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. 2003 Apr;77(8):5017–5020. doi: 10.1128/JVI.77.8.5017-5020.2003

FIG. 2.

FIG. 2.

Functionality of recombinant polymerases. (A) CAT expression in 293T cells transfected with pPOLI-CAT-RT and pcDNA expression vectors for PB1, PB2, PA (wild-type [WT] or mutants as indicated), and NP proteins. Transfections and CAT assays were performed as described earlier (4, 18) by using serially diluted cell extracts as indicated (N, undiluted; −1, 10-fold; −2, 100-fold; −3, 1,000-fold; and −4, 10,000-fold diluted). (B) Transcription initiation with ApG dinucleotide primer. Transcription reactions were carried out in vitro with nuclear extracts from cells expressing PB1, PB2, and PA (wild-type [WT] or mutants as indicated) by using a 14-nt 3′ end vRNA template in the presence of a 15-nt 5′ end vRNA as described earlier (4). The slowest-moving band represents the full-length (14-nt) transcription product. The faster-moving bands (12 and 13 nt) are most likely partial transcription products, as previously described (1). Longer exposure showed a low but detectable signal for the C453R mutant (not shown).