Figure 2
Role of pal I in MMTV gRNA dimerization, packaging, and propagation. (A) Description of the pal I mutants. The wild type pal I sequence is shown in blue and the mutations are depicted in red. (B) Typical RNA dimerization TBM and TB gels of wild type and mutant MMTV RNAs. M: monomer lane or monomer conformer; D: dimer lane or dimer conformer for each sample. (C) Transfection efficiencies of the mutants and wild type transfer vectors that were used to normalize the packaging efficiency. LUC: Luciferase activity. (D) PCR amplifications of the DNase treated cytoplasmic (panel i) and viral (panel ii) RNAs using virus specific primers. In the third panel (iii), amplification was conducted on the cDNAs obtained from cytoplasmic RNAs using primers that amplify unspliced β-actin mRNA. Multiplex amplifications were conducted in the presence of primers/competimer for 18S ribosomal RNA. The fourth panel (iv) shows PCR of cytoplasmic cDNA using primers that amplify spliced β-actin mRNA. (E) Relative packaging efficiency (RPE) of transfer vector RNAs. (F) Relative hygromycin resistance (Hygr) colony forming unit per ml (CFU/ml) for mutant transfer vectors reflecting the relative RNA propagation efficiencies. In (C), (E), and (F), the histograms represent data from at least three independent experiments (± SD). The P values for all mutants in panel (F) were significant (<0.001).