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Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7 . Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.

Cdc37 is a molecular chaperone that interacts with a range of clients and co-chaperones, forming various high molecular mass complexes. Cdc37 sequence homology among species is low. High homology between yeast and metazoan proteins is restricted to the extreme N-terminal region, which is known to bind clients that are predominantly protein kinases. We show that despite the low homology, both Saccharomyces cerevisiae and human Cdc37 are able to substitute for the Schizosaccharomyces pombe protein in a strain deleted for the endogenous cdc37 gene. Expression of a construct consisting of only the N-terminal domain of S. pombe Cdc37, lacking the postulated heat-shock protein (Hsp) 90-binding and homodimerization domains, can also sustain cellular viability, indicating that Cdc37 dimerization and interactions with the cochaperone Hsp90 may not be essential for Cdc37 function in S. pombe. Biochemical investigations showed that a small proportion of total cellular Cdc37 occurs in a high molecular mass complex that also contains Hsp90. These data indicate that the N-terminal domain of Cdc37 carries out essential functions independently of the Hsp90-binding domain and dimerization of the chaperone itself.

The cdc22+ gene of the fission yeast, Schizosaccharomyces pombe , encodes the large subunit of ribonucleotide reductase, and is periodically expressed during the mitotic cell cycle, transcript abundance reaching a maximum at the G1-S boundary. This regulation of expression is controlled by a transcription factor complex called DSC1, which binds to MCB motifs (ACGCGT) present in the promoter of cdc22+. cdc22+ has a complex pattern of MCBs, including two clusters of four motifs each, one of which is located within the transcribed region. We show that both clusters of MCBs contribute to the regulation of cdc22+ expression during the cell cycle, each having a different role. The MCB cluster within the transcribed region has the major role in regulating cdc22+, as its removal results in loss of transcription. The upstream cluster, instead, controls cell cycle-specific transcription through a negative function, as its removal results in expression of cdc22+ throughout the cell cycle. Both MCB clusters bind DSC1. We show that the interaction of DSC1 with the MCB cluster within the transcribed region has a high "on-off" rate, suggesting a mechanism by which DSC1 could activate expression, and still allow RNA polymerase to pass during transcription. Finally, we show that both clusters are orientation-dependent in their function. The significance of these results, in the context of MCB-mediated regulation of G1-S expression in fission yeast, is discussed.

Wis1 is a mitogen-activated protein kinase kinase (MAPKK) that regulates mitosis and mediates stress responses in the fission yeast, Schizosaccharomyces pombe. wis1Δ strains are viable but stress-sensitive and show a mitotic delay. At high temperatures, wis1Δ cells cease division but cellular growth continues. Mutations that suppress the heat sensitivity of a wis1Δ strain were isolated and map to two apparently novel loci, sow1 (for suppressor of wis1Δ) and sow2. In addition to suppressing wis1Δ heat sensitivity, sow1 and sow2 can suppress wis1Δ osmosensitivity and cell cycle defects. sow1 and sow2 mutants in a wis1+ background were able to grow at higher temperatures than wild-type and sow1 showed a mitotic advance. The sow genes may therefore define a novel connection between stress tolerance and cell cycle control. Copyright © 2000 John Wiley & Sons, Ltd.

Direct interaction between DNA polymerase delta and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. We show that the 54 kDa subunit of DNA polymerase delta from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C-terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21(Cip1) protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B-subunit of polymerase delta, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding.

Highly purified DNA polymerase δ from the fission yeast Schizosaccharomyces pombe is a complex of at least four distinct subunits. Genes encoding three of these (pol3 + /cdc6 + , cdc1 + and cdc27 + ) have been characterised previously. Here we describe the isolation and characterisation of cdm1 + , the gene encoding the smallest (22kDa) subunit of the Pol δ complex. Over-expression of cdm1 + , which encodes a 160 amino-acid protein with no significant sequence similarity to proteins in current databases, is able to rescue cells carrying temperature-sensitive mutations in either pol3 + /cdc6 + , cdc1 + or cdc27 + . Cells deleted for cdm1 + are viable, indicating that cdm1 + is non-essential for mitotic growth, and are no more sensitive to a variety of DNA replication inhibitors and DNA damaging agents than are wild-type cells. In addition, over-expression of cdm1 + suppresses the temperature-sensitive cdc24-M38 mutant suggesting that cdc24 + may also have a role in DNA polymerase δ function.

