FIG. 2.

Characterization of CNV replicase activity in enriched membrane fractions derived from yeast or N. benthamiana protoplasts. (A) PAGE analysis of the (³²P-labeled) in vitro replicase products on the endogenous templates, which are present in the enriched membrane fractions, obtained from yeast (top) and from protoplasts (bottom). Time of incubation, shown on the top of the gel in the time course experiments, started after the addition of galactose to the media for yeast or with the addition of incubation media after electroporation for N. benthamiana protoplasts. Arrows indicate the bands that correspond to products obtained on the various endogenous RNAs. (B) Relative RdRp activity of enriched membrane fractions. For quantification, we measured the intensity of ³²P-labeled DI-72 and gCNV RNA bands (see panel A) obtained with yeast- and plant-derived CNV replicase preparations by using a phosphorimager. The gels shown in panel A were exposed for the same time, and 100% value represents the signal obtained after 30 h of incubation in each experiment. Circles and triangles represent data obtained with CNV replicase derived from protoplasts and yeast, respectively. (C) RNA blot showing plus-strand (+) and minus-strand (−) levels in the in vitro CNV replicase products on endogenous templates. Unlabeled T7 RNA polymerase transcripts of DI-72(+) and DI-72(−) (400 ng of each) for yeast-derived samples and gCNV(+) and gCNV(−) for protoplasts-derived samples were blotted on the membrane as shown between the blots. Time points for harvesting the yeast and protoplast samples for isolation of membrane fractions are shown on the top. The blotted RNAs were hybridized with denatured ³²P-labeled RNA probes, which were generated by the CNV replicase in vitro on the endogenous templates present in the enriched membrane fractions from yeast or protoplast (see panel A). (D) Relative amounts of plus- and minus-stranded RNAs in the in vitro replicase products. Total replicase products at each time point were taken as 100% (left panel), or the amount of plus-stranded replicase product at the 30 h time point was taken as 100% (right panel). Solid and dotted lines represent plus- and minus-stranded products, while circles and triangles represent data obtained with preparations from protoplasts and yeast, respectively.