A STUDY OF THE EFFECTS OF ELEVATED TEMPERATURES ON THE GROWTH AND INHERITANCE OF SACCHAROMYCES CEREVISIAE (thesis)

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.

Download Full-text

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1208-1216.1987 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1208-1216 ◽

Cited By ~ 2

Author(s):

D J Hurt ◽

S S Wang ◽

Y H Lin ◽

A K Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Elevated Temperatures ◽

Trna Gene ◽

Single Copy ◽

Molecular Study ◽

Trna Splicing ◽

Intervening Sequences ◽

Copy Gene

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.

Download Full-text

Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

Biochemistry and Cell Biology ◽

10.1139/bcb-2020-0151 ◽

2020 ◽

Vol 98 (5) ◽

pp. 624-630 ◽

Cited By ~ 2

Author(s):

Yanrui Zhu ◽

Matthew D. Berg ◽

Phoebe Yang ◽

Raphaël Loll-Krippleber ◽

Grant W. Brown ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Genetic Disease ◽

Elevated Temperatures ◽

Temperature Sensitive ◽

Wild Type ◽

Standard Genetic Code ◽

Gene Encoding ◽

Chromatid Cohesion ◽

Sensitive Allele

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

Download Full-text

Comparison of the mitochondrial endonucleases from Neurospora crassa and Saccharomyces cerevisiae

Canadian Journal of Microbiology ◽

10.1139/m85-123 ◽

1985 ◽

Vol 31 (7) ◽

pp. 654-656 ◽

Cited By ~ 1

Author(s):

Richard G. von Tigerstrom ◽

Sheilah Stelmaschuk

Keyword(s):

Saccharomyces Cerevisiae ◽

Neurospora Crassa ◽

Mode Of Action ◽

Elevated Temperatures ◽

Physiological Role ◽

Intracellular Location ◽

Substrate Specificities ◽

Structural Differences ◽

Specific Activities

The endonucleases from Neurospora crassa and Saccharomyces cerevisiae are not closely related antigenically. They also differ with respect to their activity at pH 8, their degree of hydrophobicity, and their sensitivity to elevated temperatures. However, the two nucleases have similar specific activities, are inhibited by EDTA, and have nearly identical substrate specificities. Since the enzymes also have the same mode of action and intracellular location, these similarities may indicate that they have the same physiological role despite their structural differences.

Download Full-text

Mid2 Is a Putative Sensor for Cell Integrity Signaling in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.3969 ◽

1999 ◽

Vol 19 (6) ◽

pp. 3969-3976 ◽

Cited By ~ 146

Author(s):

Mathumathi Rajavel ◽

Bevin Philip ◽

Benjamin M. Buehrer ◽

Beverly Errede ◽

David E. Levin

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Surface ◽

Vegetative Growth ◽

Elevated Temperatures ◽

Transmembrane Domain ◽

Wall Stress ◽

Cell Lysis ◽

Mitogen Activated Protein Kinase ◽

Primary Role ◽

Cell Integrity

ABSTRACTHcs77 is a putative cell surface sensor for cell integrity signaling inSaccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39°C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated theMID2gene as a dosage suppressor of the cell lysis defect of anhcs77null mutant.MID2encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog ofMID2, designatedMTL1, which provides a partially redundant function withMID2for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.

Download Full-text

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1208 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1208-1216 ◽

Cited By ~ 61

Author(s):

D J Hurt ◽

S S Wang ◽

Y H Lin ◽

A K Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Elevated Temperatures ◽

Trna Gene ◽

Single Copy ◽

Molecular Study ◽

Trna Splicing ◽

Intervening Sequences ◽

Copy Gene

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.

Download Full-text

Homologous Subunits of 1,3-Beta-Glucan Synthase Are Important for Spore Wall Assembly in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00200-06 ◽

2006 ◽

Vol 6 (2) ◽

pp. 143-156 ◽

Cited By ~ 51

Author(s):

Satoru Ishihara ◽

Aiko Hirata ◽

Satoru Nogami ◽

Anne Beauvais ◽

Jean-Paul Latge ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Elevated Temperatures ◽

Spore Wall ◽

Regulatory Subunit ◽

Major Constituent ◽

Growth Defect ◽

Temperature Sensitive ◽

Beta Glucan ◽

Glucan Synthase ◽

Wall Assembly

ABSTRACT During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-β-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-β-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-β-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-β-glucan synthase.

