Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation

1995 ◽  
Vol 108 (3) ◽  
pp. 895-905 ◽  
Author(s):  
K. Kumada ◽  
S. Su ◽  
M. Yanagida ◽  
T. Toda
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Fission Yeast ◽  
Nitrogen Starvation ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Septum Formation ◽  
Family Protein ◽  
Chromosome Separation

Fission yeast nuc2+ gene encodes a protein of a tetratricopeptide repeat (TPR) family which is conserved throughout evolution. We previously showed that nuc2 is required for exit from the mitotic metaphase. In this study, we present evidence which shows that nuc2 has two additional roles in the cell cycle. We showed that the nuc2 mutant is sterile even at the permissive temperature and septation occurs in the absence of chromosome separation at the restrictive temperature. The nuc2 mutant fails to arrest at the G1 phase upon nitrogen starvation at the permissive temperature which is a prerequisite for conjugation. Upon starvation, however, the nuc2 mutant ceased division normally and induced starvation-dependent gene expression. Therefore, the nuc2 mutant is deficient only for failure to block DNA replication upon starvation. At the lower restrictive temperature, the nuc2 mutant showed a ‘cut’ phenotype where septation and cytokinesis takes place without the completion of mitosis. Ectopic overexpression of the nuc2+ gene caused multiple rounds of S and M phases in the complete absence of septum formation. We propose that nuc2 is a novel cell cycle regulator essential for three events; firstly for exit from mitosis, secondly for DNA replication restraint under nutrient starvation and thirdly for inhibition of septation and cytokinesis until the completion of mitosis.

scholarly journals Slm9, a Novel Nuclear Protein Involved in Mitotic Control in Fission Yeast

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 623-631
Author(s):  
Junko Kanoh ◽  
Paul Russell
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Elongation ◽  
Nuclear Protein ◽  
G1 Arrest ◽  
Permissive Temperature ◽  
Cdc2 Kinase ◽  
Synthetic Lethal ◽  
Cycle Arrest ◽  
Novel Protein

Abstract In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis. Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1. Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2. To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25°). One of the genes, slm9, encodes a novel protein of 807 amino acids. Slm9 is most similar to Hir2, the histone gene regulator in budding yeast. Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle. The slm9 disruptant is delayed at the G2-M transition as indicated by cell elongation and analysis of DNA content. Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation. The slm9 mutant is defective in recovery from G1 arrest after nitrogen starvation. The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation.

scholarly journals Spindle Pole Body Duplication in Fission Yeast Occurs at the G1/S Boundary but Maturation Is Blocked until Exit from S by an Event Downstream of Cdc10+

2004 ◽  
Vol 15 (12) ◽  
pp. 5219-5230 ◽  
Author(s):  
Satoru Uzawa ◽  
Fei Li ◽  
Ye Jin ◽  
Kent L. McDonald ◽  
Michael B. Braunfeld ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Specific Inhibitor ◽  
Spindle Pole ◽  
Feedback Mechanism ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Pole Body ◽  
In Cells

The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1–2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase–specific inhibitor of SPB maturation whose expression is under control of cdc10+. This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.

scholarly journals MEIOTIC RECOMBINATION AND DNA SYNTHESIS IN A NEW CELL CYCLE MUTANT OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1978 ◽  
Vol 90 (1) ◽  
pp. 49-68
Author(s):  
Yona Kassir ◽  
Giora Simchen
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Division ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Essential Function ◽  
Normal Meiosis

ABSTRACT Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

scholarly journals Functions of Fission Yeast Orp2 in DNA Replication and Checkpoint Control

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 599-607
Author(s):  
Joan Kiely ◽  
S B Haase ◽  
Paul Russell ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Control ◽  
Cycle Arrest

Abstract orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.

scholarly journals Loss of the TOR Kinase Tor2 Mimics Nitrogen Starvation and Activates the Sexual Development Pathway in Fission Yeast

2007 ◽  
Vol 27 (8) ◽  
pp. 3154-3164 ◽  
Author(s):  
Tomohiko Matsuo ◽  
Yoko Otsubo ◽  
Jun Urano ◽  
Fuyuhiko Tamanoi ◽  
Masayuki Yamamoto
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Nitrogen Source ◽  
Sexual Development ◽  
Functional Relationship ◽  
Nitrogen Starvation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Tor Kinase ◽  
Two Temperature

ABSTRACT Fission yeast has two TOR (target of rapamycin) kinases, namely Tor1 and Tor2. Tor1 is required for survival under stressed conditions, proper G1 arrest, and sexual development. In contrast, Tor2 is essential for growth. To analyze the functions of Tor2, we constructed two temperature-sensitive tor2 mutants. Interestingly, at the restrictive temperature, these mutants mimicked nitrogen starvation by arresting the cell cycle in G1 phase and initiating sexual development. Microarray analysis indicated that expression of nitrogen starvation-responsive genes was induced extensively when Tor2 function was suppressed, suggesting that Tor2 normally mediates a signal from the nitrogen source. As with mammalian and budding yeast TOR, we find that fission yeast TOR also forms multiprotein complexes analogous to TORC1 and TORC2. The raptor homologue, Mip1, likely forms a complex predominantly with Tor2, producing TORC1. The rictor/Avo3 homologue, Ste20, and the Avo1 homologue, Sin1, appear to form TORC2 mainly with Tor1 but may also bind Tor2. The Lst8 homologue, Wat1, binds to both Tor1 and Tor2. Our analysis shows, with respect to promotion of G1 arrest and sexual development, that the loss of Tor1 (TORC2) and the loss of Tor2 (TORC1) exhibit opposite effects. This highlights an intriguing functional relationship among TOR kinase complexes in the fission yeast Schizosaccharomyces pombe.

