Expression of luciferase under the control of HPV 16 URR in HeLa cells. The promoter activities of the H1, K2, K4, R1, O2, and S1 URR (black bars) were assessed in the context of the prototype URR, which was assigned the value 1 (white bar). The promoter activities of H1, K2, and K4 altered by site-directed mutagenesis are designated by hatched bars. The change in the YY1 motif at nt 7792 explained 9-fold of the 11-fold increase associated with K2 (shown by the difference between pALuc16K2 and pALuc16K2YY1P); the change in overlapping Oct-1/PEF-1 sites at nt 7676 was also significant, as shown by the almost twofold reduction in activity between pALuc16K2 and pALuc16K2Oct-1P. The variation in the PSM at nt 7894 upregulated the promoter threefold (as shown by the difference between pALuc16K4 and pALuc16K4PSMP). Variations in the YY1 site and TEF-1 sites at nt 7743 and 7786 in K4 and H1, respectively, did not alter promoter activity (pALuc16H1 activity was similar to pAluc16H1TEF-1P activity, and pALuc16K4 activity was similar to pALuc16K4YY1P activity).

Autoradiograph showing the binding of HeLa nuclear extract to ³²P-labeled AAV P5-60 YY1 (lane 1) and HPV 16 prototype oligonucleotide nt 7790 to 7804 not competed (lane 2) or competed with a 200-fold (lanes 3 and 6), 100-fold (lanes 4 and 7), or 50-fold (lanes 5 and 8) excess of unlabeled prototype oligonucleotides (16P7792) with a C at nt 7792 (lanes 3 to 5) or unlabeled mutant oligonucleotide (16M7792) with a T at nt 7792 (lanes 6 to 8). The unlabelled YY1 prototype (16P7792) and mutant (16M7792) oligonucleotides competed almost equally effectively for binding with the labelled prototype. Evidence that the band represents YY1 binding is provided in lane 9, where polyclonal YY1 immunoglobulin G (Santa Cruz H-414 sc1703x) has almost completely abolished binding. FP, free probe.

Autoradiograph showing the binding of HeLa nuclear extract to ³²P-labeled prototype oligonucleotide nt 7667 to 7693. Evidence that the uppermost band in lane 2 represents Oct-1 binding is provided in lane 1 by the addition of polyclonal Oct-1 antibody (Santa Cruz C-21 sc232x), which has abolished binding of the labelled prototype. The effect of the C-to-A mutation at nt 7676 on binding to Oct-1 and an uncharacterized ladder of faster-migrating complexes is shown by competition with a 200-fold (lanes 3 and 8), 100-fold (lanes 4 and 9), 50-fold (lanes 5 and 10), 25-fold (lanes 6 and 11) and 12.5-fold (lanes 7 and 12) excess of unlabeled prototype (16P7676) (lanes 3 to 7) or unlabelled mutant oligonucleotide (16M7676) (lanes 8 to 12). FP, free probe. The unlabelled Oct-1/PEF-1 prototype (16P7676) and mutant (16M7676) oligonucleotides competed equally effectively with labeled prototype for binding of Oct-1.

EMSA autoradiograph showing the binding of HeLa nuclear extract to ³²P-labeled prototype oligonucleotide nt 7893 to 7915. Lane 1 shows two slowly migrating complexes, C1 and C2, analogous to those reported by O'Connor et al. (13) and believed to represent the monomer and dimer forms of the PSM-BP, respectively. The effect of the A-to-C mutation at nt 7894 is shown by competition with a 200-fold (lanes 2 and 6), 100-fold (lanes 3 and 7), 50-fold (lanes 4 and 8), and 25-fold (lanes 5 and 9) excess of unlabeled prototype (16P7894) (lanes 2 to 5) or unlabeled mutant oligonucleotide (16M7894) (lanes 6 to 9). The PSM-BP bound to both prototype and mutant oligonucleotides, but the unlabeled mutant competed for binding to the dimeric form of PSM-BP (to give C2) less effectively than the prototype did. Binding of the monomer form of PSM-BP (to give C1) was unaffected. Evidence of the specificity of C1 and C2 is provided in lane 10 by competition with a 200-fold excess of an unrelated unlabelled oligonucleotide (16P7676), where binding of both forms of the PSM-BP has been largely unaffected. UC, uncharacterized bands; FP, free probe.