Read-through activation in Neo-GFP cell lines.

Abstract

<p>A. Nuclear RNA prepared from pooled cultures of cell lines isolated after electroporation with 1 µg of DNA was assayed by hybridization and protection from nuclease S1 digestion. Expression of <i>E1a-neo,</i> read-through transcription (RT), and <i>E1b-gfp</i> produced the specific protected bands indicated in the diagram below the autoradiographic data. The arrows indicate the relative positions of the transcripts in the template (the uncertain end of the read-through transcript is indicated by the dashed line). The probe is indicated by a line with the position of the 5′-end label shown as an asterisk. The region of the probe protected by each transcript is indicated as double-stranded (DNA-RNA hybrid). The position of divergence of the sequence of the probe for read-through transcription from the read-through RNA product is shown by the loss of DNA-RNA hybrid formation. Variation in migration of the RT and E1a-GFP products probably was caused by sequence differences at the junction site produced during plasmid construction. Lane designations: 1: HeLa cells; 2: culture derived from pNeoE1bGFP; 3: culture derived from pNeoGGTE1bGFP; 4: culture derived from pNeoDPME1bGFP. The positions of size markers (not shown) are indicated on the left of the autoradiograms. B. <i>E1b-GFP</i> RNA levels from two experiments (Expt 1 is shown in A) were quantified and normalized to the quantity of <i>E1a-Neo</i> RNA. The results are expressed relative to the pNeoE1bGFP value (1.00).</p