IN-VIVO DNA EXPRESSION OF FUNCTIONAL BROME MOSAIC-VIRUS RNA REPLICONSIN SACCHAROMYCES-CEREVISIAE

To facilitate manipulation of brome mosaic virus (BMV) RNA replicons i n Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA r eplication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminate d by a self-cleaving ribozyme at or near their natural 3' ends, In gal actose-induced yeast harboring such plasmids, expression of BMV RNA re plication proteins la and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher t han those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-e ncoded subgenomic mRNA for coat protein, Although the GAL1 promoter in itiated transcription from multiple sites, la and 2a selectively ampli fied RNA3 with the authentic viral 5' end, As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be transl ated directly from DNA-derived RNA3 transcripts, so their expression d epended on la-and 2a-directed subgenomic mRNA synthesis, In yeast in w hich DNA transcription of B3CAT, an RNA3 derivative with the chloramph enicol acetyltransferase (CAT) gene replacing the coat gene, was induc ed, CAT activity remained near background levels in the absence of la and 2a but increased over 500,000-fold when la and 2a were expressed, Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeas t URA3 gene replacing the coat gene, conferred uracil-independent grow th to ura3(-) yeast only after la and 2a expression and galactose indu ction. Once its la-and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after re pression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid, These findings should be useful for many experimental purposes.