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.

In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30 degrees C but not at 20 degrees C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mks1 and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30 degrees C and prevent the cells from growing at 8 degrees C. Elimination of M double-stranded RNA from a cold-sensitive ski- strain results in the loss of cold sensitivity. ski- [KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-k1] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski- strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI+ products.     

Plasmids controlling exclusion of the K2 killer double-stranded RNA plasmid of yeast

Saccharomyces strains of two types (K₁⁺R₁⁺ and K₂⁺R₂⁺) kill each other and K^?R^?-sensitive strains by secreting protein toxins. K₁ killer strains carry a 1.25 × 10⁶ dalton double-stranded RNA plasmid, [KIL-k₁], while K₂ killers have a 1.0 × 10⁶ dalton double-stranded RNA plasmid, [KIL-k₁]. Mating [KIL-k₁] haploids with [KIL-k₂] haploids yields only [KIL-k₁] diploids, that is, [KIL-k₁] excludes [KIL-k₂]. [EXL], a new non-Mendellan genetic element from a nonkiller strain, excludes [KIL-k₂] but does not exclude [KIL-k₂]. A second new non-Mendelian genetic element, called [NEX], when present prevents [EXL] from excluding [KIL-k₂]. [NEX] does not prevent [KIL-k₁] or [KIL-s₁] (a suppressive mutant of [KIL-k₁]) from excluding [KIL-k₂]. A chromosomal gene, called MKT1, is needed for maintenance of [KIL-k₂] if [NEX] is present. In the absence of [NEX], [KIL-k₂] does not need MKT1. [KIL-k₁] does not need MKT1 even if [NEX] is present. [EXL] replication depends on at least the products of MAK1, MAK3, MAK10and PET18. [NEX]replication depends on MAK3 but is independent of MAK4, MAKE, MAK27 and MKT1.     

Two new double-stranded RNA molecules showing non-mendelian inheritance and heat inducibility in Saccharomyces cerevisiae.

Certain strains of Saccharomyces cerevisiae were found to have a complex nuclear defect (designated clo-) that makes cells unable to maintain some L-B and some L-C double-stranded RNAs at 25 degrees C. The clo- strains were not defective in maintenance of L-A, M1, or M2 double-stranded RNAs. Most clo-strains lacking L and M carry small amounts of two double-stranded RNA species intermediate in size between L and M and denoted T (2.7 kilobase pairs) and W (2.25 kilobase pairs). Some strains carry both T and W, some carry neither, and some carry only W; no strains carrying only T have been found. Both T and W show 4+:0 segregation in meiosis and efficient transmission by cytoplasmic mixing (cytoduction), indicating that they are non-Mendelian genetic elements. T and W do not cross-hybridize with each other or with L-A, L-B, L-C, M1, M2, or chromosomal DNA. T and W are apparently distinct from other known non-Mendelian genetic elements (2mu DNA, [rho], [psi], 20S RNA, [URE3]). In most strains the copy number of both T and W is increased about 10-fold by the growth of cells at 37 degrees C. This heat inducibility of T and W is under control of a cytoplasmic gene. T and W double-stranded RNAs are not found in a purified L-containing virus-like particle preparation from a strain containing L-B, T, and W double-stranded RNAs. The role, if any, of T or W in the killer systems is not known.     

Phenethyl Alcohol Resistance in ESCHERICHIA COLI. III. a Temperature-Sensitive Mutation (dnaP) Affecting DNA Replication

A temperature-sensitive DNA replication mutant of E. coli K-12 was isolated among the mutants selected for phenethyl alcohol resistance at low temperatures. This mutation, designated as dnaP18, affects sensitivity of the cell to phenethyl alcohol, sodium deoxycholate and rifampicin, presumably due to an alteration in the membrane structure. At high temperatures (e.g., 42 degrees ), synthesis of DNA, but not RNA or protein, is arrested, leading to the formation of "filaments" in which no septum formation is apparent. Nucleoids observed under electron microscope seem to become dispersed and DNA fibrils less condensed, which may explain the loss of viability under these conditions. Genetic analyses, including reversion studies, indicate that a recessive dnaP mutation located between cya and metE on the chromosome is responsible for both alterations of the membrane properties and temperature sensitivity. The dnaP18 mutation does not affect growth of phage T4 or lambda under conditions where host DNA replication is completely inhibited. Kinetic studies of DNA replication and cell division in this mutant after the temperature shift from 30 to 42 degrees , and during the subsequent recovery at 30 degrees , accumulated evidence suggesting that DNA replication comes to a halt at 42 degrees upon completion of a cycle already initiated before the temperature shift. Since the recovery of DNA synthesis after exposure to 42 degrees does not depend on protein or RNA synthesis or other energy-requiring processes, the product of the mutant dnaP gene appears to be reversibly inactivated at 42 degrees . Taken together with the recessive nature of the present mutation, it was suggested that one of the membrane proteins involved in initiation of DNA replication is affected in this mutant.     

