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. 2005 Mar;79(6):3664–3674. doi: 10.1128/JVI.79.6.3664-3674.2005

FIG. 7.

FIG. 7.

Illustration of cDNA clones used for in vitro transcription of BBSV sat-RNAs. The pMBS-M4 monomeric cDNA plasmid was used for in vitro transcription of the full-length (+) sat-RNA and was modified for transcription of three mutant (Mut) RNAs as follows. Mut-1 contained a 5-nt (5′-TACCC-3′) deletion at the 3′ end of the sat-RNA that was generated by use of the BB-S3 and BB-S12 primers (Table 1) to amplify pMBS-M4. Mut-2 had a 3′ end extension of 39 bases from pMD18-T, produced by digesting pMBS-M4 with EcoRI prior to in vitro transcription. Mut-3 consisted of a three-G extension at the 3′ end of the sat-RNA that was obtained by PCR amplification of pMBS-M4 with the BB-S3 and BB-S2 primers. The pUBS-Md3 monomeric cDNA plasmid contained a 5-nt (5′-GAAAG-3′) deletion from the 5′ end of the sat-RNA produced by amplification with the BB-S11 and BB-S2 primers (Table 1). The plasmid pMBS-M41 lacked the T7 promoter sequence and contained a full-length sat-RNA. The pUBS-R35 plasmid contained the monomeric sat-RNA cDNA in the reverse orientation, produced by amplification with the BB-S7 and BB-S4 primers (Table 1), for in vitro transcription of a negative-sense sat-RNA probe. pMBS-D10 had the full-length dimeric sat-RNA cDNA insert, and the dimeric pMBS-D17 plasmid had 5-nt deletions at the 3′ end (5′-TACCC-3′) and at the 5′ end (5′-GAAAG-3′) of the junction region. The pMBS-D34 dimeric plasmid had a 3′ extension at the junction region consisting of 15 nt (5′-GATCCTCTAGAGATT-3′) derived from the pMD18-T vector. The dimer in pMBS-D6 contained a deletion (5′-GAAAG-3′) at the 5′ end of the junction region. Plasmid pUBS-D21 had a deletion (5′-GAAAG-3′) at the 5′-proximal end of the dimeric cDNA sequence. The dimeric sat-RNA sequence in pMBS-D50 had a deletion (5′-TACCC-3′) at the 3′-proximal end. The plasmid pUBS-D5 contained deletions (5′-TACCC-3′) at the 3′ ends of the cDNAs of each sat-RNA monomer and of the junction region and had an insertion (5′-GGGGAT-3′) from the vector. The pMBS-T6 plasmid contained a trimeric sat-cDNA insert, and pMBS-Tet7 had the tetrameric sequence. pMBS and pUBS nomenclature corresponds to plasmids in the pMD18-T and pUC18 backgrounds, respectively.