scholarly journals Fission Yeast dim1+ Encodes a Functionally Conserved Polypeptide Essential for Mitosis

1997 ◽  
Vol 137 (6) ◽  
pp. 1337-1354 ◽  
Author(s):  
Lynne D. Berry ◽  
Kathleen L. Gould
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Nuclear Division ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Sensitive Mutant ◽  
Evolutionarily Conserved ◽  
Mutant Cells

In a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D217N, we have isolated a novel temperature-sensitive mutant, dim1-35. When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events. Deletion of dim1 from the Schizosaccharomyces pombe genome produces a lethal G2 arrest phenotype. Lethality is rescued by overexpression of the mouse dim1 homolog, mdim1. Likewise, deletion of the Saccharomyces cerevisiae dim1 homolog, CDH1, is lethal. Both mdim1 and dim1+ are capable of rescuing lethality in the cdh1::HIS3 mutant. Although dim1-35 displays no striking genetic interactions with various other G2/M or mitotic mutants, dim1-35 cells incubated at restrictive temperature arrest with low histone H1 kinase activity. Morevoer, dim1-35 displays sensitivity to the microtubule destabilizing drug, thiabendazole (TBZ). We conclude that Dim1p plays a fundamental, evolutionarily conserved role as a protein essential for entry into mitosis as well as for chromosome segregation during mitosis. Based on TBZ sensitivity and failed chromosome segregation in dim1-35, we further speculate that Dim1p may play a role in mitotic spindle formation and/or function.

scholarly journals In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells.

1990 ◽  
Vol 110 (2) ◽  
pp. 417-425 ◽  
Author(s):  
H Masuda ◽  
T Hirano ◽  
M Yanagida ◽  
W Z Cande
Keyword(s):  
Fission Yeast ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Sensitive Mutant ◽  
Vitro Assay ◽  
Chromosome Separation ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Mutant Cells

To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.

scholarly journals The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function

1999 ◽  
Vol 19 (4) ◽  
pp. 2535-2546 ◽  
Author(s):  
Lynne D. Berry ◽  
Anna Feoktistova ◽  
Melanie D. Wright ◽  
Kathleen L. Gould
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Chromosome Segregation ◽  
State Level ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Anaphase Promoting Complex ◽  
Synthetic Lethal ◽  
C Complex ◽  
Mutant Cells

ABSTRACT The Schizosaccharomyces pombe dim1 + gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6mutant. At the restrictive temperature of 36°C, lid1-6mutant cells arrest with a “cut” phenotype similar to that ofcut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein ∼20S complex; the presence of lid1p in this complex depends upon functionalcut9 +. lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity oflid1 +. Further, lid1 +function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.

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

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1387-1397 ◽  
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.

scholarly journals Genes Involved in Sister Chromatid Separation and Segregation in the Budding Yeast Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 453-470
Author(s):  
Sue Biggins ◽  
Needhi Bhalla ◽  
Amy Chang ◽  
Dana L Smith ◽  
Andrew W Murray
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Mitotic Spindle ◽  
Budding Yeast ◽  
Temperature Sensitive ◽  
Chromosome Behavior ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sister Chromatids ◽  
Marked Chromosome ◽  
Sister Chromatid Separation

Abstract Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair α-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

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

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

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.

scholarly journals A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Masaki Okazaki ◽  
Kazunori Kume ◽  
Ngang Heok Tang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cross Linker ◽  
Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus

1991 ◽  
Vol 11 (11) ◽  
pp. 5718-5726
Author(s):  
A Rowley ◽  
R A Singer ◽  
G C Johnston
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cycle Performance ◽  
Carboxyl Terminus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Negative Effects ◽  
Yeast Saccharomyces Cerevisiae ◽  
Acid Protein ◽  
Mutant Cells

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family.

1993 ◽  
Vol 4 (10) ◽  
pp. 1003-1015 ◽  
Author(s):  
S Miyake ◽  
N Okishio ◽  
I Samejima ◽  
Y Hiraoka ◽  
T Toda ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Positive Role ◽  
A Cell ◽  
Cold Sensitive ◽  
Single Nucleus ◽  
Yeast Genes ◽  
Cell Cycle Block ◽  
Mutant Cells

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.

scholarly journals Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.

1996 ◽  
Vol 133 (1) ◽  
pp. 111-124 ◽  
Author(s):  
H A Sundberg ◽  
L Goetsch ◽  
B Byers ◽  
T N Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Immunofluorescent Staining ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nonpermissive Temperature ◽  
Carboxy Terminus ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Mutant Cells

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.

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