FIG. 4.

Selection of yeast colonies dependent on NoV RNA replication. Panel A is a schematic. A DNA plasmid piH2N for RNA polII-mediated expression of negative-sense NoV RNA2 encoding the yeast HIS3 gene is shown at the top; symbols for sequence elements are as described for Fig. 2A. The open arrowhead labeled “Int.” indicates the yeast ACT1 intron, cloned in the antisense orientation relative to RNA2. Below, horizontal lines represent the N2H RNA with polarity indicated. The open box represents the HIS3 ORF. Panel B shows the results of selecting for HIS3-positive colonies following transient induction in galactose-containing medium. In the first four experiments, the RNA1 plasmids encoded either WT RNA1, RNA1 containing a frame-shifted polymerase (fs), an arginine-to-glutamine mutation at position 59 of the B2 ORF (R59Q), or a B2 null mutant (B2−−). In experiments 5 and 6, the RNA1 plasmids encoded the R59Q mutant in the context of a U-to-C mutation at position 1274 (U/C, experiment 5) or a 4-nt mutation that eliminates RNA3 synthesis (RNA3−, experiment 6). Yeast cells containing piH2N in addition to plasmids encoding the indicated RNA1 were induced for 2 days on galactose-containing liquid medium. An aliquot of each, containing an equal number of cells, was used to seed cultures of dextrose-containing liquid medium lacking histidine, and 2 days later, aliquots of this culture were spotted onto dextrose-containing solid medium lacking histidine. Plates were incubated at 30°C for 4 days and photographed. The image colors were digitally reversed to improve contrast.