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1+andwis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1+encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50mutant and its suppression by wis genes.

The Schizosaccharomyces pombe wis1 + gene is essential for cell survival under stress conditions. The MAPKK homologue Wis1 is required for activation of the MAPK homologue Spc1, and integrity of the Wis1–Spc1 pathway is required for survival in extreme conditions of heat, osmolarity, oxidation or limited nutrition. We show here that Wis4, a protein kinase of a new MAPKKK class, phosphorylates Wis1 in vitro and activates it in vivo . Win1 is also required for full activation of Wis1, and Win1 rather than Wis4 mediates the osmotic stress signal. Surprisingly, the pathway can still be activated by heat or oxidative stress independently of the phosphorylation of two conserved Wis1 residues. Evidence is presented that the Pyp1 protein tyrosine phosphatase, which dephosphorylates Spc1, is central to this alternative activation mechanism.

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.

The suc22+ gene of Schizosaccharomyces pombe encodes the small subunit of ribonucleotide reductase. Two transcripts that hybridise to suc22+ have previously been described: a constitutive transcript of 1.5 kb, and a transcript of approximately 1.9 kb that is induced when DNA replication is blocked by hydroxyurea. In this paper we show that both transcripts derive from the suc22+ gene, are polyadenylated, and have transcription initiation sites separated by approximately 550 nucleotides. The absence of translation initiation codons and predicted intron splice sites within this 550 nucleotide region suggests strongly that both transcripts encode the same protein. Under normal growth conditions, the larger suc22+ transcript is present at a very low level. This low level expression is periodic during the cell cycle, showing a pattern similar to that of other genes under regulation by MCB elements with a maximum in G1/S phase. Consistent with this, there are MCB elements upstream of the initiation site of the transcript. This pattern of expression contrasts with the continuous expression, at a much higher level, of the smaller suc22+ transcript. The larger suc22+ transcript is induced by exposure of cells to 4-nitroquinoline oxide (4-NQO),a UV-mimetic agent that causes DNA damage. The transcriptional response to 4-NQO is observed in cells previously arrested in G2 by a cdc2ts mutation, demonstrating that induction can occur outside S phase. We show that the rad1+ gene, part of the mitotic checkpoint, is required for induction of the large transcript. Exposure of cells to heat shock also induces the suc22+ large transcript: a consensus heat shock element has been identified upstream of the large transcript start site.

The wis1 protein kinase of Schizosaccharomyces pombe is a member of the MAP kinase kinase family. Loss of wis1 function has previously been reported to lead to a delay in the G2-mitosis transition, loss of viability in stationary phase, and hypersensitivity to osmotic shock. It acts at least in part by activating the MAP kinase homologue sty1; loss-of-function sty1 mutants share many phenotypes with wis1 deletion mutants. We show here that, in addition, loss of wis1 function leads to defective conjugation, and to suppression of the hyperconjugation phenotype of the pat1-114 mutation. Consistent with this, the induction of the mei2 gene, which is normally induced by nitrogen starvation, is defective in wis1 mutants. In wild-type cells, nitrogen starvation leads to mei2 induction through a fall in intracellular cyclic AMP (cAMP) level and activity of the cAMP-dependent protein kinase. We show here that wis1 function is required for mei2 induction following nitrogen starvation. Expression of the fbp1 gene is negatively regulated by cAMP in response to glucose limitation: induction of fbp1 also requires wis1 and sty1 function. Loss of wis1 is epistatic over increased fbp1 expression brought about by loss of adenylate cyclase (git2/cyr1) or cAMP-dependent protein kinase (pka1) function. These observations can be explained by a model in which the pka1 pathway negatively regulates the wis1 pathway, or the two pathways might act independently on downstream targets. The latter explanation is supported, at least as regards regulation of cell division, by the observation that loss of function of the regulatory subunit of the cAMP-dependent protein kinase (cgs1) brings about a modest increase in cell length at division in both wis1+ and wis1 delta genetic backgrounds.

