Figure 2

Splicing activity assays of SD sites within the CAEV env gene. A, Schematic representation of constructs used for splicing activity assays. Reporter constructs were based on the vector pKCR3 which contained the β-globin intron flanked by its splicing sequences inserted between the early promoter and poly-A site of SV40. CAEV sequences are included in open boxes. In all constructs, the β-globin SD site was replaced by CAEV sequences containing the SD⁶¹²³ (grey box) and SD⁶¹⁴⁰ (hatched box) sites. In plasmids pKRmB1 and pKRB1, the β-globin SA site was substituted by the 3' end viral genome containing the SA⁸⁵¹⁴ site. The positions of the primers used for PCR amplification of cDNA are indicated (horizontal arrows). The positions of probes MarN2 and MarS used in southern blot analysis are indicated. The MarN PCR primer used in experiment reported in Fig. 4 is indicated. B, RT-PCR analysis of RNAs extracted from transfected 293T cells. cDNAs were PCR amplified using primer pairs PK5 and PK3, or PK5 and M3b, as indicated. PCR products were resolved on an agarose gel and visualized by ethidium bromide staining. Lane M, DNA size markers. C, Southern blot analysis of transcripts from cells transfected with pKRmB1 and pKRB1 plasmids. PCR-amplified cDNAs were fractionated through a 2.5% agarose gel, blotted to nylon, and hybridized to probes MarN2 (left panel) and MarS (right panel).