Analysis of the transcription initiation site of the CAT gene in pTEX(−)pR77CAT and pTEX(−)p5′KMP11CAT transfectants

<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> and Primer extension analyses were performed employing cytoplasmic RNA or the purified polyadenylated fraction of RNA from pTEX(−)pR77CAT (A) and pTEX(−)p5′KMP11CAT (C) transfectants, along with a radiolabeled MCS primer that maps 3–19 nucleotides downstream of the Pr77 and 5′UTRKMP11 sequences in the pTEX(−)pR77CAT and pTEX(−)p5′KMP11CAT vectors respectively. Reactions were subsequently resolved in urea-polyacrylamide gels. The sequencing of pTEX(−)pR77CAT (A) and pTEX(−)p5′KMP11CAT (C) plasmids was performed following the Sanger method using an MCS antisense primer; samples were loaded onto the left side of the gels as size standards. The sequences corresponding to L1TcPr77 (Pr77), to the splice leader (SL) and to KMP11 5′UTR are indicated at both sides of the gels. Arrows indicate the position of the extended products. The radiolabeled molecular weight (MW) marker is expressed in bp. and Deduced sequence of the extended products in the primer extension assays(B) The Pr77 sequence (Pr77) of the pTEX(−)pR77CAT transfectants (D) Sequence of the splice leader (SL) and the 5′UTR of KMP11 in pTEX(−)p5′KMP11CAT transfectants. The splicing acceptor site is indicated by an arrow. The oligonucleotide used for primer extension analysis and employed for DNA sequencing is underlined. The SL sequence is indicated in italics