Download Full-text

Killer toxin of Saccharomyces cerevisiae Y500-4L active against Fleischmann and Itaiquara commercial brands of yeast

Revista de Microbiologia ◽

10.1590/s0001-37141999000300012 ◽

1999 ◽

Vol 30 (3) ◽

pp. 253-257 ◽

Cited By ~ 2

Author(s):

Giselle A.M. Soares ◽

Hélia H. Sato

Keyword(s):

Saccharomyces Cerevisiae ◽

Elevated Temperatures ◽

Killer Toxin ◽

Toxin Production ◽

Chromosomal Dna ◽

Killer Activity ◽

Killer Yeast ◽

Torulopsis Glabrata ◽

Hansenula Anomala ◽

Killer Yeasts

The strain Saccharomyces cerevisiae Y500-4L, previously selected from the must of alcohol producing plants and showing high fermentative and killer capacities, was characterized according to the interactions between the yeasts and examined for curing and detection of dsRNA plasmids, which code for the killer character. The killer yeast S. cerevisiae Y500-4L showed considerable killer activity against the Fleischmann and Itaiquara commercial brands of yeast and also against the standard killer yeasts K2 (S. diastaticus NCYC 713), K4 (Candida glabrata NCYC 388) and K11 (Torulopsis glabrata ATCC 15126). However S. cerevisiae Y500-4L showed sensitivity to the killer toxin produced by the standard killer yeasts K8 (Hansenula anomala NCYC 435), K9 (Hansenula mrakii NCYC 500), K10 (Kluyveromyces drosophilarum NCYC 575) and K11 (Torulopsis glabrata ATCC 15126). No M-dsRNA plasmid was detected in the S. cerevisiae Y500-4L strain and these results suggest that the genetic basis for toxin production is encoded by chromosomal DNA. The strain S. cerevisiae Y500-4L was more resistant to the loss of the phenotype killer with cycloheximide and incubation at elevated temperatures (40oC) than the standard killer yeast S. cerevisiae K1.

Download Full-text

Yeast Isw1p Forms Two Separable Complexes In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.23.1.80-91.2003 ◽

2003 ◽

Vol 23 (1) ◽

pp. 80-91 ◽

Cited By ~ 93

Author(s):

Jay C. Vary, ◽

Vamsi K. Gangaraju ◽

Jun Qin ◽

Carolyn Church Landel ◽

Charles Kooperberg ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Regulation ◽

Chromatin Remodeling ◽

Elevated Temperatures ◽

Cellular Processes ◽

Biochemical Assays ◽

Associated Proteins ◽

Chromatin Remodeling Complexes ◽

Specific Activities

ABSTRACT There are several classes of ATP-dependent chromatin remodeling complexes, which modulate the structure of chromatin to regulate a variety of cellular processes. The budding yeast, Saccharomyces cerevisiae, encodes two ATPases of the ISWI class, Isw1p and Isw2p. Previously Isw1p was shown to copurify with three other proteins. Here we identify these associated proteins and show that Isw1p forms two separable complexes in vivo (designated Isw1a and Isw1b). Biochemical assays revealed that while both have equivalent nucleosome-stimulated ATPase activities, Isw1a and Isw1b differ in their abilities to bind to DNA and nucleosomal substrates, which possibly accounts for differences in specific activities in nucleosomal spacing and sliding. In vivo, the two Isw1 complexes have overlapping functions in transcriptional regulation of some genes yet distinct functions at others. In addition, these complexes show different contributions to cell growth at elevated temperatures.

Download Full-text

Genes Encoding Ribosomal Proteins Rps0A/B of Saccharomyces cerevisiae Interact With TOM1 Mutants Defective in Ribosome Synthesis

Genetics ◽

10.1093/genetics/157.3.1107 ◽

2001 ◽

Vol 157 (3) ◽

pp. 1107-1116

Author(s):

Amy L Tabb ◽

Takahiko Utsugi ◽

Clavia R Wooten-Kee ◽

Takeshi Sasaki ◽

Steven A Edling ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Proteins ◽

Elevated Temperatures ◽

Synergistic Effects ◽

Ribosomal Subunit ◽

Hect Domain ◽

Ribosome Synthesis ◽

Ubiquitin Protein Ligase ◽

40S Ribosomal Subunit ◽

Genes Encoding

Abstract The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

Download Full-text