Yeast killer dsRNA plasmids are transcribedin vivoto produce full and partial-length plus-stranded RNAs

Abstract

In vivo transcripts of the L (4.5 kb) and M (1.9 kb) dsRNA plasmids were examined in type I killers of Saccharomyces cerevisiae. Transcripts for both plasmids include full-length (l,m) and partial-length (la,ma) single-stranded species. Both L-dsRNA transcripts (l,la) have in vitro mRNA activity for L-P1, previously shown to be identical to ScV-P1, the 88,000 dalton major capsid protein of the virus-like particles containing L- and M1-dsRNAs. 1, but not 1a, is bound to poly(U)-sepharose and may be polyadenylated. Other L-dsRNA gene products and their transcripts may exist. For M1-dsRNA, both species (m, ma) have in vitro mRNA activity for M1-P1, the 32,000 dalton pre-protoxin encoded by M1-dsRNA. Both m and ma are bound to poly(U)-Sepharose and ma is probably a 5' terminal fragment of m. A functional model for M1-dsRNA killer plasmid structure is presented.

Saccharomyces cerevisiae killer virus transcripts contain template-coded polyadenylate tracts.

“…2), confirming the results of Bostian et al (8). Therefore, both the in vitro and the two major in vivo transcripts of M dsRNA elute with polyadenylated RNAs having a poly(A) length of 70 to 100 residues (37 ) was applied to a 2.5-ml column of poly(U)-Sepharose which was previously equilibrated in 50 mM Tris-hydrochloride (pH 7.5)-0.7 M NaCI-10 mM disodium-EDTA-25% formamide.…”

“…50 residues (35). Bostian et al (8) have previously reported similar poly(U)-Sepharose binding by the in vivo full-length m and subgenomic ma transcripts, which they propose is due to 3'-terminal poly(A) tracts. They propose that these may be post-transcriptional additions, although they also note that the poly(A) tract for the ma species might also be produced by "chattering" of the virion transcriptase at the A,U-rich "bubble" region.…”

“…In vivo, M dsRNA is transcribed to produce both full-length (1,830 bases) and subgenomic (approximately 1,200 bases) transcripts (8; this work). Yeast mRNA synthesized in vivo which is homologous to M dsRNA has been shown to program the in vitro synthesis of M-p32, the putative killer toxin precursor (8).…”

“…RESULTS Polarity of in vivo M dsRNA-specific transcripts. Killer cells grown in glucose synthesize both full-length (1,830 bases) and subgenomic (1,200 bases) M-specific transcripts which bind to poly(U)-Sepharose (8). Figure 1 shows that poly(A)-enriched RNA from ethanol-grown cells of strain A364A x S7 contains these full-length and subgenomic transcripts, as detected by hybridization with radioactive M dsRNA minus strands.…”

Coupling of killer virus transcription with translation in yeast cell-free extracts

“…The uncharacterized low Mr RNA band (arrow) was found exclusively at the top of the sucrose gradient. The new band was not retained on an oligo(dT)-cellulose column, unlike the m transcript (Hannig et al, 1984) or the subgenomic ma transcript produced in vivo (Bostian et al, 1983).…”