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FIG. 1. Replication and recombination of TBSV replicon RNA in yeast from the yTHC collection. (A) Schematic representation of the DI-AU-FP replicon RNA with four noncontiguous regions (RI to RIV) derived from TBSV genomic RNA and the artificial AU-FP region to promote recombination (58). (B and C) The scheme of launching DI-AU-FP replication and regulation of host gene expression from the Tet-titratable promoter. P33 and p92 replication proteins are expressed constitutively from the ADH1 promoter, whereas DI-AU-FP is expressed from the regulatable GAL1 promoter. Replication (and probably recombination) of DI-AU-FP takes place in the cytoplasm (on peroxisomal membrane surfaces). The expression of a particular host gene occurs in the absence of doxycycline, and it is switched off in the presence of doxycycline. (D) (Top). Ethidium bromide-stained agarose gel of total RNA extracts obtained from the parental yeast strains (grown without doxycycline [–DOX] or with doxycycline [+DOX]) showing the accumulation of DI-AU-FP replicon RNA and the recombinant RNA. The yeast cells coexpressed DI-AU-FP RNA, and p33/p92 replication proteins. (Bottom). Northern blot analysis of total RNA extracts with a ³²P-labeled RNA probe specific for RIII/IV of DI-AU-FP. The samples are the same as in the top panel. The unmarked faint bands represent degradation products (5' truncated RNAs that migrate fast) and additional recombinants (slow migrating).

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