Fig. 4.

In vitro copying of sgRNA3a templates with BMV RdRp. Equimolar amounts of sgRNA3a constructs JS-0 (lane 2), JS-14 (lane 3), JS-21 (lane 4), 5′-580 (lane 5) and 5′-380 (lane 6) were copied in standard BMV replicase reactions as the only templates. The radioactive products were separated in a 1.0% formamide/formaldehyde agarose gel and blotted to nylon membrane followed by autoradiography. Bands representing the copying products are marked by thick arrows. Lane 1, ³²P-labeled mixture of JS-21 and sgRNA4 transcripts as size standards. In addition, lanes 2 to 10 contain the sgRNA4 loading marker, added post-copying reaction. The densitometric quantification results are shown below. The open bars represent the relative density of bands corresponding to the in vitro copying products of JS-0 to JS-21 (which was taken as 100%) after normalizing to the internal sgRNA4 standard. Lanes 7 to 10, analysis of the ds nature of the copying products of JS-14 and JS-21 RNAs by S1 nuclease digestion (lanes 8 and 10, respectively) versus undigested products (lanes 7 and 9).