We identified the phhl + gene that encodes a MAP kinase as the effector of Wis1 MAP kinase kinase in fission yeast, which is highly homologous with HOG1 of S. cerevisiae . Heterothalic phh1 dsiruptant is phenotypically indistinguishable from wis1 deletion mutant, both displaying the same extent of partial sterility and enhanced sensitivity to a variety of stress. In phh1 disruptant, nitrogen starvation-induced expression of ste11 + , a key controller of sexual differentiation, is markedly diminished. Ectopic expression of ste11 + effectively restores fertility, but not stress resistance, to the phh1 disruptant. These data show that stress signal, mediated by a MAP kinase, is required for efficient start of sexual differentiation.

In this paper we describe properties of the cdc10-C4 mutant of the fission yeast Schizosaccharomyces pombe. The cdc10+ gene encodes a component of the DSC1Sp/MBF transcription complex, which is required for cell-cycle regulated expression at G1-S of several genes via cis-acting MCB (MIuI cell cycle box) elements. At permissive temperatures cdc10-C4 causes expression of MCB-regulated genes through the whole cell cycle, which in asynchronously dividing cells is manifested in overall higher expression levels. This overexpression phenotype is cold sensitive: in cdc10-C4 cells, MCB genes are expressed offprogressively higher levels at lower temperatures. In heterozygous cdc10-C4/cdc10+ diploid strains, MCB-regulated genes are not overexpressed, suggesting that loss, rather than alteration, of function of the cdc10-C4 gene product is the reason for unregulated target gene expression. Consistent with this, the cdc10-C4 mutant allele is known to encode a truncated protein. We have also overexpressed the region of the cdc10 protein absent in cdc10-C4 under the control of an inducible promoter. This induces a G1 delay, and additionally causes a reduction of the overexpression of MCB genes in cdc10-C4 strains. These results suggest that DSC1Sp/MBF represses, as well as activates, MCB gene expression during the cell cycle.

Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 wee1ts cdc25ts triple mutant strains. These genes were designated wis1(+)-wis5+ for win suppressing, and do not correspond to win1+ or any of the previously characterised mitotic control genes. None of the wis genes is capable of suppressing the cdc phenotype of cdc25ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function. wis1+ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis. wis4+ appears to be a specific suppressor of the win1-1 mutation. wis2+ and wis3+ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1ts and cdc25ts, suggesting that the wis2+ and wis3+ products may interact with elements central to the mitotic control.

In the budding yeast Saccharomyces cerevisiae, cell-cycle control over DNA synthesis occurs partly through the coordinate expression in late G1 phase of many, if not all, of the genes required for DNA synthesis. A cis-acting hexamer element ACGCGT (an MluI restriction site) is responsible for coordinating transcriptional regulation of these genes at the G1/S phase boundary and we have identified a binding activity, DSC1, that recognizes these sequences in a cell-cycle-dependent manner. In the distantly related fission yeast Schizosaccharomyces pombe, only one of the known DNA synthesis genes, cdc22+, which encodes a subunit of ribonucleotide reductase, is periodically expressed in late G1 (ref. 6). The promoter region of cdc22+ has two MluI sites and five related sequences, suggesting that similar controls over DNA synthesis genes could occur in fission yeast. We report here a binding activity in fission yeast that is very similar to DSC1 in budding yeast. We also show that the fission yeast cdc10+ gene product, which is required for Start and entry into S phase, is a component of this binding activity.