Structure and expression of the M2 genomic segment of a type 2 killer virus of yeast.

The M2 double-stranded (ds) RNA species encodes toxin and resistance functions in Saccharomyces cerevisiae strains with the K2 killer specificity. RNA sequence analysis reveals the presence of a large open reading frame on the larger heat-cleavage product of M2 dsRNA, which is translated in vitro to yield a 28 kd polypeptide as a major product. The postulated translation initiator AUG triplet is located within a stem and loop structure near the 5' terminus of the positive strand, which also contains plausible 18S and 5.8S ribosomal RNA binding sites. These features may serve to regulate the translation of the K2 toxin precursor. The M1 (from type 1 yeast killers) and M2 dsRNA species lack extensive sequence homology, although specific features are shared, which may represent structural elements required for gene expression and replication.     

Denaturation and renaturation of Penicillium chrysogenum mycophage double-stranded ribonucleic acid in tetraalkylammonium salt solutions

The base composition dependence of double-stranded ribonucleic acid (RNA) melting was studied by observing the structure and widths of melting transitions for Penicillium chrysogenum mycophage RNA as well as differences in melting temperatures of two RNAs of different base composition. Double-stranded RNA melting is independent of base compositions in 3.5 M Et4NCl and 4.6 M Me4NCl, where the melting temperatures are 25 and 92 degrees C, respectively. Double-stranded RNA renaturation rate constants are reported in Et4NCl solutions. The nucleation rate constant is about 10 times lower than that for double-stranded deoxyribonucleic acid. Analyses of renaturation kinetics results lead to the conclusion that each of the three similar but separable RNA segments of Penicillium chrysogenum mycophage is unique.     

Deletion of a yeast small nuclear RNA gene impairs growth.

We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.     

Co-curing of plasmids affecting killer double-stranded RNAs of Saccharomyces cerevisiae: [HOK], [NEX], and the abundance of L are related and further evidence that M1 requires L.

We describe two sets of plasmid-plasmid interactions in the yeast Saccharomyces cerevisiae. [HOK], [EXL], [NEX], and [KIL-k1] are genetically defined plasmids, and M1 and L are biochemically defined double-stranded RNA plasmids. We show that (i) [HOK], [NEX], and the abundance of L are related, and (ii) under submaximal curing conditions, all colonies retaining M1 also retain L. There are three pieces of evidence that either [NEX] required [HOK] for replication or [NEX] and [HOK] are on the same plasmid. The evidence is as follows. (i) The great majority of strains containing [HOK] also contain [NEX]. However, two [HOK] [NEX-o] strains do exist. (ii) Growth at 39 degrees C or growth at 34 degrees C with 3% ethanol or 2-propanol cures [HOK] and [NEX]. In a [HOK] [NEX] strain, the two plasmids are always co-cured. (iii) [HOK] and [NEX] are both maintained in mak4, mak6, and mak27 strains (mak = maintenance of [KIL-k1]), but not in mak3, mak10, and pet18 strains. Strains containing [HOK] and [NEX] have about fourfold more L double-stranded RNA than their isochromosomal, cured derivatives. In addition, a cytoductant which has acquired [HOK] and [NEX] has fourfold more L than its parent. These results are consistent with either [HOK] being a form of L or [HOK] increasing the copy number of L. Using a K1 killer strain in which L, as well as M1, could be cured by growth at 38 degrees C, we examined the distribution of loss of M1 and L under conditions giving 98% M-o colonies and at least 50% L-o colonies. No M1L-o colonies were observed, supporting the previous suggestion by others that M1 requires L.     

