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R B Wickner

Publications and source records attributed to R B Wickner.

At least 127 records · Page 7Linked to original sources
PubMed1983

Evidence for a new chromosome in Saccharomyces cerevisiae.

The current yeast map has 16 chromosomes, each originally defined by a centromere-linked gene unlinked to previously defined centromere markers. We examined four genes, cly2, KRB1, AMY2, and tsm0115, each centromere linked, but previously thought to be not on chromosomes I to XVI. We found that AMY2 is linked to cly2, and both are on chromosome II. tsm0115 is on the left arm of chromosome XVI. We confirm the earlier evidence that KRB1 is not on chromosomes I through XVI. This gene thus defines a new chromosome XVII. We also report meiotic linkage of met4 and pet8 (on chromosome XIV), confirming the connection between the petx-kex2 fragment of XIV and the centromere of XIV.

Chromosome Mapping↗
PubMed1983

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.

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.

Gene Expression Regulation↗
PubMed1982

Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes [HOK], [NEX] and [EXL] are on one of these dsRNAs.

[HOK], [NEX] and [EXL] are non-Mendelian genes affecting the K1 and K2 killer systems of Saccharomyces cerevisiae. T1 fingerprints of L double-stranded RNA from [HOK]-, [NEX]- and [EXL]-containing cells and their heat-cured derivatives indicate that: there are three distinct double-stranded RNAs, L-A, L-B and L-C; [HOK], [NEX] and [EXL] are all located on L-A; there are three functional variants of L-A that produce the [HOK] [NEX], [HOK] [EXL] or [EXL] phenotypes; L-A is compatible with L-B or L-C; and there are additional sequences present in lower copy number. Although their fingerprint patterns are unrelated, solution hybridization shows that L-B and L-C share sequence homology. Strains carrying L-A as the major double-stranded RNA or only L-B or only L-C all have similarly sedimenting (160S) virus-like particles with RNA polymerase activity. Virus-like particles from strains with L-A all have proteins of 81,000 and 180,000 daltons that are absent from isogenic strains cured of L-A. Virus-like particles from strains with only L-B or only L-C both have major proteins of 77,000 and 73,000 daltons.

DNA, Fungal↗
PubMed1982

Ribosomal protein L3 is involved in replication or maintenance of the killer double-stranded RNA genome of Saccharomyces cerevisiae.

Ability to secrete the K1 (or K2) toxin protein and immunity to that toxin [the K1 (or K2) killer trait] are determined by a double-stranded (ds) RNA, called M1 (or M2), whose replication and maintenance depend on at least one of the larger (L) ds RNAs and 29 chromosomal genes, called MAK genes (maintenance of killer). The location of the MAK8 gene near TCM1 (trichodermin resistance) on the yeast map suggested the possible identity of these two genes. Of six independently isolated tcm1 mutants, five were clearly mak-, and the sixth was weakly mak-. In each case, the mak- phenotype and the trichodermin-resistant phenotypes cosegregated in meiosis and showed the expected tight linkage to pet17. The mak- mutations in the trichodermin-resistant strains did not complement mak8-1, indicating that MAK8 and TCM1 are the same gene. The mak8-1 mutation does not make strains resistant to trichodermin, and one tcm1 mutation is only slightly mak-. Whereas tcm1 mutants lose M1 or M2 ds RNA, they do not lose L ds RNA. Because TCM1 codes for ribosomal protein L3 [Fried, H. M. & Warner, J. R. (1981) Proc. Natl. Acad. Sci, USA 78, 238--242], we conclude that ribosomal protein L3 is involved in the replication and maintenance of M ds RNA. Mutations in cyh2 or cry1, producing resistance to cycloheximide and crytopleurine due to mutant ribosomal proteins, do not produce a mak- phenotype. In analogy with bacterial ribosome assembly mutants, yeast low-temperature-sensitive (lts) mutants may have defective ribosomes. We thus examined mutants for an effect on the killer system. An lts5 mutant, unable to grow at 5 degrees C, also has a mak- phenotype (at 30 degrees C) that cosegregates in meiosis with the lts- phenotype. Mutations in seven other lts genes do not result in the mak- phenotype.

