Journal of General Virology

Fig. 3. Accumulation and maintenance of the B3URA3 RNA replicon and 3a-disrupted derivatives in yeast. (a, b) As an alternative to inefficient transfection of yeast with in vitro transcripts, Ura⁺ 1a2a⁺ yeast strains bearing B3URA3 (wt, lane 1), B3URA3-3a^fs (3a^fs, lane 2) or B3URA3-3a (3a, lane 3) as free RNA replicons were obtained by induction from DNA plasmids followed by plasmid curing, as follows: 1a2a⁺ yeast cells were transformed with a DNA plasmid (see Fig. 1) expressing the desired B3URA3 derivative from the galactose-inducible, glucose-repressible GAL1 promoter, and then were cultured in synthetic medium lacking histidine, leucine and tryptophan to select for the 1a and 2a expression plasmids and B3URA3 plasmid, respectively, and containing galactose to induce transcription of the B3URA3 derivative. Ura⁺ cells with URA3 expressed from the BMV RNA replicon were selected by plating these yeast on synthetic medium lacking uracil, histidine, leucine and tryptophan and containing glucose to repress plasmid transcription of the B3URA3 derivative (Ishikawa et al., 1997). These [Ura⁺ His⁺ Leu⁺ Trp⁺] colonies were then cultured in synthetic glucose medium lacking uracil, histidine and leucine but containing tryptophan to remove selection for the B3URA3 derivative plasmid. [Ura⁺ His⁺ Leu⁺ Trp^–] cells were isolated from these cultures and Southern blot analysis confirmed that the DNA plasmids encoding B3URA3 derivatives were lost and that no B3URA3 DNA sequences were present in any other form. These [Ura⁺ His⁺ Leu⁺ Trp^–] yeast cells were extracted (Janda & Ahlquist, 1993) to yield total RNA, which was denatured with glyoxal, separated by electrophoresis in 1 % agarose, blotted onto a nylon membrane (Hybond-N), fixed by ultraviolet irradiation and hybridized with BMV RNA-specific probes. The probe to detect BMV-related positive-strand RNAs in (a) was ³²P-labelled RNA complementary to the conserved 200 nucleotides at the 3´ end of BMV RNA. The probe to detect BMV-related negative-strand RNAs in (b) was ³²P-labelled RNA having the sequence of the conserved 200 nucleotides at the 3´ end of BMV RNA. Lanes 4 (marked 'none') contain RNA from 1a2a⁺ yeast without B3URA3 RNA. The positions of positive- and negative-strand RNA3 bands are shown by brackets. In lane 3 for B3URA3-3a in (a), the band just below subgenomic RNA4 is believed to represent a degradation product of B3URA3-3a RNA. An asterisk marks an accumulation of background RNAs swept ahead of a ribosomal RNA band. (c) The specific yeast strains analysed in (a) and (b), containing B3URA3 (), B3URA3-3a^fs () or B3URA3-3a () RNA replicons, were successively subcultured in medium containing uracil and analysed as in Fig. 2. Circles represent data for parallel subcultures of one of the B3URA3-containing cell lines of Fig. 2 (obtained by direct transfection of 1a2a⁺ yeast with B3URA3 in vitro transcripts). The difference in slope between Fig. 2 and Fig. 3 may represent experimental variation derived from slight differences in culture conditions. Equivalent results were obtained in another experiment using independent isolates of 1a2a⁺ yeast bearing B3URA3-3a^fs or B3URA3-3a.

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