Nucleoside diphosphokinase, an enzyme with step changes in activity during the cell cycle of the fission yeast Schizosaccharomyces pombe. II. Dissociation of the steps from the DNA-division cycle after induction synchronization

1989 ◽  
Vol 93 (1) ◽  
pp. 185-189
Author(s):  
J. Creanor ◽  
J.M. Mitchison
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Doubling Time ◽  
Phase Relations ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Normal Cycle ◽  
Short Cycle ◽  
Cycle Times

Synchrony was induced in cultures of the mitotic mutant cdc2.33 of Schizosaccharomyces pombe by shifting up an asynchronous culture to the restrictive temperature for a period of 3.5-4.5 h and then shifting down to the permissive temperature. The resulting synchronous divisions had short cycle times, down to 50% of the normal cycle. The oscillatory control of nucleoside diphosphokinase activity was also synchronized by the shift-down and the activity rose in a step pattern. Unlike the situation in the normal cycle, this step pattern was dissociated from the shortened cell cycle and had a longer period and different phase relations. It may be that the normal entrainment or coupling between the cell cycle and the activity control fails if the cell cycle is too short. The period of the activity control (equal to the protein doubling time at the restrictive temperature) appears to be temperature-compensated.

scholarly journals The origin recognition complex in silencing, cell cycle progression, and DNA replication.

1995 ◽  
Vol 6 (6) ◽  
pp. 741-756 ◽  
Author(s):  
S Loo ◽  
C A Fox ◽  
J Rine ◽  
R Kobayashi ◽  
B Stillman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Origin Recognition Complex ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Gene Encoding ◽  
Atp Binding Site ◽  
Origin Recognition

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.

scholarly journals EFFECTS OF THE MITOTIC CELL-CYCLE MUTATION cdc4 ON YEAST MEIOSIS

Genetics ◽  
1977 ◽  
Vol 86 (1) ◽  
pp. 57-72
Author(s):  
G Simchen ◽  
J Hirschberg
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Dna ◽  
Mitotic Cell ◽  
Restrictive Temperature ◽  
Mitotic Cell Cycle ◽  
Sporulation Medium ◽  
Permissive Temperature ◽  
Yeast Meiosis ◽  
High Degree

ABSTRACT The mitotic cell-cycle mutation cdc4 has been reported to block the initiation of nuclear DNA replication and the separation of spindle plaques after their replication. Meiosis in cdc4/cdc4 diploids is normal at the permissive temperature (25°) and is arrested at the first division (one-nucleus stage) at the restrictive temperature (34° or 36°). Arrested cells at 34° show a high degree of commitment to recombination (at least 50% of the controls) but no haploidization, while cells arrested at 36° are not committed to recombination. Meiotic cells arrested at 34° show a delayed and reduced synthesis of DNA (at most 40% of the control), at least half of which is probably mitochondrial. It is suggested that recombination commitment does not depend on the completion of nuclear premeiotic DNA replication in sporulation medium.—Transfer of cdc4/cdc4 cells to the restrictive temperature at the onset of sporulation produces a uniform phenotype of arrest at a 1-nucleus morphology. On the other hand, shifts of the meiotic cells to the restrictive temperature at later times produce two additional phenotypes of arrest, thus suggesting that the function of cdc4 is required at several points in meiosis (at least at three different times).

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast

1999 ◽  
Vol 112 (14) ◽  
pp. 2313-2321 ◽  
Author(s):  
L. Cerutti ◽  
V. Simanis
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Spindle Pole Body ◽  
Mitotic Cell ◽  
Spindle Pole ◽  
The Other ◽  
Septum Formation ◽  
Spindle Pole Bodies ◽  
Interphase Cells ◽  
Early Mitosis

In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is induced by a signal transduction network involving several protein kinases and a GTPase switch. One of the roles of the spg1p GTPase is to localise the cdc7p protein kinase to the poles of the mitotic spindle, from where the onset of septation is thought to be signalled at the end of mitosis. Immunofluorescence studies have shown that cdc7p is located on both spindle pole bodies early in mitosis, but only on one during the later stages of anaphase. This is mediated by inactivation of spg1p on one pole before the other. The GAP for spg1p is a complex of two proteins, cdc16p and byr4p. Localisation of cdc16p and byr4p by indirect immunofluorescence during the mitotic cell cycle showed that both proteins are present on the spindle pole body in interphase cells. During mitosis, byr4p is seen first on both poles of the spindle, then on only one. This occurs prior to cdc7p becoming asymmetric. In contrast, the signal due to cdc16p decreases to a low level during early mitosis, before being seen strongly on the same pole as byr4p. Double staining indicates that this is the opposite pole to that which retains cdc7p in late anaphase. Examination of the effect of inactivating cdc16p at various stages of the cell cycle suggests that cdc16p, together with cdc2p plays a role in restraining septum formation during interphase. The asymmetric inactivation of spg1p is mediated by recruitment of the cdc16p-byr4p GAP to one of the poles of the spindle before the other, and the asymmetry of the spindle pole bodies may be established early during mitosis. Moreover, the spindle pole bodies appear to be non-equivalent even after division has been completed.

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