Figure 7

Fractionation experiments indicate that Rev and Rex have little effect on export or stability of unspliced RmRE-containing reporter transcripts. A. Integrity of cytoplasmic and nuclear fractions obtained from transfected Jurkat cells. Jurkat cells were subjected to transfection by electroporation and, after 48 hr, cells were fractionated. Fractions were used for RNA extraction and subjected to Northern blotting prior to staining with methylene blue and photography. The nuclear ribosomal precursors (arrows on the left) and cytoplasmic mature ribosomal RNAs (arrows on the right) are indicated. B. Semi-quantitative RT-PCRs of fractionated RNA obtained from Jurkat cells transfected with the HIV-based reporter vector pDM128. Cells were co-transfected with pDM128 and either pEGFPN3 control vector (no Rev) or RevGFP (Rev) expression plasmids as indicated by minus or plus signs. After 48 hr, cells were fractionated, and RNA samples were extracted and subjected to RT-PCRs using primers for the cat gene or glyceraldehyde-3-phosphate dehydrogenase (gapdh). Fractions (FR) used for RNA extraction are indicated as nuclear (N) or cytoplasmic (C). PCRs performed in the absence of reverse transcriptase (RT) are indicated (lanes 1–4) (equivalent to 2 μl of a diluted cDNA reaction). Either 2 μl (lanes 5–8 and 13–16) or 4 μl (lanes 9–12 and 17–20) of the diluted cDNAs were used for RT-PCRs as indicated in Methods to show that the reactions were performed in the linear range. Samples were analyzed on a 1.5% agarose gel using either 5 (gapdh) or 15 μl (cat) of the 50 μl reaction. Markers (M) are given in basepairs (bp). C. Semi-quantitative RT-PCR assays of the MMTV-based reporter plasmids in the presence or absence of RemGFP, RevGFP, and RexGFP. Semi-quantitative RT-PCRs were performed using RNA extracted from transfected Jurkat cells and primers specific for the Renilla luciferase (Rluc) or gapdh genes. Left and right panels show results of two different transfection experiments. M = DNA markers (in bp); P = HMRluc plasmid positive control; H₂0 = PCR without added cDNA. D. Quantitation of RT-PCR results from cytoplasmic fractions of cells transfected with the MMTV-based reporter plasmid. Stained RT-PCR products from panel C were quantitated using ImageJ software and normalized for RNA amounts and integrity using gapdh expression. The normalized RNA levels obtained from each reporter plasmid (in the presence of the control EGFP expression vector only) were assigned values of 1, and the other samples have been reported relative to these values. These results are representative of at least three different transfection experiments.