Fission yeast temperature‐sensitive mutants cut3‐477 and cut14‐208 fail to condense chromosomes but small portions of the chromosomes can separate along the spindle during mitosis, producing phi‐shaped chromosomes. Septation and cell division occur in the absence of normal nuclear division, causing the cut phenotype. Fluorescence in situ hybridization demonstrated that the contraction of the chromosome arm during mitosis was defective. Mutant chromosomes are apparently not rigid enough to be transported poleward by the spindle. Loss of the cut3 protein by gene disruption fails to maintain the nuclear chromatin architecture even in interphase. Both cut3 and cut14 proteins contain a putative nucleoside triphosphate (NTP)‐binding domain and belong to the same ubiquitous protein family which includes the budding yeast Smc1 protein. The cut3 mutant was suppressed by an increase in the cut14+ gene dosage. The cut3 protein, having the highest similarity to the mouse protein, is localized in the nucleus throughout the cell cycle. Plasmids carrying the DNA topoisomerase I gene partly suppressed the temperature sensitive phenotype of cut3‐477, suggesting that the cut3 protein might be involved in chromosome DNA topology.

Disorder in sister chromatid separation can lead to genome instability and cancer. A temperature-sensitive S. pombe mis6-302 frequently loses a minichromosome at 26 degrees C and abolishes equal segregation of regular chromosomes at 36 degrees C. The mis6+ gene is essential for viability, and its deletion results in missegregation identical to mis6-302. Mis6 acts before or at the onset of S phase, and mitotic missegregation defects are produced only after the passage of G1/S at 36 degrees C. Mis6 locates at the centromeres throughout the cell cycle. In the mutant, positioning of the centromeres becomes abnormal, and specialized chromatin in the inner centromeres, which give the smear micrococcal nuclease pattern in wild type, is disrupted. The ability to establish correct biorientation of sister centromeres in metaphase cells requires the Mis6-containing chromatin and originates during the passage of G1/S.

High-fidelity chromosome transmission is fundamental in controlling the quality of the cell division cycle. The spindle pole-to-pole distance remains constant from metaphase to anaphase A. We show that fission yeast sister centromere-connecting proteins, Mis6 and Mis12, are required for correct spindle morphogenesis, determining metaphase spindle length. Thirty-five to sixty percent extension of metaphase spindle length takes place in mis6 and mis12 mutants. This may be due to incorrect spindle morphogenesis containing impaired sister centromeres or force unbalance between pulling by the linked sister kinetochores and kinetochore-independent pushing. The mutant spindle fully extends in anaphase, although it is accompanied by drastic missegregation by aberrant sister centromere separation. Hence, metaphase spindle length may be crucial for segregation fidelity. Suppressors of mis12 partly restore normal metaphase spindle length. In mis4 that is defective in sister chromatid cohesion, metaphase spindle length is also long, but anaphase spindle extension is blocked, probably due to the activated spindle checkpoint. Extensive missegregation is caused in mis12 only when Mis12 is inactivated from the previous M through to the following M, an effective way to avoid missegregation in the cell cycle. Mis12 has conserved homologs in budding yeast and filamentous fungi.

CENP-A, the centromere-specific histone H3 variant, plays a crucial role in organizing kinetochore chromatin for precise chromosome segregation. We have isolated Ams2, a Daxx-like motif-containing GATA factor, and histone H4, as multicopy suppressors of cnp1-1, an S. pombe CENP-A mutant. While depletion of Ams2 results in the reduction of CENP-A binding to the centromere and chromosome missegregation, increasing its dosage restores association of a CENP-A mutant protein with centromeres. Conversely, overexpression of CENP-A or histone H4 suppresses an ams2 disruptant. The intracellular amount of Ams2 thus affects centromeric nucleosomal constituents. Ams2 is abundant in S phase and associates with chromatin, including the central centromeres through binding to GATA-core sequences. Ams2 is thus a cell cycle-regulated GATA factor that is required for centromere function.

CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In ⌬ams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in ⌬ams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in ⌬ams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged. INTRODUCTIONThe kinetochore is a multiprotein-DNA complex that is indispensable for chromosome segregation and that normally forms on a single chromosomal locus, the centromere (Cleveland et al., 2003). Lack of a kinetochore or formation of multiple kinetochores on a chromosome may have deleterious effects on mitosis (Karpen and Allshire, 1997;Amor and Choo, 2002;Henikoff and Dalal, 2005). CENP-A represents the most likely candidate for the epigenetic "mark" responsible for the maintenance of centromere identity (Black et al., 2004. Because reformation of CENP-A-containing nucleosomes after DNA synthesis is thought to be a prerequisite for mitotic kinetochore assembly, precise targeting of CENP-A into a single, restricted locus on each chromosome before cell division is essential for cell viability . At least three components that affect CENP-A localization, the Mis16 -Mis18 complex (Hayashi et al., 2004;Fujita et al., 2007), the Mis6 -Sim4 complex (Takahashi et al., 2000;Pidoux et al., 2003), and Ams2 GATA-type transcription factor (Chen et al., 2003a), have been identified in the fission yeast Schizosaccharomyces pombe, which is an ideal model organism in which to study complex centromere structure and function (Takahashi et al., 1992;Karpen and Allshire, 1997).Which phase of the cell cycle is used for CENP-A incorporation remains controversial. During S phase, canonical core histones have been suggested to be deposited into duplicated DNAs in a semiconservative manner (Tagami et al., 2004;Natsume et al., 2007). Experiments using fluorescence recovery after photobleaching demonstrated that CENP-A of the budding yeast Saccharomyces cerevisiae is recruited to centromeres coincident with DNA synthesis (Pearson et al., 2004), presumably reflecting disassembly and reassembly of cent...

Fission yeast Cid13 and budding yeast Trf4/5 are members of a newly identified nucleotidyltransferase family conserved from yeast to man. Trf4/5 are thought to be essential DNA polymerases. We report that Cid13 is a poly(A) polymerase. Unlike conventional poly(A) polymerases, which act in the nucleus and indiscriminately polyadenylate all mRNA, Cid13 is a cytoplasmic enzyme that specifically targets suc22 mRNA that encodes a subunit of ribonucleotide reductase (RNR). cid13 mutants have reduced dNTP pools and are sensitive to hydroxyurea, an RNR inhibitor. We propose that Cid13 defines a cytoplasmic form of poly(A) polymerase important for DNA replication and genome maintenance.

Abstract. Two fission yeast temperature-sensitive mutants, cut6 and lsdl, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron-dense nuclear materials and contained highly compacted chromatin. The cut6 + and lsdl + genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsdl ÷ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsdl. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of nonchromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters. N'UCLEAR division proceeds during the M-phase of the cell cycle and ensures that daughter nuclei will receive an equal set of chromosomes before the onset of cytokinesis. The mechanism and regulation of nuclear division are crucially important in the cell's transmission of genetic information to a descendant (Murray and Hunt, 1993;Koshland, 1994;Holm, 1994;Yanagida, 1995). Nuclear division has to be properly coordinated with cytokinesis (e.g., Fankhauser et al., 1993). If cytokinesis is triggered before the completion of nuclear division, the resulting cells may lose viability due to aberrant cytokinesis. In the fission yeast Schizosaccharomyces pombe, temperature-sensitive (ts) 1 mutants showing the occurrence of such uncoordinated cytokinesis have been isolated and called cut (cell untimely torn) mutants (Hirano et al., 1986;Samejima et al., 1993 K. Takahashi's present address is Department of Molecular Biology, University of Geneva, Switzerland.1. Abbreviations used in this paper: DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; SPB, spindle pole body; ts, temperature-sensitive; YPD, yeast extract/peptone/dextrose medium. sequent cytokinesis take place although the preceding nuclear division is absent or abnormal. The cut + gene products are thus implicated in the proper coordination between nuclear division and cytokinesis.In this study, the phenotypes of two fission yeast mutants, cut6 (Hirano et al., 1986) and lsdl (isolated in the present study), and the gene products of cut6 ÷ and lsdl + were investigated. These mutants show infrequently the cut phenotype and very frequently a striking nuclear division phenotype resulting in daughter nuclei of unequal size (designated lsd, lar...

Glucose transporters play a pivotal role in glucose homeostasis. The fission yeast high-affinity glucose transporter Ght5 is regulated with regard to transcription and localization via CaMKK and TOR pathways. These results clarify the evolutionarily conserved mechanisms underlying glucose homeostasis that prevent hyperglycemia in humans.