Cdc37 is a molecular chaperone whose clients are predominantly protein kinases, many of which are important in cell-cycle progression. Temperature-sensitive mutants of cdc37 in Schizosaccharomyces pombe are lethal at the restrictive temperature, arresting cell division within a single cell cycle. These mutant cells elongate during incubation at the restrictive temperature, consistent with a cell-cycle defect. The cell-cycle arrest arises from defective function of the mutant Cdc37 proteins rather than a reduction in Cdc37 protein levels. Around 80% of the arrested, elongated cells contain a single nucleus and replicated (2C) DNA content, indicating that these mutants arrest the cell cycle in G2 or mitosis (M). Cytological observations show that the majority of cells arrest in G2. In fission yeast, a G2 cell-cycle arrest can arise by inactivation of the cyclin-dependent kinase (Cdk) Cdc2 that regulates entry into mitosis. Studies of the cdc37 temperature-sensitive mutants show a genetic interaction with some cdc2 alleles and overexpression of cdc2 rescues the lethality of some cdc37 alleles at the restrictive temperature, suggesting that Cdc2 is a likely client for the Cdc37 molecular chaperone. In cdc37 temperature-sensitive mutants at the restrictive temperature, the level of Cdc2 protein remains constant but Cdc2 protein kinase activity is greatly reduced. Inactivation of Cdc2 appears to result from the inability to form complexes with its mitotic cyclin partner Cdc13. Further evidence for Cdc2 being a client of Cdc37 in S. pombe comes from the identification of genetic and biochemical interactions between these proteins.

Cdc37 is a molecular chaperone that interacts with a range of clients and co-chaperones, forming various high molecular mass complexes. Cdc37 sequence homology among species is low. High homology between yeast and metazoan proteins is restricted to the extreme N-terminal region, which is known to bind clients that are predominantly protein kinases. We show that despite the low homology, both Saccharomyces cerevisiae and human Cdc37 are able to substitute for the Schizosaccharomyces pombe protein in a strain deleted for the endogenous cdc37 gene. Expression of a construct consisting of only the N-terminal domain of S. pombe Cdc37, lacking the postulated heat-shock protein (Hsp) 90-binding and homodimerization domains, can also sustain cellular viability, indicating that Cdc37 dimerization and interactions with the cochaperone Hsp90 may not be essential for Cdc37 function in S. pombe. Biochemical investigations showed that a small proportion of total cellular Cdc37 occurs in a high molecular mass complex that also contains Hsp90. These data indicate that the N-terminal domain of Cdc37 carries out essential functions independently of the Hsp90-binding domain and dimerization of the chaperone itself.

Recent investigations have revealed hitherto unsuspected roles for small, non-coding RNA molecules and regulated pre-mRNA splicing during meiosis in fission yeast.

The identification of a Schizosaccharomyces pombe homologue of the cdc37 gene is described. The gene product is most similar to the budding yeast homologue, but shows similarity to metazoan Cdc37 proteins, with a region of high similarity at the extreme N-terminus. Gene transplacement experiments in diploid cells followed by tetrad dissection show that the gene is essential. Depletion of the gene product after switching off expression of cdc37 from the regulatable nmt81 promoter results in cessation of growth and division. The cells arrest heterogeneously, with a significant proportion showing mitotic defects; paradoxically, a proportion of the cells show a short-cell phenotype consistent with an advanced cell cycle.

Replication factor C (RF-C) is a five subunit DNA polymerase (Pol) delta/straightepsilon accessory factor required at the replication fork for loading the essential processivity factor PCNA onto the 3'-ends of nascent DNA strands. Here we describe the genetic analysis of the rfc2 +gene of the fission yeast Schizosaccharomyces pombe encoding a structural homologue of the budding yeast Rfc2p and human hRFC37 proteins. Deletion of the rfc2 + gene from the chromosome is lethal but does not result in the checkpoint-dependent cell cycle arrest seen in cells deleted for the gene encoding PCNA or for those genes encoding subunits of either Pol delta or Pol straightepsilon. Instead, rfc2 Delta cells proceed into mitosis with incompletely replicated DNA, indicating that the DNA replication checkpoint is inactive under these conditions. Taken together with recent results, these observations suggest a simple model in which assembly of the RF-C complex onto the 3'-end of the nascent RNA-DNA primer is the last step required for the establishment of a checkpoint-competent state.