Maintenance of double-stranded telomeric repeats as the critical determinant for cell viability in yeast cells lacking Ku

The Saccharomyces cerevisiae Ku complex, while important for nonhomologous DNA end joining, is also necessary for maintaining wild-type telomere length and a normal chromosomal DNA end structure. Yeast cells lacking Ku can grow at 23 degrees C but are unable to do so at elevated temperatures due to an activation of DNA damage checkpoints. To gain insights into the mechanisms affected by temperature in such strains, we isolated and characterized a new allele of the YKU70 gene, yku70-30(ts). By several criteria, the Yku70-30p protein is functional at 23 degrees C and nonfunctional at 37 degrees C. The analyses of telomeric repeat maintenance as well as the terminal DNA end structure in strains harboring this allele alone or in strains with a combination of other mutations affecting telomere maintenance show that the altered DNA end structure in yeast cells lacking Ku is not generated in a telomerase-dependent fashion. Moreover, the single-stranded G-rich DNA on such telomeres is not detected by DNA damage checkpoints to arrest cell growth, provided that there are sufficient double-stranded telomeric repeats present. The results also demonstrate that mutations in genes negatively affecting G-strand synthesis (e.g., RIF1) or C-strand synthesis (e.g., the DNA polymerase alpha gene) allow for the maintenance of longer telomeric repeat tracts in cells lacking Ku. Finally, extending telomeric repeat tracts in such cells at least temporarily suppresses checkpoint activation and growth defects at higher temperatures. Thus, we hypothesize that an aspect of the coordinated synthesis of double-stranded telomeric repeats is sensitive to elevated temperatures.     

The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein

MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W). The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein. The C-terminal region is lysine-rich and is necessary for mak11-complementing activity. The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane. We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation. The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients. The fusion protein could be released from the membrane fraction by salt washing. Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight. While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.     

Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR.

The porcine group C rotavirus (Cowden strain) NSP3 protein (the group C equivalent of the group A gene 7 product, formerly called NS34) shares homology with known double-stranded RNA-binding proteins, such as the interferon-induced, double-stranded RNA-dependent protein kinase PKR. A clone of NSP3, expressed both in vitro and in COS-1 cells, led to the synthesis of minor amounts of a product with an M(r) of 45,000 (the expected full-length M(r) of NSP3) and major amounts of products with M(r)s of 38,000 and 8,000. Restriction enzyme digestion analysis prior to expression in vitro and amino-terminal sequence analysis suggest that the products with M(r)s of 38,000 and 8,000 are cleavage products of the protein with an M(r) of 45,000. The full-length protein and the product with an M(r) of 8,000, both of which contain the motif present in double-stranded RNA-binding proteins, bound specifically to double-stranded RNA. The products with M(r)s of 45,000 and 8,000 were also detected in Cowden strain-infected MA104 cells. NSP3 products expressed in COS-1 cells were capable of inhibiting activation of the double-stranded RNA-dependent protein kinase similar to other double-stranded RNA-binding proteins, and NSP3 products expressed in HeLa cells were capable of rescuing the replication of an interferon-sensitive deletion mutant of vaccinia virus.     

The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I

The paramyxovirus Sendai (SV), is a well-established inducer of IFN-alphabeta gene expression. In this study we show that SV induces IFN-alphabeta gene expression normally in cells from mice with targeted deletions of the Toll-IL-1 resistance domain containing adapters MyD88, Mal, Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), and TRIF-related adaptor molecule TLR3, or the E3 ubiquitin ligase, TNFR-associated factor 6. This TLR-independent induction of IFN-alphabeta after SV infection is replication dependent and mediated by the RNA helicase, retinoic acid-inducible gene-I (RIG-I) and not the related family member, melanoma differentiation-associated gene 5. Furthermore, we characterize a RIG-I-like RNA helicase, Lgp2. In contrast to RIG-I or melanoma differentiation-associated gene 5, Lgp2 lacks signaling caspase recruitment and activation domains. Overexpression of Lgp2 inhibits SV and Newcastle disease virus signaling to IFN-stimulated regulatory element- and NF-kappaB-dependent pathways. Importantly, Lgp2 does not prevent TLR3 signaling. Like RIG-I, Lgp2 binds double-stranded, but not single-stranded, RNA. Quantitative PCR analysis demonstrates that Lgp2 is present in unstimulated cells at a lower level than RIG-I, although both helicases are induced to similar levels after virus infection. We propose that Lgp2 acts as a negative feedback regulator of antiviral signaling by sequestering dsRNA from RIG-I.     

Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease.

