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. 2018 Mar 15;10(3):131. doi: 10.3390/v10030131

Figure 7.

Figure 7

Accumulation of parental B3Δ5′ and B3Δ3′ in spherules and layers. (A) Features of CUP1-driven B3Δ5′ and B3Δ3′ in relation to B3URA3. Labels are as in Figure 1A. Illustration of DNA-dependent transcription of B3Δ5′, (−) negative-strand RNA synthesis and subgenomic RNA4 transcription. (B) Accumulation of positive (+)- and negative (−)-strand B3Δ5′ after transient transcription for nine yeast generations (72 to 96 h) in liquid cultures containing galactose and 500 μM CuSO4. 1a and 2apol were expressed from the ADH1 or the GAL1 promoter. The form of the BMV replication complex induced is indicated. Equal amounts of cells were harvested, and equal amounts of RNA analyzed by Northern blotting. Positive (+)- and negative (−)- strand RNA was detected using a using a 32-P labeled probe targeting 3′3a or URA3, respectively. The black arrowhead points to transcripts that have not been cleaved by the ribozyme. Ethidium bromide staining of 18S rRNA is indicated at the bottom. The histogram illustrates B3Δ5′ accumulation in each condition relative to ADH1 spherules. Bars represent the average and standard error of three biological replicates. Positive-strand B3Δ5′ accumulated to higher levels (p < 0.01) in GAL1 spherules and GAL1 layers than in other conditions. Negative-strand B3Δ5′ accumulated to higher levels (p < 0.01) in GAL1 layers that in other conditions. (C) Accumulation of positive (+)- and negative (−)-strand B3Δ5′ and B3Δ3′, and replication proteins. Cultures were grown and samples were processed as in (B) except that duplicate samples were collected for total RNA or protein extraction. B3URA3 was included as size marker. (+) B3URA3 and (-) B3URA3 indicate B3URA3 of positive and negative polarity, respectively. shB3URA3 is a transcript formed after premature termination of transcription at the oligo (A) tract (Sullivan and Ahlquist, 1999) and is approximately the same size as B3Δ3′. Empty arrowheads point to intermolecular RNA recombinants matching the size of B3URA3. The middle panel shows accumulation of replication proteins 1a and 2apol in conditions inducing the formation of spherules or layers. 1a and 2apol were detected by immunoblotting, and 3-phosphoglycerate kinase (PGK) was used as loading control. The histogram illustrates B3Δ5′ or B3Δ3′ accumulation in each condition relative to ADH1 spherules. Bars represent the average and standard error of three biological replicates. Positive-strand B3Δ5′ accumulated to higher levels (p < 0.01) in GAL1 spherules and GAL1 layers that in other conditions. Positive-strand B3Δ3′ accumulated to higher levels (p < 0.01) in GAL1 layers than in other conditions.