Studies on DNA replication in S. pombe have provided powerful insights into the way in which the genome of this model eukaryote is replicated and how the replication process is controlled. These studies have been facilitated by the simplicity and range of methods available in this organism for physiological and genetic analysis of DNA replication mutants. In the future, continued focus on the analysis of such mutants, coupled with increasingly sophisticated biochemical investigation of the processes of DNA replication in both wild-type and mutant cells, will ensure continued rapid progress in this area.

The Wis1 protein kinase in Schizosaccharomyce pombe (S. pombe) is involved in regulating the G2-M transition. Overexpression of the wis1+ gene advances mitosis in a dosage-dependent manner. Strains deleted for wis1+ are viable, but the cells are elongated, indicating delayed mitosis. The wis1+ deletion strains show very poor viability in stationary phase and altered conjugation activity, implicating wis1+ in several aspects of the S. pombe life cycle. With regard to the domain structure, the N-terminal domain shows no overall similarity with proteins in current databases, although it is rich in Ser, particularly in the 100 most N-terminal residues, Thr, Pro, and Glu. Clusters of these amino acids (PEST-rich sequences) are characteristic of some unstable proteins. Wis1 is highly similar in sequence throughout its catalytic domain to Pbs2 and Ste7 of Saccharomyces cerevisiae, Byr1 of S. pombe, and Mek/MAP kinase kinases of vertebrates, all of which are elements or presumptive elements of signal transduction pathways.

SUMMARY The fission yeast cut5+ (identical to rad4+) gene is essential for S phase. Its temperature-sensitive (ts) mutation causes mitosis while S phase is inhibited: dependence of mitosis upon the completion of S phase is abolished. If DNA is damaged in mutant cells, however, cell division is arrested. Thus the checkpoint control system for DNA damage is functional, while that for DNA synthesis inhibition is not in the cut5 mutants. Transcription of the cut5+ gene is not under the direct control of cdc10+, which encodes a transcription factor for the START of cell cycle. The transcript level does not change during the cell cycle. The protein product has four distinct domains and is enriched in the nucleus. Its level does not alter during the cell cycle. The N-domain is important for cut5 protein function: it is essential for complementation of ts cut5 mutations and its overexpression blocks cell division. Furthermore, it resembles the N-terminal repeat domain of proto-oncoprotein Ect2, which, in the C-domain, contains a regulator-like sequence for small G proteins. We discuss a hypothesis that the cut5 protein is an essential component of the checkpoint control system for the completion of DNA synthesis. The restraint of mitosis until the completion of S phase is mediated by the cut5 protein, which can sense the state of chromosome duplication and negatively interacts with M phase regulators such as cdc25 and cdc2.

The cell division cycle gene cdc27+ of the fission yeast Schizosaccharomyces pombe is required for the transition from G2 into mitosis. Genetic and physiological experiments suggest a close relationship between cdc27+ and the cdc2+ gene, a key regulator of mitosis in yeast and also in higher eukaryotic cells. We isolated the cdc27+ gene by complementation of a temperature-sensitive cdc27 mutant. The DNA sequence of this gene predicts a 1116 nucleotide open reading frame split by five short introns, ranging in size from 49 to 74 nucleotides. Analysis of cDNA clones confirmed the structure of the gene. The deduced cdc27+ gene product consists of 372 amino acids with a predicted Mr of 43 kDa. No homology of the predicted protein with known proteins could be found, thus the cdc27+ gene encodes a novel function required for the G2-M transition. Northern analysis revealed two mRNAs of 1.4 and 2.2 kb transcribed from this gene, the smaller transcript being approximately tenfold more abundant than the larger. The level of cdc27+ mRNAs remained constant through the cell cycle indicating that the time of action of the cdc27+ gene, which is known to be regulated by elements of the mitotic control, is not determined by periodic accumulation of its transcripts.

