Summary auto-generated
This study characterizes the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1, a single-stranded DNA plant virus with a circular genome of approximately 1 kb. Using Northern hybridization and 3' RACE analysis, the researchers identified two polyadenylated RNA transcripts from BBTV DNA-1 in infected banana plants and transgenic tobacco. The larger transcript corresponds to the previously identified major replication protein (Rep) open reading frame (ORF) of 856 nucleotides. Polyadenylation occurs at nucleotide 963, immediately following the stop codon, with the polyadenylation signals contained within the coding region. The second, unexpected transcript derives from a small ORF completely internal to the major Rep ORF in a -2 reading frame. This internal ORF encodes a putative 5 kilodalton protein of unknown function and possesses a large 272-base untranslated region. Both transcripts were also detected in transgenic tobacco plants where the Rep ORF was expressed under a cauliflower mosaic virus promoter, confirming that both transcripts use viral polyadenylation signals. A conserved upstream termination element (A/TTGTAA) was identified in multiple BBTV isolates.
Key findings
- Two polyadenylated mRNAs are transcribed from BBTV DNA-1: one from the major rep gene and one from a small internal ORF encoding a 5 kDa protein
- The major Rep ORF transcript terminates at nucleotide 963 with polyadenylation signals within the coding region immediately after the stop codon
- The internal ORF is completely contained within the major Rep ORF in a -2 frame and has a large 272-nucleotide untranslated region
- Both transcripts maintain identical 3' end sequences when expressed in transformed tobacco plants, indicating viral control of polyadenylation
- A conserved upstream element (A/TTGTAA) was identified in all BBTV DNA-1 isolates, similar to other plant viruses
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Abstract
We have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1. Northern hybridization and 3' RACE analysis identified two poly- adenylated RNAs associated with BBTV DNA-1. Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep). An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF. Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF. This encoded a putative 5 kDa protein of unknown function. Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1. This Rep ORF was inserted 3' of a cauliflower mosaic virus 35S promoter and 5' of a vegetable storage protein terminator. The transcripts mapped from these tobacco plants were identical at the 3' end to the transcripts from BBTV infected banana plants. The site of polyadenylation for the Rep ORF was at base 963 immediately 3' of the translational stop codon confirming that the polyadenylation signals for this transcript were all within the ORF. However, the internal ORF had a large untranslated region of 272 bases with its site of polyadenylation at nucleotide 803 and a polyadenylation signal 3' of the translational stop codon. A possible upstream termination signal (A/TTGTAA) was identified and was conserved within BBTV DNA-1 sequences from different international isolates.