Journal:

Article Title: Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase

doi:

Figure Lengend Snippet: Immobilised affinity purified polymerase is transcriptionally active. Nuclear extracts from Vac3P or VacT7 infected HeLa cells were mixed with SA-beads coated with the 5′-v16B oligonucleotide. The SA-beads were then resuspended in transcription buffer containing nucleotides, the indicated primer and 3′-vRNA template. Transcription products incorporated [α- 32P]GTP and were resolved on an 18% 7 M urea denaturing polyacrylamide gel and visualised by autoradiography for (A) 16 or (B) 120 h. (A) Lane 1: ApG primed reaction. Lane 2, globin mRNA primed reaction. Lane 3, ApG primed reaction with the omission of the 5′-v16B oligonucleotide. Lane 4, ApG primed reaction with the omission of 3′V template RNA. Lane 5, unprimed reaction. Lane 6, globin mRNA primed reaction using VacT7 nuclear extract. Lane 7, ApG primed reaction using VacT7 extract. (B) Co-expression of PB2 is required for unprimed, ApG primed, globin mRNA primed transcription. Lanes 1–3, SA beads coated with Vac3P. Lanes 4–6, SA beads coated with VacPB1/PA. Lane 7, SA beads coated with VacT7. Lane 8, Vac3P extract plus 5′-v16B (omitting bead selection procedure). Lanes 1 and 4, unprimed transcription. Lanes 2, 5, 7 and 8, ApG primed transcription. Lanes 3 and 6, globin mRNA primed. Lane 4, VacPB1/PA, unprimed synthesis.

Article Snippet: A synthetic RNA oligonucleotide (5′V16B) was synthesised (Cruachem Ltd) with the sequence: 5′-AGUAGAAACAAGGGUG(CH 2 ) 12 -biotin.

Techniques: Affinity Purification, Infection, Autoradiography, Expressing, Selection