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. 2000 Jan;20(1):113–125. doi: 10.1128/mcb.20.1.113-125.2000

FIG. 2.

FIG. 2

Use of G-less cassette templates for transcriptional analysis of HPV-11 E6 promoter activity. (A) HPV-11 G-less cassettes. Plasmids p7072-70GLess/I+ and p7072-70GLess/I− contain the HPV-11 URR spanning nucleotides 7072 to 7933/1 to 70 linked to either a G-less cassette of 388 nucleotides preceded by the adenovirus MLP Inr element (I+) or another G-less cassette of 377 nucleotides without the MLP Inr (I−). Plasmids p7862-70GLess/I+ and p7862-70GLess/I− containing a truncated HPV-11 URR from nucleotide 7862 to nucleotide 70 without CEI and CEII were similarly linked to G-less cassettes with or without the MLP Inr. The compilation of cis-acting elements and trans-acting factors in the HPV-11 URR is mainly based on published information (28, 44, 72). The boundaries of CEI (28) and CEII (13) and the origin of replication (7) are indicated by brackets. (B) Start site mapping of HPV-11 and MLP G-less cassette templates. In vitro transcription reactions were performed with HeLa nuclear extracts with either an adenovirus MLP-driven G-less cassette template (pMLΔ53 [42]), HPV-11 p7072-70GLess/I+ and p7862-70GLess/I+ templates (both give the same start sites and are thus indicated as HPV/I+), or p7072-70GLess/I− and p7862-70GLess/I− templates (both give the same start sites and are thus indicated as HPV/I−). The transcription start sites (arrows), MLP Inr (underlined), and TATA boxes (brackets) are shown on the left of each panel with intensities of relative start sites denoted by dots. (C) Comparison of nucleotide sequences surrounding the core promoter elements of the MLP and HPV templates. HPV-11 nucleotides are in boldface while vector or MLP sequences are in regular type. Brackets indicate the boundaries of the TATA boxes with lines above the sequence demarking the MLP initiator element. The promoter-proximal E2-binding sites (E2-BS 3 and E2-BS 4) are underlined, and the nucleotides (open arrows) corresponding to HPV-11 number designations are indicated in parentheses. (D) E2-mediated repression of the homologous E6 promoter. In vitro transcription was performed with HeLa nuclear extracts with 50 ng of each HPV and pMLΔ53 templates, in the absence (−) or presence of increasing amounts of baculovirus-expressed E2 protein.