FIG. 3.

RNase protection assays to characterize the 5′ and 3′ ends of ORF 71- and 72-associated transcripts. (A) A 373-nucleotide ³²P-labeled antisense riboprobe (KSHV nucleotides 123,568 to 123,874 plus vector sequence) was transcribed in vitro using T7 RNA polymerase. Undigested probe is shown in lane 1. Probe was hybridized with 10 μg of total RNA from KSHV-negative human HeLa cells (lane 2), Dox-treated TRExBCBL1-Rta cells (lane 3), mock-treated TRExBCBL1-Rta cells (lane 4), BC-1 cells (lane 5), and BC3 cells (lane 6). After hybridization and RNase digestion, protected fragments were resolved on an 8 M urea-8% polyacrylamide gel and visualized by autoradiography. An open arrow indicates full-length probe. Fragment sizes were calculated from a DNA sequencing ladder run in adjacent lanes (not shown). (B) A 388-nucleotide antisense riboprobe (lane 1) corresponding to KSHV nucleotides 121,820 to 122,205 (plus 2 additional nucleotides), was hybridized to total RNA isolated from HeLa (lane 2) and Dox- or mock-treated TRExBCBL1-Rta cells (lanes 3 and 4).