Identification of the polyA addition site of the APH-2 transcript. (A) RNA samples obtained from 293T cells transfected with the pHTLV-2 ΔEcoRI plasmid (lane 2) were analyzed by 3′ RLM-RACE using the 21-3 primer. Lane 1 represents PCR amplification in the absence of any cDNA. M indicates 100-bp marker. (B) Position of the polyA addition site next to a consensus polyA signal and a GT-rich consensus sequence. The sequences of the APH-2 mRNA and of the 3′ polyA tail are shown. ■ represents the coding portion of the APH-2 spliced transcript. HTLV-2 sequences retrieved from GenBank were also compared using MO as a reference sequence. Comparisons were focused on the AATAAA polyA signal (position 5185 on the sense strand), the cleavage site deduced from our 3′RACE results and the GT-rich sequence. (C) PolyA⁺ RNA was extracted from pHTLV-2 ΔEcoRI-transfected 293T cells (lane 1) or from nontransfected cells (lane 2) and was run on a 1.5% agarose gel. After migration and transfer, the membrane was hybridized with a probe corresponding to the coding segment of the APH-2 cDNA and signals were revealed with a PhosphorImager. An RNA marker was migrated in parallel and corresponding molecular weights are indicated on the left side of the panel.