scholarly journals Killer systems in Saccharomyces cerevisiae: three distinct modes of exclusion of M2 double-stranded RNA by three species of double-stranded RNA, M1, L-A-E, and L-A-HN.

1983 ◽  
Vol 3 (4) ◽  
pp. 654-661 ◽  
Author(s):  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Killer Toxin ◽  
Mendelian Inheritance ◽  
Double Stranded Rna ◽  
Heat Curing ◽  
Natural Variants

M1 and M2 double-stranded RNAs (dsRNAs) code for the K1R1 and K2R2 killer toxin and resistance functions, respectively. Natural variants of a larger dsRNA (L-A) carry various combinations of the [EXL], [HOK], and [NEX] genes, which affect the K1 and K2 killer systems. Other dsRNAs, the same size as L-A, called L-B and L-C, are often present with L-A. We show that K1 killer strains have [HOK] and [NEX] but not [EXL] on their L-A (in disagreement with Field et al., Cell 31:193-200, 1982). These strains also carry other L-size molecules detectable after heat-curing has eliminated L-A. The exclusion of M2 dsRNA observed on mating K2 strains with K1 strains is due to the M1 dsRNA (not the L-A dsRNA as claimed by Field et al.) in the K1 strains. Four independent mutants of a [KIL-k2] [NEX-o] [HOK-o] strain were selected for resistance to [EXL] exclusion of M2 ([EXLR] phenotype). The [EXLR] phenotype showed non-Mendelian inheritance in each case, and these mutants had simultaneously each acquired [HOK]. The mutations were located on L-A and not on M2, and did not confer resistance to M1 exclusion of M2.

Killer systems in Saccharomyces cerevisiae: three distinct modes of exclusion of M2 double-stranded RNA by three species of double-stranded RNA, M1, L-A-E, and L-A-HN

1983 ◽  
Vol 3 (4) ◽  
pp. 654-661
Author(s):  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Killer Toxin ◽  
Mendelian Inheritance ◽  
Double Stranded Rna ◽  
Heat Curing ◽  
Natural Variants

M1 and M2 double-stranded RNAs (dsRNAs) code for the K1R1 and K2R2 killer toxin and resistance functions, respectively. Natural variants of a larger dsRNA (L-A) carry various combinations of the [EXL], [HOK], and [NEX] genes, which affect the K1 and K2 killer systems. Other dsRNAs, the same size as L-A, called L-B and L-C, are often present with L-A. We show that K1 killer strains have [HOK] and [NEX] but not [EXL] on their L-A (in disagreement with Field et al., Cell 31:193-200, 1982). These strains also carry other L-size molecules detectable after heat-curing has eliminated L-A. The exclusion of M2 dsRNA observed on mating K2 strains with K1 strains is due to the M1 dsRNA (not the L-A dsRNA as claimed by Field et al.) in the K1 strains. Four independent mutants of a [KIL-k2] [NEX-o] [HOK-o] strain were selected for resistance to [EXL] exclusion of M2 ([EXLR] phenotype). The [EXLR] phenotype showed non-Mendelian inheritance in each case, and these mutants had simultaneously each acquired [HOK]. The mutations were located on L-A and not on M2, and did not confer resistance to M1 exclusion of M2.

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

1984 ◽  
Vol 4 (1) ◽  
pp. 181-187
Author(s):  
M Wesolowski ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mendelian Inheritance ◽  
Chromosomal Dna ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Genetic Elements ◽  
Virus Like Particle ◽  
Particle Preparation ◽  
Mendelian Genetic ◽  
Efficient Transmission

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.

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

1984 ◽  
Vol 4 (1) ◽  
pp. 181-187 ◽  
Author(s):  
M Wesolowski ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mendelian Inheritance ◽  
Chromosomal Dna ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Genetic Elements ◽  
Virus Like Particle ◽  
Particle Preparation ◽  
Mendelian Genetic ◽  
Efficient Transmission

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.

scholarly journals A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 70 ◽  
Author(s):  
Angela Crabtree ◽  
Emily Kizer ◽  
Samuel Hunter ◽  
James Van Leuven ◽  
Daniel New ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Next Generation Sequencing ◽  
Killer Toxin ◽  
Next Generation ◽  
High Quality ◽  
Yeast Saccharomyces Cerevisiae ◽  
Single Strain ◽  
Double Stranded Rna ◽  
Generation Sequencing ◽  
K1 Toxin

Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3′ polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae. The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast.

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins

1990 ◽  
Vol 10 (4) ◽  
pp. 1373-1381
Author(s):  
J Tao ◽  
I Ginsberg ◽  
N Banerjee ◽  
W Held ◽  
Y Koltin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fungal Pathogen ◽  
Secretory Pathway ◽  
Rna Virus ◽  
Killer Toxin ◽  
Ustilago Maydis ◽  
Cleavage Sites ◽  
Double Stranded Rna ◽  
Killer Toxins ◽  
Covalently Linked

There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.

scholarly journals Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

1990 ◽  
Vol 10 (4) ◽  
pp. 1373-1381 ◽  
Author(s):  
J Tao ◽  
I Ginsberg ◽  
N Banerjee ◽  
W Held ◽  
Y Koltin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fungal Pathogen ◽  
Secretory Pathway ◽  
Rna Virus ◽  
Killer Toxin ◽  
Ustilago Maydis ◽  
Cleavage Sites ◽  
Double Stranded Rna ◽  
Killer Toxins ◽  
Covalently Linked

There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.

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.

scholarly journals Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae.

1990 ◽  
Vol 265 (28) ◽  
pp. 17274-17280
Author(s):  
M Tokunaga ◽  
A Kawamura ◽  
K Kitada ◽  
F Hishinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Killer Toxin ◽  
Linear Dna ◽  
Dna Plasmid
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