scholarly journals [HOK], A NEW YEAST NON-MENDELIAN TRAIT, ENABLES A REPLICATION-DEFECTIVE KILLER PLASMID TO BE MAINTAINED

Genetics ◽  
1982 ◽  
Vol 100 (2) ◽  
pp. 159-174
Author(s):  
Reed B Wickner ◽  
Akio Toh-E
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Double Stranded Rna ◽  
Protein Toxin ◽  
Defective Mutant ◽  
Chromosomal Genes ◽  
Killer Plasmid ◽  
New Yeast ◽  
Lower Copy ◽  
Host Strains

ABSTRACT The K1 killer plasmid, [KIL-k1], of Saccharomyces cerevisiae is a 1.25 × 106 dalton linear double-stranded RNA plasmid coding for a protein toxin and immunity to that toxin. The [KIL-sd1] plasmid is a replication-defective mutant of [KIL-k1] that depends on one of the recessive chromosomal superkiller (ski  -) mutations for its maintenance (Toh-e and Wickner 1979). This report concerns a means by which [KIL-sd1] can be stably maintained in a SKI  + host. Strains carrying a plasmid we call [HOK] (helper of killer) stably maintain [KIL-sd1]. [HOK] segregates 4 [HOK]:0 in meiotic crosses and is efficiently transferred by cytoplasmic mixing (heterokaryon formation). [HOK] depends for its maintenance on the products of PET18, MAK3, and MAK10, three chromosomal genes needed to maintain [KIL-k1], but is independent of 10 other MAK genes and of MKT1. [HOK] is not mitochondrial DNA and is unaffected by agents which convert ψ+ strains to ψ-. [HOK] is also distinct from the previously described plasmids [URE3], 20S RNA, 2 µ DNA, and [EXL]. Strains lacking [HOK] consistently have a four-fold lower copy number of L double-stranded RNA than strains carrying [HOK].

scholarly journals TWENTY-SIX CHROMOSOMAL GENES NEEDED TO MAINTAIN THE KILLER DOUBLE-STRANDED RNA PLASMID OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1978 ◽  
Vol 88 (3) ◽  
pp. 419-425
Author(s):  
Reed B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Double Stranded Rna ◽  
Plasmid Maintenance ◽  
Protein Toxin ◽  
Yeast Strains ◽  
Chromosomal Genes ◽  
Complementation Groups ◽  
Killer Plasmid

ABSTRACT The double-stranded RNA killer plasmid gives yeast strains carrying it both the ability to secrete a protein toxin and immunity to that toxin. This report describes a new series of mutants in chromsomal genes needed for killer plasmid maintenance (mak genes). These mutants comprise 12 complementation groups. There are a total of at least 26 mak genes. Each mak gene product is needed for plasmid maintenance in diploids as well as in haploids. None of these mak mutations prevent the killer plasmid from entering the mak  - spores in the process of meiotic sporulation. Complementation between mak mutants can be performed by mating meiotic spores from a makx/α plasmid-carrying diploid with a maky haploid. If x = y, about half the diploid clones formed lose the killer plasmid. If x # y, complementation occurs, and all of the diploid clones are killers.

scholarly journals TWO CHROMOSOMAL GENES REQUIRED FOR KILLING EXPRESSION IN KILLER STRAINS OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1976 ◽  
Vol 82 (3) ◽  
pp. 429-442
Author(s):  
Reed B Wickner ◽  
Michael J Leibowitz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Mutant Phenotype ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Killer Strain ◽  
Chromosomal Genes ◽  
Complementation Groups ◽  
Killer Plasmid ◽  
Type Strains

ABSTRACT The killer character of yeast is determined by a 1.4 × 106 molecular weight double-stranded RNA plasmid and at least 12 chromosomal genes. Wild-type strains of yeast that carry this plasmid (killers) secrete a toxin which is lethal only to strains not carrying this plasmid (sensitives). —— We have isolated 28 independent recessive chromosomal mutants of a killer strain that have lost the ability to secrete an active toxin but remain resistant to the effects of the toxin and continue to carry the complete cytoplasmic killer genome. These mutants define two complementation groups, kex1 and kex2. Kex1 is located on chromosome VII between ade5 and lys5. Kex2 is located on chromosome XIV, but it does not show meiotic linkage to any gene previously located on this chromosome. —— When the killer plasmid of kex1 or kex2 strains is eliminated by curing with heat or cycloheximide, the strains become sensitive to killing. The mutant phenotype reappears among the meiotic segregants in a cross with a normal killer. Thus, the kex phenotype does not require an alteration of the killer plasmid. —— Kex1 and kex2 strains each contain near-normal levels of the 1.4 × 106 molecular weight double-stranded RNA, whose presence is correlated with the presence of the killer genome.

Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein

1988 ◽  
Vol 8 (2) ◽  
pp. 938-944
Author(s):  
H Uemura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Efficient Production ◽  
Double Stranded Rna ◽  
Transfection Experiment ◽  
Heat Curing ◽  
Viral Particles ◽  
Chromosomal Genes ◽  
Viruslike Particles ◽  
Chromosomal Mutations

For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required. The requirement for some of these genes can be completely suppressed by a cytoplasmic element, [B] (for bypass). We have isolated a mutant unable to maintain [B] (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A. The heat curing of [B] always coincided with the loss of L-A. To confirm that [B] is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without [B] activity and transfected these purified particles into a strain which did not have either L-A or M1. The transfectants harboring L-A and M1 from a [B] strain showed the [B] phenotype, but the transfectants with L-A and M1 from a [B-o] strain did not show the [B] phenotype. Furthermore, the transfectants having L-A from a [B] strain and M1 from a [B-o] strain also showed the [B] phenotype. Therefore, we concluded that [B] is a property of a variant of L-A. In the transfection experiment, we also proved that the superkiller phenotype of the [B] strain is a property of L-A and that L-A with [B] activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles. These data suggest that MAK genes whose mutations are suppressed by [B] are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.

scholarly journals Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein.

1988 ◽  
Vol 8 (2) ◽  
pp. 938-944 ◽  
Author(s):  
H Uemura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Efficient Production ◽  
Double Stranded Rna ◽  
Transfection Experiment ◽  
Heat Curing ◽  
Viral Particles ◽  
Chromosomal Genes ◽  
Viruslike Particles ◽  
Chromosomal Mutations

For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required. The requirement for some of these genes can be completely suppressed by a cytoplasmic element, [B] (for bypass). We have isolated a mutant unable to maintain [B] (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A. The heat curing of [B] always coincided with the loss of L-A. To confirm that [B] is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without [B] activity and transfected these purified particles into a strain which did not have either L-A or M1. The transfectants harboring L-A and M1 from a [B] strain showed the [B] phenotype, but the transfectants with L-A and M1 from a [B-o] strain did not show the [B] phenotype. Furthermore, the transfectants having L-A from a [B] strain and M1 from a [B-o] strain also showed the [B] phenotype. Therefore, we concluded that [B] is a property of a variant of L-A. In the transfection experiment, we also proved that the superkiller phenotype of the [B] strain is a property of L-A and that L-A with [B] activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles. These data suggest that MAK genes whose mutations are suppressed by [B] are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.

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

1986 ◽  
Vol 6 (5) ◽  
pp. 1552-1561
Author(s):  
R Esteban ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Major Coat Protein ◽  
Double Stranded Rna ◽  
Dsrna Molecule ◽  
Protein Toxin ◽  
New Type ◽  
Viruslike Particles ◽  
M1 Rna

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

scholarly journals MUTANTS OF THE KILLER PLASMID OF SACCHAROMYCES CEREVISIAE DEPENDENT ON CHROMOSOMAL DIPLOIDY FOR EXPRESSION AND MAINTENANCE

Genetics ◽  
1976 ◽  
Vol 82 (2) ◽  
pp. 273-285
Author(s):  
Reed B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperature ◽  
Mating Type ◽  
Chromosomal Gene ◽  
Wild Type ◽  
Opposite Mating Type ◽  
Diploid Clone ◽  
Wild Type Phenotype ◽  
Chromosomal Genes ◽  
Killer Plasmid

ABSTRACT Mutants of the killer plasmid of Saccharomyecs cerevisiaehave been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.—Haploids carrying these mutant plasmids (called d for diploid-dependent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K+R+) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.—Sporulation of the phenotypically K+R+ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.

