clone Cells; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D008745:Methylation; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012191:Retroviridae Proteins / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004279:DNA, Viral; D015683:Gene Products, gag; D012191:Retroviridae Proteins; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes