FIG. 1.

Determination of the transcription initiation site of the UXP pre-mRNA by primer extension analysis (A) and RNase protection assay (B). Total cytoplasmic RNAs isolated from mock- or Ad5-infected A549 cells at 24 or 32 h p.i. were used in the assays. (A) The primer used was a 5′-end-labeled oligonucleotide complementary to the first exon of the UXP mRNA. The 5′-end nucleotide of the primer is located 30 bp downstream of the initiation codon, ATG, of the UXP gene. The dideoxy sequence of a plasmid carrying Ad5 bp 30967 to 31418 with the same primer is shown as a size standard. The arrow on the right indicates the major extension product. The extension product in control (Ctrl) RNA was from kanamycin RNA, and its corresponding primer was included in the kit. 5′-end labeled φX174 DNA/HinfI was used as marker (lane M). (B) For RNase protection, the labeled riboprobe corresponding to Ad5 nt 30967 to 31418 was used. The arrow on the left indicates the major protected product. The number next to the arrow on the left indicates the size of the protected product and the location of the initiation site on the Ad5 genome. Torula yeast RNA (yRNA) was used as negative-control RNA. 5′-end-labeled φX174 DNA/HinfI was used as the marker (lane M).