The osmotic integrity of the yeast cell requires a functional PKC1 gene product.

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.

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Sld2, Which Interacts with Dpb11 inSaccharomyces cerevisiae, Is Required for Chromosomal DNA Replication

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.6102 ◽

1998 ◽

Vol 18 (10) ◽

pp. 6102-6109 ◽

Cited By ~ 107

Author(s):

Yoichiro Kamimura ◽

Hiroshi Masumoto ◽

Akio Sugino ◽

Hiroyuki Araki

Keyword(s):

Dna Replication ◽

Temperature Shift ◽

Growth Defect ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Chromosomal Dna ◽

Autonomously Replicating Sequence ◽

Complementation Groups ◽

Synthetically Lethal ◽

Dna Polymerase Ii

ABSTRACT The DPB11 gene, which genetically interacts with DNA polymerase II (ɛ), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint inSaccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal withdpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11suppressed the temperature-sensitive growth defect caused bysld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.

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Suppressors of a Saccharomyces cerevisiae pkc1 mutation identify alleles of the phosphatase gene PTC1 and of a novel gene encoding a putative basic leucine zipper protein.

Genetics ◽

10.1093/genetics/141.4.1275 ◽

1995 ◽

Vol 141 (4) ◽

pp. 1275-1285 ◽

Cited By ~ 13

Author(s):

K N Huang ◽

L S Symington

Keyword(s):

Protein Kinase ◽

Leucine Zipper ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Growth Defect ◽

Temperature Sensitive ◽

Gene Encoding ◽

Basic Leucine Zipper ◽

Mapk Cascades ◽

Synthetically Lethal

Abstract The PKC1 gene product, protein kinase C, regulates a mitogen-activated protein kinase (MAPK) cascade, which is implicated in cell wall metabolism. Previously, we identified the pkc1-4 allele in a screen for mutants with increased rates of recombination, indicating that PKC1 may also regulate DNA metabolism. The pkc1-4 allele also conferred a temperature-sensitive (ts) growth defect. Extragenic suppressors were isolated that suppress both the ts and hyperrecombination phenotypes conferred by the pkc1-4 mutation. Eight of these suppressors for into two complementation groups, designated KCS1 and KCS2. KCS1 was cloned and found to encode a novel protein with homology to the basic leucine zipper family of transcription factors. KCS2 is allelic with PTC1, a previously identified type 2C serine/threonine protein phosphatase. Although mutation of either KCS1 or PTC1 causes little apparent phenotype, the kcs1 delta ptc1 delta double mutant fails to grow at 30 degrees. Furthermore, the ptc1 deletion mutation is synthetically lethal in combination with a mutation in MPK1, which encodes a MAPK homologue proposed to act in the PKC1 pathway. Because PTC1 was initially isolated as a component of the Hog1p MAPK pathway, it appears that these two MAPK cascades share a common regulatory feature.

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THE SELECTION OF S. CEREVISIAE MUTANTS DEFECTIVE IN THE START EVENT OF CELL DIVISION

Genetics ◽

10.1093/genetics/95.3.561 ◽

1980 ◽

Vol 95 (3) ◽

pp. 561-577 ◽

Cited By ~ 4

Author(s):

Steven I Reed

Keyword(s):

Cell Division ◽

Genetic Analysis ◽

Linkage Map ◽

Nuclear Genes ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mating Pheromone ◽

Preliminary Characterization ◽

Complementation Groups ◽

Selection Of

ABSTRACT Thirty-three temperature-sensitive mutations defective in the start event of the cell division cycle of Saccharomyces cereuisiae were isolated and subjected to preliminary characterization. Complementation studies assigned these mutations to four complementation groups, one of which, cdc28, has been described previously. Genetic analysis revealed that these complementation groups define single nuclear genes, unlinked to one another. One of the three newly identified genes, cdc37, has been located in the yeast linkage map on chromosome IV, two meiotic map units distal to hom2.—Each mutation produces stage-specific arrest of cell division at start, the same point where mating pheromone interrupts division. After synchronization at start by incubation at the restrictive temperature, the mutants retain the capacity to enlarge and to conjugate.

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RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4314 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4314-4326 ◽

Cited By ~ 21

Author(s):

C Mann ◽

J Y Micouin ◽

N Chiannilkulchai ◽

I Treich ◽

J M Buhler ◽

...

Keyword(s):

Cell Division ◽

Amino Acid ◽

Rna Polymerase ◽

Essential Gene ◽

Temperature Sensitive ◽

G1 Arrest ◽

Restrictive Temperature ◽

Amino Acid Residues ◽

Gene Encoding ◽

Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

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The SIT4 protein phosphatase functions in late G1 for progression into S phase

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2133-2148.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2133-2148

Author(s):

A Sutton ◽

D Immanuel ◽

K T Arndt

Keyword(s):

Cell Cycle ◽

Protein Phosphatase ◽

Catalytic Domain ◽

Function Analysis ◽

S Phase ◽

Growth Defect ◽

Temperature Sensitive ◽

Amino Terminal ◽

Polymorphic Gene ◽

Cycle Dependent

Saccharomyces cerevisiae strains containing temperature-sensitive mutations in the SIT4 protein phosphatase arrest in late G1 at the nonpermissive temperature. Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase. While the levels of SIT4 do not change in the cell cycle, SIT4 associates with two high-molecular-weight phosphoproteins in a cell-cycle-dependent fashion. In addition, we have identified a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of SIT4 and can also partially suppress the phenotypic defects due to a null mutation in BCY1. The SSD1 protein is implicated in G1 control and has a region of similarity to the dis3 protein of Schizosaccharomyces pombe. We have also identified a gene, PPH2alpha, that in high copy number can partially suppress the growth defect of sit4 strains. The PPH2 alpha gene encodes a predicted protein that is 80% identical to the catalytic domain of mammalian type 2A protein phosphatases but also has an acidic amino-terminal extension not present in other phosphatases.