The wis1+ gene encodes a newly identified mitotic control element in Schizosaccharomyces pombe. It was isolated by virtue of its interaction with the mitotic control genes cdc25, wee1 and win1. The wis1+ gene potentially encodes a 66 kDa protein with homology to the serine/threonine family of protein kinases. wis1+ plays an important role in the regulation of entry into mitosis, as it shares with cdc25+ and nim1+/cdr1+ the property of inducing mitosis in a dosage-dependent manner. Increased levels of wis1+ expression cause mitotic initiation to occur at a reduced cell size. Loss of wis1+ function does not prevent vegetative growth and division, though wis1- cells show an elongated morphology, indicating that their entry into mitosis and cell division is delayed relative to wild type cells. wis1- cells undergo a rapid reduction of viability upon entry into stationary phase, suggesting a role for wis1+ in the integration of nutritional sensing with the control over entry into mitosis.

The suitability of fission yeast as a model for understanding the eukaryotic cell cycle has been validated in five years of exciting developments. We review recent advances in understanding the nature of the controls that regulate progression through the cell cycle and the coordination of DNA replication and mitosis.

We have isolated 150 benomyl resistant mutants of the fission yeast Schizosaccharomyces pombe. Seven of these mutants were found to be cold sensitive for mitosis. These mutants were the subject of physiological, cytological and genetical characterisation. Growth and division of the seven mutants were similar to the wild type strain at 35 °C. After shift from the permissive (35 °C) to the restrictive temperature (20 °C) the mutants became blocked in mitosis whilst cellular growth continued. Consequently, elongate cells were formed. Six of the seven benomyl resistant mutants became blocked in mitosis at 20 °C with a single aberrant nucleus. In every case the benomyl resistant and cold sensitive phenotype was due to a mutation in a single nuclear gene. These mutants were found to comprise a single genetic linkage group (ben4) and were unlinked to existing TBZ/MBC resistant mutants of S. pombe. Whilst no cross resistance was found in our mutants to TBZ, six of the seven mutants were super sensitive to the spindle poison CIPC. We believe that the phenotype exhibited by these mutants is consistent with a defective tubulin subunit.

In the fission yeast Schizosaccharomyces pombe , nutritional reduction of growth rate by supplying poor nitrogen, carbon or phosphate sources causes a decrease in cell size. The effect on cell division following three different nutritional shifts-up has been investigated. In all cases, about 20% of the cells divide at the original cell length, and then cell division stops for a period. Cell division then resumes at the new faster rate, cell length at division being characteristic of the new medium. Further investigation reveals that the first effect of the shift is to inhibit nuclear division rapidly and completely. These results are strongly suggestive of the operation of a cell size requirement for entry into nuclear division. The cell size necessary for nuclear division is set, or modulated, by the prevailing growth conditions. This model is confirmed by a nutritional shift-down, where nuclear division and cell division are stimulated after the shift. Cell length at division falls rapidly until the new shorter length is attained, when a new steady state is assumed at a slower growth rate. The control system is compared with that in bacteria, and its implications for various models proposed for the control of timing of mitosis are discussed.

The ends (telomeres) of eukaryotic chromosomes must have special features to ensure their stability and complete replication. Studies in yeast, protozoa, slime moulds and flagellates show that telomeres are tandem repeats of simple sequences that have a G-rich and a C-rich strand. Mammalian telomeres have yet to be isolated and characterized, although a DNA fragment within 20 kilobases of the telomeres of the short arms of the human sex chromosomes has been isolated. Recently we showed that a chromosome from the fission yeast Schizosaccharomyces pombe could, in some cases, replicate as an autonomous mini-chromosome in mouse cells. By extrapolation from other systems, we reasoned that mouse telomeres could be added to the S. pombe chromosome ends in the mouse cells. On setting out to test this hypothesis we found to our surprise that the telomeric probe used (containing both the S. pombe and Tetrahymena thermophila repeats) hybridized to a series of discrete fragments in normal mouse DNA and DNA from a wide range of eukaryotes. We show here that the sequences hybridizing to this probe are located at the telomeres of most, if not all, human chromosomes and are similar to the Tetrahymena telomeric-repeat component of the probe.

