Figure 7

Modulatory effect of Tat on antisense transcription. (A) Jurkat cells were transfected with 10 μg of circular or BamHI-digested pAsLTRXLuc plasmid in combination with 5 μg of pCMV-tat or empty pCMV vector. (B) 293T cells were transfected with 400 ng of circular or BstZ17I-digested pNL4.3ΔBstAsLucR-E-, 200 ng of pActin-LacZ and 200 ng of pCMV-tat or empty vector. (C) 293T cell clones and a pooled population stably transfected with pNL4.3ΔBstAsLucR-E- were transfected with 200 ng pCMV-tat or empty vector. Results are presented as fold induction compared to empty vector. (D) A pool of 293T cells stably transfected for pNL4.3ΔNar1 was transfected with 5 μg of pCMV-tat or the empty pCMV5 vector. cDNA synthesis was performed with random primers. PCR amplifications were performed to detect the presence of the HIV antisense transcript (lane 3-4-7-8) using 24-6/25-3 primers. β-actin amplification was performed as control (lane 1-2-5-6). Lanes 1, 2, 3 and 4 represent control for DNA contamination to which RNA was directly added for PCR amplification. (E) RNA from the transfected 293T cells described in Dwere also used for amplification of antisense transcripts from 24-6F-synthesized cDNA using 30-20 (anchor) primer in combination with 25-3 (lane 1, 3, 5 and 7) or 26-5 (lanes 2, 4, 6 and 8) primers. Samples were tested for cDNA cleanup efficiency (lanes 1, 2, 5 and 6). Tat expression in transfected cells is indicated above the gel for both latter panels. Luciferase activities in A, Band Crepresent the mean value of three measured samples ± S.D.