Infectivity studies on different potato spindle tuber viroid (PSTV) RNAs synthesized in vitro with the SP6 transcription system
AUTOR(ES)
Tabler, Martin
RESUMO
We have constructed two sets of clones in which one to six head-to-tail connected DNA copies of the potato spindle tuber viroid (PSTV) RNA genome were inserted into the plasmid pSP62- PL downstream of the promoter for SP6 RNA polymerase. In vitro transcription of these constructs with the promoter-specific SP6 RNA polymerase yielded the corresponding oligomeric single-stranded linear PSTV RNA molecules of (+) and (−) polarity. Except for short vector-derived terminal sequences these in vitro synthesized PSTV RNA forms are equivalent to the RNA intermediates of the PSTV replication cycle which are present in vivo only in extremely low concentrations. From each DNA template molecule up to 600 RNA copies could be transcribed in vitro and yields > 100 µg were obtained. When mechanically inoculated to tomato seedlings the PSTV (+) RNA oligomers were as infectious as the natural PSTV (+) RNA monomers. Surprisingly, the corresponding oligomeric PSTV (−) RNAs were ˜104-fold less infectious. However, when these (−) RNAs were partially protected prior to inoculation by mixing or hybridizing them with non-infectious (+) RNA fragments or by `capping' their 5' terminus, an increase in the number of infections was observed. The in vitro synthesis of infectious RNA from cloned cDNA means that, in principle, it should be possible to develop vector systems from pathogens with RNA genomes.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=554485Documentos Relacionados
- Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV)
- Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells
- Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid.
- Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious.
- Separation and infectivity of circular and linear forms of potato spindle tuber viroid.