Chromosome Mapping↗
PubMed1982

[HOK], a new yeast non-Mendelian trait, enables a replication-defective killer plasmid to be maintained.

The K1 killer plasmid, [KIL-k1], of Saccharomyces cerevisiae is a 1.25 x 10(6) 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 psi+ strains to psi-. [HOK] is also distinct from the previously described plasmids [URE3], 20S RNA, 2 mu DNA, and [EXL]. Strains lacking [HOK] consistently have a four-fold lower copy number of L double-stranded RNA than strains carrying [HOK].

Crosses, Genetic↗
PubMed1982

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.

Plasmids↗
PubMed1981

Curing of the 2 mu DNA plasmid from Saccharomyces cerevisiae.

The 2 mu DNA plasmid is often eliminated from yeast cells when they are transformed with the 2 mu DNA-LEU2-pMB9 composite plasmid pJDB219. Since pJDB219 is subsequently lost with high frequency, derivatives lacking all 2 mu DNA can be prepared from any strain.

DNA, Fungal↗
PubMed1980

Virion DNA-independent RNA polymerase from Saccharomyces cerevisiae.

The "killer" plasmid and a larger double-stranded RNA plasmid of yeast exist in intracellular virion particles. Purification of these particles from a diploid killer strain of yeast (grown into stationary growth on ethanol) resulted in co-purification of a DNA-independent RNA polymerase activity. This activity incorporates and requires all four ribonucleoside triphosphates and will not act on deoxyribonucleoside triphosphates. The reaction requires magnesium, is inhibited by sulfhydryl-oxidizing reagents and high concentrations of monovalent cation, but is insensitive to DNase, alpha-amanitin, and actinomycin D. Pyrophosphate inhibits the reaction as does ethidium bromide. Exogenous nucleic acids have no effect on the reaction. The product is mostly single-stranded RNA, some of which is released from the enzymatically active virions.

DNA-Directed RNA Polymerases↗
PubMed1980

Plasmids controlled 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 X 10(6) dalton double-stranded RNA plasmid, [KIL-k1], while K2 killers have a 1.0 X 10(6) dalton double-stranded RNA plasmid, [KIL-k2]. Mating [KIL-k1] haploids with [KIL-k2] haploids yields only [KIL-k1] diploids, that is, [KIL-k1] excludes [KIL-k2]. [EXL], a new non-Mendelian genetic element from a nonkiller strain, excludes [KIL-k2] but does not exclude [KIL-k1]. 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, MAK10 and PET18. [NEX] replication depends on MAK3 but is independent of MAK4, MAK6, MAK27 and MKT1.

Crosses, Genetic↗
PubMed1980

"Superkiller" mutations suppress chromosomal mutations affecting double-stranded RNA killer plasmid replication in saccharomyces cerevisiae.

Saccharomyces cerevisiae strains carrying a 1.5 x 10(6)-dalton double-stranded RNA genome in virus-like particles (killer plasmid) secrete a protein toxin that kills strains not carrying this plasmid. At least 28 chromosomal genes (mak genes) are required to maintain or replicate this plasmid. Recessive mutations in any of four other chromosomal genes (ski for superkiller) result in enhanced toxin production. We report that many ski- mak- double mutants are able to maintain the killer plasmid, indicating that the SKI products have an effect on plasmid replication. The ski1-1 mutation suppresses (bypasses) all mak mutations tested except mak16-1. A variant killer plasmid is described that confers the superkiller phenotype and, like chromosomal ski mutations, makes several mak genes dispensable for plasmid replication.

Genes, Lethal↗
PubMed1980

A stable plasmid carrying the yeast Leu2 gene and containing only yeast deoxyribonucleic acid.

