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. 2001 May;75(9):4184–4194. doi: 10.1128/JVI.75.9.4184-4194.2001

FIG. 1.

FIG. 1

(A) Genomic map of RTBV and experimental strategy. The lower part of the figure shows the genome map of RTBV. Viral DNA is represented by a double line, with the box marked R′ indicating the region of the genome that is transcribed twice in the terminally redundant transcript. The thick arrows outside the DNA represent the major viral ORFs (I through IV). Viral transcripts are shown as thin arrows inside the DNA, with the 6.3-kb intron in the spliced transcript encoding ORF IV indicated (dashed line). The basic expression plasmid is represented schematically in the upper part of the panel. The 35S promoter, CAT reporter gene, and RTBV/nos sequences are represented as open boxes. The RTBV, cryptic nos, and nos cleavage sites are indicated with a solid arrow, an open arrowhead, and an open arrow, respectively. The position of the antisense probe transcript for RNase protection analysis is indicated. Homologous probes were used for each construct and were transcribed from linearized plasmid using the T7 promoter present downstream of the nos sequence in the vector. The RTBV sequences inserted in RTPA-L and RTPA-S are indicated with the numbers referring to the transcription start site. Processing efficiencies are given in percent and were roughly the same in N. plumbaginifolia and rice protoplasts, with values from cryptic nos and nos sites being combined as “read through.” Values given are the average of at least three independent transfections. (B) Representative RNase protection assays of RNAs expressed from constructs RTPA-L and RTPA-S. Fragments corresponding to processing at the RTBV, cryptic nos, and nos sites are indicated with a solid arrow, an open arrowhead, and an open arrow, respectively. Signal intensities were always weaker from rice protoplast RNA (RTPA-L not shown). The positions of labeled DNA size markers (pBR322/HpaII) are indicated. The expected sizes of protected fragments at the RTBV, cryptic nos, and nos sites are 515, 818, and 932 nt, or 211, 334, and 448 nt with the RTPA-L and RTBV-S probes, respectively. Signals at the size of the full-length probe in this and other figures are discussed in the text.