FIG. 9.
Poly(A) tail repair of MIDI-C mutant RNAs during DI replication over four virus passages. (A) In vitro-transcribed MIDI-C RNAs were used as templates to establish the RT-PCR assay for poly(A) tail repair. A poly(A) tail of more than 10 A residues was necessary to amplify MIDI-C using oligo (dT)12–18 and primer JS24 (Table 1) (lanes 1 to 4). (B to E) RT-PCR analysis of RNAs from the DI replication experiment in Fig. 6. An 847-bp RT-PCR product was expected using primers oligo(dT)12–18 and JS24 (lanes 1 to 5). Primers M648-633 and M144-163 (Table 1) were used as a control to amplify a 500-bp portion of the M gene (lanes 6 to 10). PCR products were analyzed by electrophoresis through 1% agarose gels containing ethidium bromide. M denotes DNA markers corresponding to the following sizes (in kilobases) from top to bottom: (A) 1.2, 0.8, and 0.4; (B to E) 2.0, 1.2, 0.8, and 0.4. C denotes control for the size of the expected poly(A) PCR product and corresponds to 10 μl of PCR product from RT-PCR of in vitro-transcribed MIDI-C Awt RNA.