P. Pfeiffer et al., UNUSUAL STRUCTURE OF THE DOUBLE-STRANDED-RNA ASSOCIATED WITH THE 447 CYTOPLASMIC MALE-STERILITY IN VICIA-FABA, Journal of General Virology, 74, 1993, pp. 1167-1173
The 16.7 kbp dsRNA specific to the '447' cytoplasmic male sterility (C
MS) line of Vicia faba was labelled in vitro with [alpha-P-32]ATP and
poly(A) polymerase, and by T4 RNA ligase-mediated addition of [P-32]pC
p. Analysis of the reaction products under denaturing conditions revea
led in both cases extensive labelling of a 4.5 kb ssRNA, already detec
ted in previous experiments in which the RNA-dependent RNA polymerase
associated with the dsRNA was allowed to pursue RNA synthesis on prein
itiated complexes. Mobility shift analysis of total pCp-labelled dsRNA
revealed not two but three different 3' termini. The most prominent s
equencing pattern corresponded to the 4.5 kb ssRNA indicating that thi
s RNA species has a preferentially accessible, free 3' OH extremity. N
orthern blot analysis of the denatured dsRNA confirmed that the 4.5 kb
ssRNA is a subgenomic mRNA and detected its counterpart of about 12 k
b. Nearly all 16.7 kbp dsRNA molecules featured an interrupted positiv
e-sense strand, indicating a marked prevalence of transcription over r
eplication complexes. This unusual strategy of transcription by a stra
nd displacement mechanism, following initiation at an internal discont
inuity, is compared with that of other dsRNA viruses or defective viru
ses, and is discussed in relation to the expression of the CMS trait.