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. 2008 Aug 2;82(21):10724–10734. doi: 10.1128/JVI.00921-08

FIG. 4.

FIG. 4.

HPV-16 E1 transcripts originate from a TATAA-dependent promoter upstream of P97. (A) Specific transcripts were amplified by 5′ RACE, using nested primers as indicated, which detected early gene transcripts in tobacco acid pyrophosphatase (TAP)-treated total RNA isolated from SCC13 cells transiently transfected with HPV-16 plasmids. Mutations in HPV-16 ORFs and TATAA boxes (as illustrated in Fig. 1) are indicated. T, TATAA box; M, 50-bp molecular marker ladder (Invitrogen). P14/E1-specific amplification products are indicated by an arrow. The clonal (15) HFK cell line harbors stably replicating HPV-16 W12E plasmids. “PCR kit control” refers to a murine PCR template/primer set provided in the 5′ RACE kit (lane 10), while the asterisk indicates nonspecific products. (B) Reciprocal 5′RACE analysis for P97 transcription in the same RNA samples analyzed in panel A. Controls #1 (primer nt 952 to 972) and #2 (primer nt 483 to 461) also contain primer nt 22 to 38 and HPV-16 template (lanes 10 and 12) or no template (lanes 11 and 13). The asterisk indicates nonspecific products in lanes 1 to 8. (C) Map of early transcripts associated with the HPV-16 P14 and P97 transcription start sites and related splice donor and acceptor sites.