The plasmid pSLe1 is a deletion derivative of the yeast-Escherichia coli hybrid plasmid pJDB219, obtained by HindIII digestion, ligation, and transformation directly into Saccharomyces cerevisiae. pSLe1 has only yeast sequences; it contains one of the inverted repeated sequences of plasmid 2muDNA and the LEU2 gene. pSLe1 is stably maintained in yeast cells without selective pressure. pSLe1 is about half as large as 2muDNA, but pSLe1 does not displace the normal 2muDNA.

DNA Restriction Enzymes↗
PubMed1980

Cloning of the URA1 gene of Saccharomyces cerevisiae.

A 5.7-kilobase segment of Saccharomyces cerevisiae deoxyribonucleic acid which complements both the yeast ura1 and Escherichia coli pyrD mutations in dihydroorotate dehydrogenase has been cloned in plasmid YRp7.

Cloning, Molecular↗
PubMed1980

Isolation and characterization of temperature-sensitive mak mutants of Saccharomyces cerevisiae.

The K1 killer plasmid of Saccharomyces cerevisiae is a 1.5-megadalton linear double-stranded ribonucleic acid molecule. Using simplified screening and complementation procedures, we have isolated mutants in three chromosomal genes that are temperature sensitive for killer plasmid maintenance or replication. One of these genes, mak28-1, was located on chromosome X. Two of the temperature-sensitive mutants rapidly lost the wild-type killer plasmid of A364A during spore germination and outgrowth at nonpermissive temperatures, but during vegetative growth, they only lowered the plasmid copy number. These two mutants did not lose two other wild-type K1 killer plasmids, indicating a heterogeneity of the killer plasmids in laboratory yeast strains.

Chromosome Mapping↗
PubMed1979

Mapping chromosomal genes of Saccharomyces cerevisiae using an improved genetic mapping method.

A triploid (3n) strain of Saccharomyces cerevisiae was constructed carrying a standard marker on each of chromosomes 1 through XVII in the -/+/+ configuration. This is called a "supertriploid." Meiotic spores from this strain (n + approximately n/2) were mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n + approximately 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 + approximately 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 alpha 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.

Aneuploidy↗
PubMed1979

Mak mutants of yeast: mapping and characterization.

Killer strains of Saccharomyces cerevisiae are those carrying a 1.5 x 10(6)-dalton double-stranded (ds) ribonucleic acid (RNA) (M) in virus-like particles and secreting a protein toxin. Most yeast (koller or not) also carry a 3 x 10(6)-dalton dsRNA (L). We have mapped mutations in eight of the chromosomal genes needed for maintaining M (mak genes). The mak genes are widely distributed on the yeast map, with no multigene complexes. We show that mutants defective in these and other mak genes lose M dsRNA, but not L dsRNA. The mak3-1 mutation results in markedly decreased cellular levels of L dsRNA, but mak3-1 stains do not lose L dsRNA completely. Mutation of mak16 results in temperature-sensitive growth, whereas mutations in mak13, mak15, mak17, mak20, mak22, and mak27 result in slow growth at any temperature. No effect of mak mutations on mating, meiosis, sporulation, germination, homothallism, or ultraviolet sensitivity has been found. The specificity of mak mutations is discussed.

Chromosome Mapping↗
PubMed1978

Pet18: a chromosomal gene required for cell growth and for the maintenance of mitochondrial DNA and the killer plasmid of yeast.

Mutations in the pet18 gene of Saccharomyces cerevisiae (formerly denoted pets) confer three phenotypes on mutant strains: (i) inability to respire (petite), (ii) inability to maintain the double-stranded RNA killer plasmid (sensitive), and (iii) temperature sensitivity for growth. We find that pet18 mutants lack mitochondrial DNA. However, despite their inability to maintain the killer RNA plasmid and mitochondrial DNA, pet18 mutants still can carry the other yeast plasmids, [URE3--1], [PSI], and 2-micron DNA. The temperature sensitivity of the pet18 mutants is not expressed as a selective defect in total DNA, RNA, or protein synthesis.

DNA, Mitochondrial↗