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

1986 ◽  
Vol 6 (5) ◽  
pp. 1552-1561 ◽  
Author(s):  
R Esteban ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Major Coat Protein ◽  
Double Stranded Rna ◽  
Dsrna Molecule ◽  
Protein Toxin ◽  
New Type ◽  
Viruslike Particles ◽  
M1 Rna

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

scholarly journals A MUTANT KILLER PLASMID WHOSE REPLICATION DEPENDS ON A CHROMOSOMAL "SUPERKILLER" MUTATION

Genetics ◽  
1979 ◽  
Vol 91 (4) ◽  
pp. 673-682
Author(s):  
Akio Toh-E ◽  
Reed B Wickner
Keyword(s):  
Molecular Weight ◽  
Specific Form ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Virus Like Particles ◽  
Chromosomal Mutation ◽  
Recessive Mutations ◽  
Yeast Strains ◽  
Chromosomal Genes ◽  
Killer Plasmid

ABSTRACT Yeast strains carrying a 1.5 × 106 molecular weight linear double-stranded RNA in virus-like particles (M dsRKA, 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 skil-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.

scholarly journals MAPPING CHROMOSOMAL GENES OF SACCHAROMYCES CEREVISIAE USING AN IMPROVED GENETIC MAPPING METHOD

Genetics ◽  
1979 ◽  
Vol 92 (3) ◽  
pp. 803-821
Author(s):  
Reed B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Mapping ◽  
Rapid Method ◽  
Genetic Map ◽  
Mapping Method ◽  
Single Chromosome ◽  
Meiotic Analysis ◽  
Chromosomal Genes ◽  
Killer Plasmid ◽  
Standard Marker

ABSTRACT A triploid (3n) strain of Saccharomyces cereuisiae was constructed carrying a standard marker on each of chromosomes I through XVII in the ——/+J+ configuration. This is called a "supertriploid." Meiotic spores from this strain (n + ∼ n/2) were mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n + ∼ n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n + ∼ n/2) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the -/+ configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and α strains, each pair carrying the same set of markers. Using this new, more rapid method ("supertriploid mapping"), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.

scholarly journals DOMINANT CHROMOSOMAL MUTATION BYPASSING CHROMOSOMAL GENES NEEDED FOR KILLER RNA PLASMID REPLICATION IN YEAST

Genetics ◽  
1977 ◽  
Vol 87 (3) ◽  
pp. 453-469
Author(s):  
Reed B Wickner ◽  
Michael J Leibowitz
Keyword(s):  
Plasmid Replication ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Virus Like Particles ◽  
Chromosomal Mutation ◽  
Reversion Frequency ◽  
Yeast Strains ◽  
Chromosomal Genes ◽  
Chromosomal Changes ◽  
Killer Plasmid

ABSTRACT Yeast strains carrying a double-stranded RNA plasmid of 1.4-1.7 × 106 daltons encapsulated in virus-like particles secrete a toxin that kills strains lacking this plasmid. The plasmid requires at least 24 chromosomal genes (pets, and mak1 through mak23) for its replication or maintenance. We have detected dominant Mendelian mutations (called KRB1 for killer replication bypass) that bypass two chromosomal genes, mak7 and pets, normally needed for plasmid replication. Strains mutant in mak7 and carrying the bypass mutation (mak7-1 KRB1) are isolated as frequent K+R+ sectors of predominantly K-R- segregants from crosses of mak7-1 with a wild-type killer. All KRB1 mutations isolated in this way are inherited as single dominant centromere-linked chromosomal changes. They define a new centromere. KRB1 is not a translational suppressor. KRB1 strains contain a genetically normal killer plasmid and ds RNA species approximately the same in size and amount as do wild-type killers. Bypass of both mak7 and pets by one mutation suggests that these two genes are functionally related. Two properties of the inheritance of KRB1 indicate an unusually high reversion frequency: (1) Heat or cycloheximide (treatments known to cure strains of the wild-type killer plasmid) readily induce conversion of mak7-1 KRB1 strains from killers to nonkillers with concomitant disappearance of KRB1 as judged by further crosses, and (2) mating two strains of the type mak7-1 KRB1 with each other yields mostly 2 K+R+: 2 K-R- segregation, although the same KRB1 mutation and the same killer plasmid are present in both parents.

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