Fig 2. Increased level of TBSV minus-strand synthesis on recRNAs and degRNAs in yeast expressing ded1–199^ts mutant.

(A) Top and bottom panels: Northern blot analysis of (+) and (-)RNA levels, respectively, in wt and ded1–199^ts yeast.The ratios of the most abundant recRNAs versus repRNA (DI-AU-FP) and degRNA versus repRNA were calculated. See further details in Fig. 1. Note that viral RNA recombination is a chance event depending on many factors and when the first recombination event occurs, thus influencing the outcome. Quantitation is based on multiple repeats (different yeast streaks) in each experiment, and despite the different numbers on recRNAs, both Figure 1 and 2 show the same trend. (B) Top panel: In vitro RNA binding assay with purified wt Ded1p and mutants. The assay contained the ³²P-labeled DI-72 (-)repRNA (~0.1 pmol) plus increasing amount of MBP, MBP-Ded1, MBP-Ded1–95 or MBP-Ded1–199 (all were used in 40, 80 and 160 nM). The free or Ded1p-bound ssRNA was separated on nondenaturing 5% acrylamide gels. Bottom panel: The MBP-affinity purified MBP-Ded1 and mutants are analyzed on a SDS-PAGE gel.