Saccharomyces cerevisiae strains are often host to several types of cytoplasmic double-stranded RNA (dsRNA) genomes, some of which are encapsidated by the L-A dsRNA product, an 86,000-dalton coat protein. Here we present the finding that nuclear recessive mutations in the NUC1 gene, which encodes the major nonspecific nuclease of yeast mitochondria, resulted in at least a 10-fold increase in amounts of the L-A dsRNA and its encoded coat protein. The effect of nuc1 mutations on L-A abundance was completely suppressed in strains that also hosted the killer-toxin-encoding M dsRNA. Both NUC1 and nuc1 strains containing the L-A genome exhibited an increase in coat protein abundance and a concomitant increase in L-A dsRNA when the cells were grown on a nonfermentable carbon source rather than on glucose, an effect independent of the increase in coat protein due to nuc1 mutations or to the absence of M. The increase in L-A expression in nuc1 strains was similar to that observed in strains with mutations in the nuclear gene encoding the most abundant outer mitochondrial membrane protein, porin. nuc1 mutations did not affect the level of porin in the mitochondrial outer membrane. Since the effect of mutations in nuc1 was to alter the copy number of the L-A coat protein genome rather than to change the level of the M toxin genome (as do mak and ski mutations), these mutations define a new class of nuclear genes affecting yeast dsRNA abundance.     

Accumulation of pre-tRNA splicing ''2/3'' intermediates in a Saccharomyces cerevisiae mutant.

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature-sensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre-tRNA(Phe) in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate. The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed. This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15 degrees C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey, M. and Culbertson, M.R. (1988) Genetics, 118, 609-617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.     

Double-stranded cucumovirus associated RNA 5: experimental analysis of necrogenic and non-necrogenic variants by temperature-gradient gel electrophoresis.

Cucumber mosaic virus (CMV) and peanut stunt virus (PSV) each contain a fifth major RNA in the size range of 334 to 393 nucleotides. This fifth RNA is a satellite capable of modulating the expression of viral disease symptoms. It is present in infected tissue in single-stranded and double-stranded form. Nucleotide sequence variants of the double-stranded CMV-associated RNA 5 (dsCARNA 5) and PSV-associated RNA 5 (dsPARNA 5) were analysed by temperature-gradient gel electrophoresis. Gels were 5% polyacrylamide, containing 8 M urea in 8.9 mM Tris-borate buffer, with temperature differences of 25-40 degrees C establishing gradients either perpendicular or parallel to the direction of the electric field. For dsCARNA 5 two characteristic transitions were detected with increasing temperature: at temperatures between 40 degrees C and 46 degrees C a drastic retardation in electrophoretic mobility induced by partial dissociation of the duplex structure from the ends and at temperatures above 52 degrees C an abrupt increase in mobility due to complete strand dissociation. dsPARNA 5 exhibited both transitions at up to 10 degrees C higher temperatures and an additional retardation between the transitions mentioned. Seven different variants of dsCARNA 5, 4 necrogenic and 3 non-necrogenic, were analysed. Some showed only one single band, others gave rise to up to six well separated bands corresponding to six molecular species. From all experimental results a correlation between the temperature of the retardation transition and the necrogenicity of CARNA 5 was derived. The diagnostic application of the temperature-gradient gel analysis in agriculture, particularly for the use of non-necrogenic variants as biological control agents to impede CMV-infections, is discussed.     

A mutant killer plasmid whose replication depends on a chromosomal "superkiller" mutation

Yeast strains carrying a 1.5 x 10(6) molecular weight linear double-stranded RNA in virus-like particles (M dsRNA, the killer plasmid or virus) secrete a toxin that is lethal to strains not carrying this plasmid. Recessive mutations in any of four chromosomal genes (called ski1-ski4) result in increased production of toxin activity. We report here a mutation of the killer plasmid (called [KIL-sd] for ski-dependent) that makes the killer plasmid dependent for its replication on the presence of a chromosomal mutation in any ski gene. Thus, the [KIL-sd] plasmid is lost from SKI(+) strains. When the wild-type killer plasmid, [KIL-k], is introduced into a ski2-2 [KIL-o] strain, the killer plasmid changes to a [KIL-sd] plasmid. This may represent a specific form of mutagenesis or selective replication in the ski2-2 strain of [KIL-sd] variants (mutants) in the normal [KIL-k] population. The ski2-1 and ski2-3 mutations do not convert [KIL-k] to [KIL-sd], but ski2-3 does allow maintenance of the [KIL-sd] plasmid. The [KIL-sd] plasmid thus lacks a plasmid site or product needed for replication in wild-type cells.     

Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase.

The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E. coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R. (1989) Cell 58, 409-419). We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter. The purified protein possesses a distinct DNA topoisomerase activity: similar to E. coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA. Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA. A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined. These sequences differ from those recognized by E. coli DNA topoisomerase I but resemble those recognized by E. coli DNA topoisomerase III. Based on these results, the yeast TOP3 gene product can formally be termed S. cerevisiae DNA topoisomerase III. Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.