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NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2391 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2391-2403 ◽

Cited By ~ 79

Author(s):

C Costigan ◽

D Kolodrubetz ◽

M Snyder

Keyword(s):

Protein Kinase ◽

Synthetic Lethality ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Growth Defect ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mitogen Activated Protein ◽

Multiple Copies ◽

Delta Cells

The yeast SLK1 (BCK1) gene encodes a mitogen-activated protein kinase (MAPK) activator protein which functions upstream in a protein kinase cascade that converges on the MAPK Slt2p (Mpk1p). Dominant alleles of SLK1 have been shown to bypass the conditional lethality of a protein kinase C mutation, pkc1-delta, suggesting that Pkc1p may regulate Slk1p function. Slk1p has an important role in morphogenesis and growth control, and deletions of the SLK1 gene are lethal in a spa2-delta mutant background. To search for genes that interact with the SLK1-SLT2 pathway, a synthetic lethal suppression screen was carried out. Genes which in multiple copies suppress the synthetic lethality of slk1-1 spa2-delta were identified, and one, the NHP6A gene, has been extensively characterized. The NHP6A gene and the closely related NHP6B gene were shown previously to encode HMG1-like chromatin-associated proteins. We demonstrate here that these genes are functionally redundant and that multiple copies of either NHP6A or NHP6B suppress slk1-delta and slt2-delta. Strains from which both NHP6 genes were deleted (nhp6-delta mutants) share many phenotypes with pkc1-delta, slk1-delta, and slt2-delta mutants. nhp6-delta cells display a temperature-sensitive growth defect that is rescued by the addition of 1 M sorbitol to the medium, and they are sensitive to starvation. nhp6-delta strains also exhibit a variety of morphological and cytoskeletal defects. At the restrictive temperature for growth, nhp6-delta mutant cells contain elongated buds and enlarged necks. Many cells have patches of chitin staining on their cell surfaces, and chitin deposition is enhanced at the necks of budded cells. nhp6-delta cells display a defect in actin polarity and often accumulate large actin chunks. Genetic and phenotypic analysis indicates that NHP6A and NHP6B function downstream of SLT2. Our results indicate that the Slt2p MAPK pathway in Saccharomyces cerevisiae may mediate its function in cell growth and morphogenesis, at least in part, through high-mobility group proteins.

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An Essential Function of a Phosphoinositide-Specific Phospholipase C Is Relieved by Inhibition of a Cyclin-Dependent Protein Kinase in the Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/148.1.33 ◽

1998 ◽

Vol 148 (1) ◽

pp. 33-47 ◽

Cited By ~ 2

Author(s):

Jeffrey S Flick ◽

Jeremy Thorner

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Phospholipase C ◽

Growth Defect ◽

Temperature Sensitive ◽

Complete Medium ◽

Restrictive Temperature ◽

Dependent Protein Kinase ◽

Yeast Saccharomyces Cerevisiae ◽

Dependent Protein

Abstract The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the δ isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1Δ cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80Δ) mutation and growth on low-phosphate medium, also permitted growth of plc1Δ cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1Δ cells by pho80Δ does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81Δ and spl2Δ show a synthetic phenotype in combination with plc1Δ. Unlike single mutants, plc1Δ pho81Δ and plc1Δ spl2Δ double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

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RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4314-4326.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4314-4326

Author(s):

C Mann ◽

J Y Micouin ◽

N Chiannilkulchai ◽

I Treich ◽

J M Buhler ◽

...

Keyword(s):

Cell Division ◽

Amino Acid ◽

Rna Polymerase ◽

Essential Gene ◽

Temperature Sensitive ◽

G1 Arrest ◽

Restrictive Temperature ◽

Amino Acid Residues ◽

Gene Encoding ◽

Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

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Senescence Mutants of Saccharomyces cerevisiae With a Defect in Telomere Replication Identify Three Additional EST Genes

Genetics ◽

10.1093/genetics/144.4.1399 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1399-1412 ◽

Cited By ~ 18

Author(s):

Thomas S Lendvay ◽

Danna K Morris ◽

Jeannie Sah ◽

Bhuvana Balasubramanian ◽

Victoria Lundblad

Keyword(s):

Saccharomyces Cerevisiae ◽

Telomerase Activity ◽

Gene Encoding ◽

Epistasis Analysis ◽

Mutant Strains ◽

Primary Determinant ◽

Complementation Groups ◽

Telomere Replication ◽

Senescence Phenotype ◽

Novel Protein

The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cermzsiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity.

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