Strains of Schizosaccharomyces pombe carrying the wee 1 mutation divide at a reduced cell size compared with the wild-type. In this paper, we investigate the mechanism which determines the time of division and cell size at division in wee 1 strains, using three experimental approaches. The evidence suggests that the wild-type control (a cell size control over entry into nuclear division) is absent in wee 1 strains. Instead, a mechanism operates which comprises a cell size control over the initiation of S phase plus a minimum incompressible period in G2 (“timer”) from S phase to nuclear division. The elements of this second control mechanism exist in wild-type cells, though the control is not normally expressed. In particular, the G2 interval in wild-type cells is normally longer than that in wee 1 cells, but can be reduced to this minimum value by delaying S phase. Thus there are two independent controls over entry into nuclear division, one of which operates in wild-type, and the other in wee 1 cells.

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.

THERE is currently much interest in the mechanism which controls the timing of cell division. Certain features of the control have been found to be common to a variety of eukaryotes. In particular, the importance of cell size as a parameter affecting cell cycle progress has been reported for mammalian cells(1,2) and for several single-celled eukaryotes(3-6). Another feature common to several systems is that growth conditions have a direct effect on the timing of division cycle events(7-9), and on cell size(9,10). In the fission yeast Schizosaccharomyces pombe, both cell size(6) and nutritional conditions(9) have been shown to affect cycle kinetics. The organism has been used extensively as a model eukaryotic system, largely because of the ease of measuring cell size and because division occurs by binary fission(11). More recently, its genetic tractability has led to the isolation of cell division cycle (cdc) mutants(12), and also of wee mutants altered in the control coordinating growth with the division cycle(13-15). The existence of such control mutants allows a more direct approach to the investigation of the molecular basis of division control, in contrast to the indirect methods used in other systems(4,16-18). wee mutants are so far unique to S. pombe. The most conspicuous property of wee mutants is their reduced cell size(13,14). Analysis of these mutants(15,19) and other evidence(9) has shown that control over cell division timing normally acts at entry to mitosis. As the function of a number of cdc genes is specifically required for mitosis(12), interactions between wee and cdc mutants which affect mitosis might be expected. I report here that the mitotic defect caused by a defective cdc25 allele is suppressed in wee mutants. Suppression by wee1 mutants is almost complete, while the wee2.1 mutation is a less effective suppressor. The significance of these findings for genetic models of the control of mitosis is considered.

Two independent DNA sequences, of 5.6 and 2.4 kb in size, were isolated from Schizosaccharomyces pombe gene libraries on the basis of their ability to rescue the temperature-conditional lethality conferred by a cdc22 mutation. Integration of these sequences into the S.pombe genome by homologous recombination, followed by genetic mapping, demonstrated that the site of integration of the 5.6 kb fragment is tightly linked to the cdc22 locus, while that of the 2.4 kb fragment is unlinked. This shows that the 5.6 kb fragment carries the authentic cdc22+ gene while the 2.4 kb fragment carries and extragenic suppressor sequence. The cdc22 transcript was identified by Northern blot analysis and shown to be 3.3 kb in size. The level of the transcript during the cell cycle was investigated in synchronous cultures prepared by elutriation. The cdc22 transcript is cell cycle regulated, reaching a maximum level during late G1/S phase, at least 12-fold higher than the minimum level observed in mid G2.

To test the functional capacity of a fission yeast chromosome in mouse cells, a strain of the fission yeast Schizosaccharomyces pombe, ED628 Int5, was constructed. A plasmid bearing the SV2NEO gene, which can confer G418 resistance to mouse cells, was integrated at the ura4 locus on S. pombe chromosome III. S. pombe Int5 chromosomes were introduced into mouse C127 cells by PEG-facilitated protoplast fusion. Here we describe two independent G418-resistant cell lines with distinct growth characteristics, F1.1 and F7.1, and examine the structure of material derived from S. pombe Int5 chromosome III in these lines. F1.1 is shown to contain a single rearranged block of chromatin from S. pombe chromosome III integrated into a mouse chromosome, maintained in the absence of selection. In contrast, the data for F7.1 are consistent with the presence of linear, unintegrated copies of S. pombe chromosome III, which are apparently intact and maintained in an unstable but autonomous state. The unstable maintenance of this chromosome may be due to defective centromere function leading to missegregation at mitosis or to over- or underreplication.

