FIG. 5.
Effect of overexpression of selected host proteins interacting with p33 replication proteins on recombination by a TBSV repRNA in yeast. (A) Northern blotting of total RNA obtained from yeast overexpressing zz-tagged yeast proteins as shown. The host proteins were overexpressed separately from the galactose-inducible GAL1 promoter 20 h prior to launching the replication of DI-AU TBSV repRNA, a highly recombinogenic repRNA (7). Then, the yeast culture was grown in the presence of 50 μM copper sulfate at 29°C for 24 h, followed by a 1:10 dilution into the same medium. The recombinant repRNAs were measured in samples obtained from yeast cultures 48 h after the dilution step. Note that the ratio of recRNA to repRNA was calculated, since that reflects the frequency of recombination more accurately than does the amount of recRNA, which is dependent on both the frequency of recombination and the amount of the template (original parental repRNA). The ratio of recRNA to repRNA in the control yeast expressing a short peptide was set to 100%. Each experiment was performed six to eight times. (B) Western blotting showing the overexpressed zz-tagged host proteins (marked with arrows) and the tombusvirus p33 and p92pol (marked with asterisks) replication proteins.