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

Figure 2.

Figure 2

A transposed template recruitment element (RE) supports BMV RNA3 recruitment and replication. (A) Cassettes for in vivo transcription of B3URA3 and derivatives. The bracketed region represents a cDNA copy of B3URA3 flanked by a 5′-linked GAL1 promoter and a 3′-linked, self-cleaving, hepatitis delta ribozyme (Rz). Coordinates correspond to wt BMV RNA3. In B3URA3ΔIR, a B3URA3 derivative lacking the intergenic region, RE sequences (nt 1012–1200) were inserted between nt 602 and 603, creating B3URA3ΔIR + TRE. A box B-deficient TRE (B3URA3ΔIR + TREΔbox B) was created by removing nt 1094–1113; (B) Membrane association of B3URA3 derivatives in yeast expressing or lacking BMV replication protein 1a. After transcription for 72 h, equal amounts of cells were spheroplasted and lysed osmotically. Half of the lysate was processed to obtain the total RNA fraction (T). The other half was centrifuged at 10,000× g to yield a pellet (P) and supernatant (S) fractions. RNA was isolated from each fraction, and equal proportions analyzed by Northern blotting to detect positive-strand URA3 sequences. Representative blots are shown. The histogram shows the average and standard error from three replicates; (C) RNA replication in the absence of selection. Yeast was transformed with plasmids carrying B3URA3, or its derivatives, and with plasmids expressing 1a and 2apol replication proteins. Transcription was induced with galactose for 72 h, and equal amounts of cells harvested for RNA extraction. Equal amounts of total RNA were analyzed by Northern blotting with a single-stranded, 32-P labeled RNA probe complementary to positive (+)- or negative (−)-strand URA3. The solid arrowhead points to transcripts that have not been cleaved by the ribozyme. The empty arrowhead points to short transcripts formed after premature termination of transcription at the oligo(A) tract (Sullivan and Ahlquist, 1999). Ethidium bromide staining of 18S rRNA is indicated at the bottom. Negative-strand B3URA3 accumulation was quantified for three biological replicates. The histogram shows average and standard error relative to B3URA3.