The major cell cycle control in the fission yeast Schizosaccharomyces pombe acts at entry to mitosis, and involves three previously identified genes cdc2, cdc25 and wee1. The presence of a weel mutation phenotypically suppresses cdc25 mutations. This paper describes the isolation and subsequent analysis of a strain in which the suppression is reversed by the presence of a new mutation, designated win1.1. The mutation causes a slight increase in cell size at division in most genetic backgrounds. However, when combined with a wee1 mutation and cdc25.22, the win 1.1 mutation interacts strongly to generate a novel phenotype: cells are phenotypically cdc during growth on minimal medium but cdc+ when cultured on complex medium. The win1 locus is unlinked to previously identified genes involved in mitosis.

Schizosaccharomycespombe adjusts cell size in response to changes in the nutritional environment.1,2 A comparison of cell size in different media shows that cells are small under poorer growth conditions and that shifts from richer to poorer media and vice versa result in a rapid change in size. The kinetics of the response reveal that the adjustment is made at the G2 mitotic control and is achieved by either stimulating or delaying cell division.

The means by which cells control the timing of events in the cell division cycle have been investigated in depth recently. The existence of controls over the major events of DNA replication and mitosis has been demonstrated in several systems; controls of both events have been identified in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The control points have been defined on the basis of cell cycle arrest caused by certain mutations1–4 or mating pheromones5, and by growth-related parameters3,6–10. Investigation of how the component processes, generally identified by specific genes, are organized into developmental pathways11,12 has provided much information on how the proper temporal order of events is maintained, but little is known about the control of the absolute timing of the component processes in the cycle. The time of completion of a gene-controlled process in the cell cycle can be estimated from the transition or execution point of a temperature-sensitive mutant defective in the particular gene13. In the fission yeast S. pombe the transition points of most mutants defective for mitosis are temporally clustered shortly before the event14, but the mechanism which maintains this coordination has not been investigated. I report here that the transition points of three genes required for mitosis are advanced in strains carrying a wee 1 mutation, whose best known effect is to reduce cell size at mitosis15. The observed advancement can be as much as 0.4 of the cycle, implicating the wee 1+ gene in a system which controls the timing and temporal coordination of developmental steps controlled by three mitotic genes.

The p107wee1 protein kinase plays a central role in regulating the cell cycle of fission yeast. It mediates transmission of signal(s) related to the nutritional status of the cell to the p34cdc2 protein kinase, which is an active component of the MPF complex driving cells into mitosis. p107wee1 is itself subject to control by the products of other genes such as nim1+/cdr1+, win1+, and perhaps wis1+ and other wis+ genes. At present, the relationships between these genes and their possible roles in the mitotic control are unclear and must await further analysis (Fig. 5). It is likely that some of the gene products are concerned with the sensing and/or transmission of nutritional signals. p107wee1 negatively regulates the activity of p34cdc2, probably by direct tyrosine phosphorylation, and also appears to regulate the activities of the cdc1+ and cdc27+ gene products. The effects of nitrogen starvation and of wee1 mutations on conditional lethal mutations at the cdc1, cdc2, and cdc27 loci, taken together, support the largely speculative model shown in Figure 5. During the normal cycle, the balance between phosphorylated and dephosphorylated p34cdc2 changes such that at the appropriate time, p34cdc2 is activated and the cell enters mitosis. We suggest that the cdc1+ and cdc27+ products may be regulated in a similar way. Such a mechanism would ensure coordinated activation of these and perhaps other proteins required for the G2/M transition. There are, of course, many uncertainties, and these must await elucidation by biochemical and genetic analysis.

One of the most exciting aspects of scientific research is the convergence of several apparently quite diverse types of investigation. Such events are rare, and the resulting cross-fertilization demands that findings derived from different areas are substantially reassessed. A handful of recent papers 1–6 , each concerned with control of the initiation of mitosis, have brought together observations from cell types that were previously regarded as possessing quite different control systems.