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. 2002 Mar;76(5):2410–2423. doi: 10.1128/jvi.76.5.2410-2423.2002

FIG. 6.

FIG. 6.

Polyadenylated 5"HS5 LTR RNAs detected by RT-PCR. (a) DNA sequences of the U3 promoter (P) and R regions of 5"HS5 LTR. The two AATAAA motifs with heavy underlines are the TATA box in the promoter and the potential polyadenylation signal in the R region, respectively. Angled arrow, LTR transcriptional initiation site. Underlined bases in the promoter region, binding sites for transcription factors AML1 (GTGGT), CCAAT, CACCC, and GATA (21, 23, 28). Thin horizontal arrows, F1 and F2 forward primers used in the RT-PCRs to amplify LTR cDNAs. (b) The AATAAA motif in the R region did not serve as the polyadenylation signal to terminate the LTR R RNAs. Top, genomic map of the region between the 5"HS5 LTR and the HS5 site. Angled arrow, transcriptional initiation site of the LTR R RNA. ∗, locations of the AATAAA motifs in the LTR and six additional AATAAA motifs or potential polyadenylation signals between the 5"HS5 LTR and the 3" end of the HS5 site. Left-to-right arrows, polyadenylated LTR R RNAs; dotted lines, different DNA sequences present in the 3" ends of the R RNAs generated by different potential polyadenylation signals in the region. (A)n, poly(A) tails of unknown lengths. (T)33, oligo(dT) primer with 33 Ts used as the reverse primer in cDNA synthesis and PCR. Horizontal line bracketed by forward F1 and reverse (T)33 primers, PCR products generated from the cDNAs by primer pair F1-(T)33. Thick lines bracketed by arrowheads, second-round nested PCRs amplified from the F1-(T)33 PCR products by nested primer pairs F2-G1 and F3-G2. The positions of all horizontal arrows, lines, and arrowheads are aligned with the genomic map of the region at the top. (c) Gel electrophoresis of PCR and nested PCR products. Left panel, PCR bands generated by the F1-(T)33 primer pair from LTR R RNAs of placenta (lane P), Bewo (lane B), and K562 (lane K) cells. Lane M, DNA size markers, the same as in Fig. 2c. The PCR bands were generated from cDNA templates after 25 PCR cycles. Center and right panels, the nested PCR bands were amplified from the F1-(T)33 PCR products after 25 additional PCR cycles by nested primer pair F2-G1 or F3-G2.