DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae.

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

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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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MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.123.2.387 ◽

1993 ◽

Vol 123 (2) ◽

pp. 387-403 ◽

Cited By ~ 58

Author(s):

M T Brown ◽

L Goetsch ◽

L H Hartwell

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Structural Integrity ◽

Spindle Pole ◽

Chromosome Loss ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

In Vitro Mutagenesis ◽

Spindle Elongation

The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.

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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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Efficient Chromosome Biorientation and the Tension Checkpoint in Saccharomyces cerevisiae both Require Bir1

Molecular and Cellular Biology ◽

10.1128/mcb.01911-08 ◽

2009 ◽

Vol 29 (16) ◽

pp. 4552-4562 ◽

Cited By ~ 13

Author(s):

Vasso Makrantoni ◽

Michael J. R. Stark

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Chromosome Segregation ◽

Temperature Sensitive ◽

Protein Kinase Activity ◽

Restrictive Temperature ◽

Microtubule Depolymerization ◽

Spindle Poles ◽

Sister Kinetochore

ABSTRACT Accurate chromosome segregation requires the capture of sister kinetochores by microtubules from opposite spindle poles prior to the initiation of anaphase, a state termed chromosome biorientation. In the budding yeast Saccharomyces cerevisiae, the conserved protein kinase Ipl1 (Aurora B in metazoans) is critical for ensuring correct chromosomal alignment. Ipl1 associates with its activators Sli15 (INCENP), Nbl1 (Borealin), and Bir1 (Survivin), but while Sli15 clearly functions with Ipl1 to promote chromosome biorientation, the role of Bir1 has been uncertain. Using a temperature-sensitive bir1 mutant (bir1-17), we show that Bir1 is needed to permit efficient chromosome biorientation. However, once established, chromosome biorientation is maintained in bir1-17 cells at the restrictive temperature. Ipl1 is partially delocalized in bir1-17 cells, and its protein kinase activity is markedly reduced under nonpermissive conditions. bir1-17 cells arrest normally in response to microtubule depolymerization but fail to delay anaphase when sister kinetochore tension is reduced. Thus, Bir1 is required for the tension checkpoint. Despite their robust mitotic arrest in response to nocodazole, bir1-17 cells are hypersensitive to microtubule-depolymerizing drugs and show a more severe biorientation defect on recovery from nocodazole treatment. The role of Bir1 therefore may become more critical when spindle formation is delayed.

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Pga1 Is an Essential Component of Glycosylphosphatidylinositol-Mannosyltransferase II of Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0258 ◽

2007 ◽

Vol 18 (9) ◽

pp. 3472-3485 ◽

Cited By ~ 17

Author(s):

Keisuke Sato ◽

Yoichi Noda ◽

Koji Yoda

Keyword(s):

Saccharomyces Cerevisiae ◽

Essential Gene ◽

Carboxypeptidase Y ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Chain Reaction ◽

Essential Component ◽

Polymerase Chain ◽

Triton X ◽

Synthetic Intermediate

The Saccharomyces cerevisiae essential gene YNL158w/PGA1 encodes an endoplasmic reticulum (ER)-localized membrane protein. We constructed temperature-sensitive alleles of PGA1 by error-prone polymerase chain reaction mutagenesis to explore its biological role. Pulse-chase experiments revealed that the pga1ts mutants accumulated the ER-form precursor of Gas1 protein at the restrictive temperature. Transport of invertase and carboxypeptidase Y were not affected. Triton X-114 phase separation and [3H]inositol labeling indicated that the glycosylphosphatidylinositol (GPI)-anchoring was defective in the pga1ts mutants, suggesting that Pga1 is involved in GPI synthesis or its transfer to target proteins. We found GPI18, which was recently reported to encode GPI-mannosyltransferase II (GPI-MT II), as a high-copy suppressor of the temperature sensitivity of pga1ts. Both Gpi18 and Pga1 were detected in the ER by immunofluorescence, and they were coprecipitated from the Triton X-100–solubilized membrane. The gpi18ts and pga1ts mutants accumulated the same GPI synthetic intermediate at the restrictive temperature. From these results, we concluded that Pga1 is an additional essential component of the yeast GPI-MT II.

