The genomic RNA ofpotyviruses has a characteristic 5' non-translated region (5'NTR) to which a viral protein, VPg, is covalently attached. This suggests that the viral RNA lacks a conventional cap structure and thus its translation may not proceed in the same way as most cellular mRNAs. To investigate the role of the 5'NTR during translation, various derivatives of the turnip mosaic potyvirus (TuMV) leader were fused to the reporter gene fl-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit reticulocyte lysate and in planta following microprojectile DNA delivery into tobacco cell suspensions. GUS transcripts fused with the TuMV 5'NTR, whether they were capped or not, were efficiently translated, whereas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in excess over functional transcripts, translation was inhibited in a dose-dependent manner. Similarly, transcripts synthesized from the reverse complement of the 5'NTR inhibited translation to the same extent as the wild-type sequence, indicating that cap independence was not conferred by a specific sequence within the viral leader. A stable hairpin loop was placed in front or after the viral sequence. This hairpin loop normally prevented translation of control GUS transcripts but when the viral leader was positioned after it a significant level of GUS activity was measured, whether the transcripts were capped or not. On the other hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes bound to an internal site within the TuMV 5'NTR and then presumably scanned the sequence for the initiator AUG.

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PCR amplification with degenerate primers targeted to highly conserved amino acid motifs within the MYB domain was used to demonstrate that black spruce (Picea mariana) possesses a diverse MYB gene family. Amino acid sequence comparisons revealed three broad MYB subfamilies, one of which shares extensive similarity with maize C1, a central regulator of anthocyanin biosynthesis. A cDNA clone encoding a MYBR2R3 protein from P. mariana with high levels of sequence homology to maize C1 was shown to transactivate the Bz2 promoter in combination with maize R in embryonal tissues of both black spruce and larch. Functional dependence on the maize R protein, and the presence of a conserved C-terminal GIDPxTH motif, support the conservation of MYBR2R3 function in conifers, and demonstrate that the basic components of MYBR2R3-dependent transcriptional regulation have been conserved between angiosperms and gymnosperms.