Figure 4.

Replication of B3URA3 + TRE and B3URA3Δ3′3a, and intramolecular RNA recombination in spherule-forming conditions. (A) Accumulation of B3URA3 + TRE and B3URA3Δ3′3a in actively transcribing cultures in the absence of selection. In yeast expressing BMV 1a and 2a^pol, B3URA3 or its derivatives were transcribed from the GAL1 promoter in liquid cultures for 72 h (9–10 yeast generations). Equal amounts of cells were harvested for RNA extraction and analysis by Northern blotting with 32-P labeled, strand-specific URA3 probes. Representative Northern blots are shown. Arrowheads indicate transcripts before ribozyme cleavage. RNA3 and RNA4 of positive and negative polarity are indicated are indicated by (+) and (−), respectively. Ethidium bromide staining of 18S rRNA is indicated at the bottom. The histogram shows RNA accumulation relative to B3URA3, and bars represent the average and standard error of six biological replicates. B3URA3Δ3′3a accumulated to higher levels than B3URA3 and B3URA3 + TRE (p < 0.01); (B) Accumulation of B3URA3 + TRE and B3URA3Δ3′3a in Ura⁺ cells. Individual Ura⁺ colonies were grown in liquid cultures (8 ml) under uracil selection. Total RNA was extracted and processed as in (A). Positive strand RNA3 and RNA4 in B3URA3Δ3′3a accumulated to higher levels than B3URA3 and B3URA3 + TRE (p < 0.01). (C) Detection of intramolecular RNA recombinants in Ura⁺ colonies after plasmid launching of B3URA3 + TRE. B3URA3 and B3URA3Δ3′3a were processed in parallel as size markers. For each construct, twenty Ura⁺ colonies obtained after plasmid launching were individually grown in liquid cultures (8 mL) lacking uracil, the RNA extracted, and analyzed as in (A). Lane 4 shows B3URA3Δ3′3a replacing B3URA3 + TRE. Lanes 5 and 6 show both B3URA3 + TRE and B3URA3Δ3′3a. B3URA3Δ3′3a did not suffer genetic modifications (lanes 7, 8 and 9). Ethidium bromide staining of 18S rRNA is indicated at the bottom.