Electron microscopic heteroduplex analysis of "killer" double-stranded RNA species from yeast.

Abstract

Wild-type and mutant double-stranded RNA (dsRNA) species from the yeast Saccharomyces cerevisiae were studied by electron microscopic heteroduplex mapping to determine the sequence relationships among the different RNA molecules. Three mutant dsRNAs, 1.5, 1.4, and 0.73 kilobase, were found to be derived by the same internal deletion of the wild-type (1.83 kilobases) molecule. This deletion includes a segment of about 200 base pairs that was estimated to be nearly 100% A + U. In addition, the sequences of the two larger mutant RNA species are tandem, direct duplications. One of the duplicated molecules appears to have a second internal deletion that occurred after the duplication. The mutant dsRNAs are functionally similar to the defective interfering virus particles of anima viruses all of the mutant species prevent the propagation of the wild-type dsRNA when both are present in the same cell. The four dsRNAs share the same sequences at their termini, a finding that may suggest that these sequences are important for the replication of the dsRNAs.Viruses and virus-like particles containing double-stranded RNA (dsRNA) are common to many species of fungi (1). Killer strains of the yeast Saccharomyces cerevisiae possess viral particles with two separately encapsidated dsRNA species: large (L), 2.5 X 106 daltons; and medium (M), 1.18 X 106 daltons (see refs. 2 and 3 for recent reviews). These strains excrete a toxic protein that kills sensitive strains but not killer strains. The M-dsRNA was believed to produce toxin and immunity to toxin because (i) the non-Mendelian segregation of these two killer traits is paralleled by cytoplasmic transmission of the dsRNA, and (ii) strains with an alteration in either toxin production or immunity either lack the M-dsRNA or have an altered form of it (4, 5). Recently, in vitro translation experiments have proved that M encodes the killer toxin (J. Hopper, personal communication). Denatured M-dsRNA directed the synthesis of a protein that, although larger than killer toxin, crossreacted with antiserumto toxin and contained all of the tryptic peptides of toxin produced in vivo. In similar in vitro experiments, the L-dsRNA directed the synthesis of the major capsid protein of the virus-like particles (6).Nonkiller mutants have been isolated that lack the M-dsRNA but have smaller (S) dsRNA species (7). When these mutants are mated to a killer strain the S-dsRNAs prevent or "suppress" the propagation of the M-dsRNA in the resulting diploids. With continued growth, the diploid cells rapidly become nonkillers and the M-dsRNA can no longer be detected. These suppressive S-dsRNAs therefore, are analogous to defective interfering virus particles, the subgenomic nucleic acid species of animal viruses that limit the replication of the standard virus during co-infection (8).This report describes electron microscopic heteroduplex mapping experiments between the M-and S-dsRNAs. The 4224 heteroduplexes studied show that the S-dsRNAs arose by deletions of M-dsRNA sequences. The various S-...

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

“…These are thus partial-length molecules with homology to M1, like the S (suppressive) dsRNAs previously described (16) and previously shown to be in VLPs (17).…”

Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase

“…Single-stranded DNA (M13mp18; 7250 nt; Yanisch-Perron et al, 1985), dsDNA (pMal-c 2 linearized with HindIII; 6646 bp) and dsRNA (yeast killer virus; 5200 bp, 2900 bp and 2200 bp; Fried & Fink, 1978) competed similarly but less efficiently than ssRNA, with 50 % inhibition observed at ratios ranging from 22 to 45. Taken together, these results show that HC-Pro has a preference for ssRNA [with the exception of poly(A)] rather than for DNA (ss or ds) or dsRNA.…”

Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN.

“…ItsS was mapped near the centromere of chromosome XVI (30), and we show here that it is the same as mak6. Mutations in 11 other MAK genes (mak3, 4,7,8,10,11,12,16,18,19, and 27) do not cause cold sensitivity for growth (R. B. Wickner, unpublished data), and among the low temperature-sensitive mutants of Singh and Manney, only ltsS (mak6) shows the mak-phenotype (30). The ski mutants were first discovered based on their increased production of the K1 toxin (the superkiller phenotype) (18).…”

“…dsRNA was purified by cellulose chromatography and analyzed on agarose gels as previously described (18) or by the rapid method described by Fried and Fink (8). Briefly, spheroplasts produced by zymolyase were lysed by sodium dodecyl sulfate, and the extract was digested with proteinase K, extracted with phenol, and precipitated with ethanol; dsRNA was purified by cellulose chromatography and analyzed on 1% agarose gels.…”