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1 Institut de Biologie Moléculaire des Plantes, 12, rue du Général Zimmer, 67000 Strasbourg
and2 Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue Descartes, 67000 Strasbourg, France
The 16.7 kbp dsRNA specific to the ‘447’ cytoplasmic male sterility (CMS) line of Vicia faba was labelled in vitro with [
-32P]ATP and poly(A) polymerase, and by T4 RNA ligase-mediated addition of [32P]pCp. Analysis of the reaction products under denaturing conditions revealed in both cases extensive labelling of a 4.5 kb ssRNA, already detected in previous experiments in which the RNA-dependent RNA polymerase associated with the dsRNA was allowed to pursue RNA synthesis on preinitiated complexes. Mobility shift analysis of total pCp-labelled dsRNA revealed not two but three different 3' termini. The most prominent sequencing pattern corresponded to the 4.5 kb ssRNA, indicating that this RNA species has a preferentially accessible, free 3' OH extremity. Northern blot analysis of the denatured dsRNA confirmed that the 4.5 kb ssRNA is a subgenomic mRNA and detected its counterpart of about 12 kb. Nearly all 16.7 kbp dsRNA molecules featured an interrupted positive-sense strand, indicating a marked prevalence of transcription over replication complexes. This unusual strategy of transcription by a strand displacement mechanism, following initiation at an internal discontinuity, is compared with that of other dsRNA viruses or defective viruses, and is discussed in relation to the expression of the CMS trait.
Received 2 November 1992;
accepted 14 January 1993.
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