FIG. 8.
Subcellular distribution of unspliced and spliced chimeric RNA. (A) Schematic representation of chimeric β-globin/HIV-1 introns. Exons are depicted as boxes, and introns are shown as lines. Empty boxes and lines denote sequences of the parental plasmid, pSVβ; black boxes and lines denote HIV-1 sequences. The chloramphenicol acetyltransferase (CAT) open reading frame (not shown) is located further downstream in the second exon. β 5′ss, β-globin 5′ss; 3′ss, HIV-1 tat/rev intron 3′ss. The HIV-1-derived sequences in pSVHTSB include 236 nt of the tat/rev intron and 85 nt of the HIV-1 downstream exon. pSVCTSB differs from pSVHTSB only in the deletion of ∼200 nt of intron sequence as indicated. Also shown are the probes used for the detection of the unspliced and spliced RNA from the constructs. Homologous sequence spanned from the EcoRI site within the CAT gene to 25 nt 3′ of the 5′ss. The β probe was used to probe RNA from pSVβ. The HT probe was used to analyze RNA from both pSVHTSB and pSVCTSB. (B) S1 nuclease protection analysis of subcellular RNA (5 μg of either nuclear [N] or cytoplasmic [C] RNA) isolated from COS-7 cells transfected with pSVβ, pSVHTSB, or pSVCTSB. 5′-end-labeled probes used for S1 analysis span part of the CAT gene and the entire HIV-1 segment and contain heterologous sequences (wavy line) at their 3′ ends. Bands corresponding to unspliced (U) and spliced (S) protection products of pSVHTSB (HT) or pSVCTSB (CT) are indicated.