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end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.120.1.55 ◽

1993 ◽

Vol 120 (1) ◽

pp. 55-65 ◽

Cited By ~ 247

Author(s):

S Raths ◽

J Rohrer ◽

F Crausaz ◽

H Riezman

Keyword(s):

Saccharomyces Cerevisiae ◽

Lucifer Yellow ◽

Fluid Phase ◽

Cell Surface Receptor ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Dependent Manner ◽

Fluid Phase Endocytosis ◽

Alpha Factor

alpha-factor, one of two peptide hormones responsible for synchronized mating between MATa and MAT alpha-cell types in Saccharomyces cerevisiae, binds to its cell surface receptor and is internalized in a time-, temperature-, and energy-dependent manner (Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). After internalization, alpha-factor is delivered to the vacuole via vesicular intermediates and degraded there consistent with an endocytic mechanism (Singer, B., and H. Riezman. 1990. J. Cell Biol. 110:1911-1922; Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). We have isolated two mutants that are defective in the internalization process. Both mutations confer a recessive, temperature-sensitive growth phenotype upon cells that cosegregates with their endocytosis defect. Lucifer yellow, a marker for fluid-phase endocytosis, shows accumulation characteristics in the mutants that are similar to the uptake characteristics of 35S-alpha-factor. The endocytic defect in end4 cells appears immediately upon shift to restrictive temperature and is reversible at permissive temperature if new protein synthesis is allowed. Furthermore, the end4 mutation only affects alpha-factor internalization and not the later delivery of alpha-factor to the vacuole. Other vesicle-mediated processes seem to be normal in end3 and end4 mutants. END3 and END4 are the first genes shown to be necessary for the internalization step of receptor-borne and fluid-phase markers in yeast.

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CDP1, a Novel Saccharomyces cerevisiae Gene Required for Proper Nuclear Division and Chromosome Segregation

Genetics ◽

10.1093/genetics/144.4.1387 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1387-1397 ◽

Cited By ~ 4

Author(s):

Pamela K Foreman ◽

Ronald W Davis

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Nuclear Division ◽

Immunofluorescent Staining ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Mitotic Spindles ◽

Wild Type ◽

New Gene ◽

Mutant Cells

To identify new gene products involved in chromosome segregation, we isolated Saccharomyces cerevisiae mutants that require centromere binding factor I (Cbf1p) for viability. One Cbf1p-dependent mutant (denoted cdp1-1) was selected for further analysis. The CDP1 gene encodes a novel 125-kD protein that is notably similar to previously identified mouse, human and Caenorhabditis elegans proteins. CDP1Δ and cdp1-1 mutant cells were temperature sensitive for growth. At the permissive temperature, cdp1-1 and cdp1Δ cells lost chromosomes at a frequencies ∼20-fold and ∼110-fold higher than wild-type cells, respectively. These mutants also displayed unusually long and numerous bundles of cytoplasmic microtubules as revealed by immunofluorescent staining. In addition, we occasionally observed improperly oriented mitotic spindles, residing entirely within one of the cells. Presumably as a result of undergoing nuclear division with improperly oriented spindles, a large percentage of cdp1 cells had accumulated multiple nuclei. While cdp1 mutant cells were hypersensitive to the microtubule-disrupting compound thiabendazole, they showed increased resistance to the closely related compound benomyl relative to wild-type cells. Taken together, these results suggest that Cdp1p plays a role in governing tubulin dynamics within the cell and may interact directly with microtubules or tubulin.

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ARE MITOTIC FUNCTIONS REQUIRED IN MEIOSIS?

Genetics ◽

10.1093/genetics/76.4.745 ◽

1974 ◽

Vol 76 (4) ◽

pp. 745-753

Author(s):

G Simchen

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Division ◽

Mitotic Cell ◽

Intragenic Recombination ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mitotic Cell Cycle ◽

Permissive Temperature ◽

Meiotic Behavior ◽

Group I

ABSTRACT Sporulation of diploid yeasts (Saccharomyces cerevisiae), homozygous or heterozygous for temperature-sensitive mitotic cell-cycle mutations, was examined at the restrictive and permissive temperatures. Twenty genes, represented by 32 heterozygotes and 60 homozygotes, were divided into three groups, showing (i) normal sporulation, (ii) no sporulation at the restrictive temperature but normal sporulation at the permissive temperature, (iii) no sporulation at both temperatures. Group (i) as well as several other strains were tested for their meiotic behavior with regard to intragenic recombination and haploidization. The conclusion reached was that all the mitotic nuclear-division and DNA-synthesis functions were required in meiosis. The only cell-division mutations not to affect meiosis were in three cytokinesis loci and in one budemergence locus.

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Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5512 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5512-5522 ◽

Cited By ~ 18

Author(s):

Phuay-Yee Goh ◽

Uttam Surana

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Genetic Interaction ◽

S Phase ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Anaphase Promoting Complex ◽

Essential Function ◽

Additional Role ◽

Wd40 Repeats

ABSTRACT Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G1/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4(cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1cells. However, the previously characterized mutationcdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G2/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G2/M transition when released from hydroxyurea-induced S-phase arrest. A second function forCDC4 in late S or G2 phase was further confirmed by the observation that cells lacking the CDC4gene are arrested both at G1/S and at G2/M. We subsequently isolated additional temperature-sensitive mutations in theCDC4 gene (such as cdc4-12) that render the mutant defective in both G1/S and G2/M transitions at the restrictive temperature. While the G1/S block in both cdc4-12 and cdc4Δ mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G2/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.

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