Figure 1.

Construction of the pTP deletion mutant virus. The RNA polymerase II transcription map for Ad5 is schematically presented at the top. Early region primary transcripts (except for E2B, for which only the primary coding region is presented) are indicated with open arrowheads. Late region mRNAs are presented with solid arrowheads with the primary transcripts for IVa2 and pIX indicated. The coding regions of the major late transcript are presented (L1–L5) along with the tripartite leader segments (TPL1–3) found on each of the mRNAs derived from the major late transcript. The left end of the viral chromosome through 29.4 map units (enlarged within the dashed lines) was used to make plasmid p0-29.4_ΔpTP. Bacterial plasmid sequence is indicated by thin lines, the adenovirus sequence by a thicker line, and the regions of the pTP gene deleted by thickest lines in the enlargement (indicated by dashed lines) of the region encoding the main exon of pTP. The XbaI site within the E1A gene was replaced with a BstBI adaptor and the KpnI site within the E1B gene was deleted by site-specific mutation. p0-29.4_ΔpTP DNA was digested with XbaI and XmnI and ligated with the 29.4–100 map unit fragment of dl308 DNA that had been digested with XbaI and purified by centrifugation twice on 10–40% sucrose gradients. The ligation mixture was used to transfect 293-pTP₂ cells that stably express pTP (11) and 293-pTP₁₄ cells that inducibly express pTP at relatively high concentration (12) using Ca₃(PO₄)₂ precipitation. A schematic representation of dl308_ΔpTP with early region transcripts including the splicing pattern for pTP (dashed lines) and sites for a limited number of restriction enzymes is presented below.