FIG. 4.

Processing at the RTBV poly(A) site in a promoter-proximal position gives rise to SS-RNA in transfected protoplasts. (A) Constructs consisting of the RTBV (to −681) or CaMV 35S (to −343) upstream promoter sequences, the RTBV leader sequence (either complete [wt] or deleted between +8 and +83 [Δ]) and the CAT reporter gene fused to RTBV ORF I are shown. All constructs are terminated by the CaMV polyadenylation signal. The location of the homologous region of the antisense probe used for RNase A/T₁ mapping, and the extent of protected fragments corresponding to SS and RT transcripts, are indicated. (B and C) RNase protection analysis. Total RNA isolated from transfected rice (B) or N. plumbaginifolia (C) protoplasts was subjected to RNase A/T₁ protection analysis with an antisense probe transcribed from RTPA-L. Protected fragments corresponding to RTBV SS and RT transcripts for the four constructs used are indicated, with the percentage of SS shown below the gels. ∗, Values with the RTBV-Δ construct in N. plumbaginifolia were too low to quantify reliably. Other values given are the average from two independent experiments (variation was within 10% of the mean). i.c., internal control; pDES7 (see Materials and Methods).