Nuclear run-on assay to identify the RNA polymerase responsible for transcription of the L1Tc retroelement Detection of nascent RNAs sensitive and resistant to RNA polymerase inhibitors by dot blotting

<p><b>Copyright information:</b></p><p>Taken from "The L1Tc non-LTR retrotransposon of contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts"</p><p></p><p>Nucleic Acids Research 2007;35(7):2199-2214.</p><p>Published online 16 Mar 2007</p><p>PMCID:PMC1874656.</p><p>© 2007 The Author(s)</p> Nascent transcripts from epimastigotes in the logarithmic phase of growth (non-treated) were hybridized to the following membrane-bound cloned DNAs: 18S rDNA (18S) (), the α-tubulin gene (pTcα3) (), the tRNA gene (tRNA), the sequences coding for full-length L1Tc (L1Tc) or L1Tc endonuclease (NL1Tc), L1Tc reverse transcriptase (RT), and the L1Tc nucleic acid chaperone protein (C2-L1Tc). Run-on was also performed with nuclei incubated in the presence of 1mg/ml of α-amanitin, 0.6% of sarkosyl and 8 μM of tagetitoxin. Quantification of the L1Tc nascent RNAs from untreated nuclei and nuclei treated with different RNA polymerase inhibitors. The percentage (%) of the nascent transcripts sensitive and resistant to different RNA polymerase inhibitors was calculated relative to the total amount of the nascent transcripts from untreated nuclei detected in each case. Scanning of the autoradiograms was performed with an Image Quant program (Molecular Dynamics)