RESUMEN

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.

Asunto(s)

Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Proteínas de Drosophila/genética , Genes Fúngicos , Chaperonas Moleculares/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/fisiología , ADN de Hongos/genética , Proteínas de Drosophila/fisiología , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Chaperonas Moleculares/fisiología , Datos de Secuencia Molecular , Fenotipo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Homología de Secuencia de Aminoácido

RESUMEN

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.

Asunto(s)

Proteínas de Ciclo Celular/genética , Regiones Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Transactivadores/metabolismo , Factores de Transcripción , Transcripción Genética/genética , Secuencia de Bases , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Huella de ADN , ARN Polimerasas Dirigidas por ADN , Fase G1 , GTP Fosfohidrolasas , Regulación Fúngica de la Expresión Génica , Glicoproteínas de Membrana/genética , Proteínas de la Membrana , Datos de Secuencia Molecular , Ribonucleótido Reductasas , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/genética

RESUMEN

Wis1 is a mitogen-activated protein kinase kinase (MAPKK) that regulates mitosis and mediates stress responses in the fission yeast, Schizosaccharomyces pombe. wis1Delta strains are viable but stress-sensitive and show a mitotic delay. At high temperatures, wis1Delta cells cease division but cellular growth continues. Mutations that suppress the heat sensitivity of a wis1Delta strain were isolated and map to two apparently novel loci, sow1 (for suppressor of wis1Delta) and sow2. In addition to suppressing wis1Delta heat sensitivity, sow1 and sow2 can suppress wis1Delta 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.

Asunto(s)

Ciclo Celular/genética , Genes Fúngicos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Ciclo Celular/fisiología , Genes Supresores/genética , Calor , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Mutagénesis , Schizosaccharomyces/enzimología , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/efectos de la radiación

RESUMEN

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.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , ADN Polimerasa III/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Sitios de Unión , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Datos de Secuencia Molecular , Unión Proteica , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Alineación de Secuencia , Transducción de Señal , Ubiquitina-Proteína Ligasas

RESUMEN

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.

Asunto(s)

Replicación del ADN , ADN de Hongos/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , ADN Polimerasa II/metabolismo , ADN Polimerasa III/metabolismo , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Genes Fúngicos , Genes Letales , Antígenos de Histocompatibilidad Menor , Mitosis , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína de Replicación C , Proteínas de Schizosaccharomyces pombe , Homología de Secuencia de Aminoácido , Ubiquitina-Proteína Ligasas

RESUMEN

Highly purified DNA polymerase delta 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 delta 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 delta function.

Asunto(s)

ADN Polimerasa III/genética , Factores de Intercambio de Guanina Nucleótido , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Recuento de Células/efectos de los fármacos , Proteínas de Ciclo Celular/genética , División Celular/genética , Cromosomas/genética , Clonación Molecular , Proteínas Fúngicas/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/genética , Datos de Secuencia Molecular , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Schizosaccharomyces/crecimiento & desarrollo , Análisis de Secuencia de ADN , Supresión Genética/genética , Temperatura

RESUMEN

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+ and wis4+, 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 win1 mutations. 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 wee1-50 mutant and its suppression by wis genes.

Asunto(s)

Genes Fúngicos , Quinasas Quinasa Quinasa PAM , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular/genética , Mapeo Cromosómico , Activación Enzimática , Quinasas de Proteína Quinasa Activadas por Mitógenos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Concentración Osmolar , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/química , Mapeo Restrictivo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

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.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Regulación Fúngica de la Expresión Génica , Quinasas Quinasa Quinasa PAM , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Ciclo Celular/genética , Secuencia Conservada , Activación Enzimática , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Presión Osmótica , Estrés Oxidativo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

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.

Asunto(s)

Ciclo Celular , Replicación del ADN , Origen de Réplica , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/genética , División Celular , Replicación del ADN/efectos de los fármacos , ADN de Hongos/análisis , Citometría de Flujo/métodos , Genes Fúngicos , Genes Letales , Técnicas Genéticas , Genoma Fúngico , Hidroxiurea/farmacología , Cinética , Mutagénesis , Mutágenos/farmacología , Micología/métodos , Schizosaccharomyces/citología

RESUMEN

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.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/metabolismo , Alelos , Secuencia de Aminoácidos , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Cromosomas Fúngicos , Clonación Molecular , ADN Polimerasa III , Eliminación de Gen , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas de Schizosaccharomyces pombe , Homología de Secuencia de Aminoácido , Ubiquitina-Proteína Ligasas

RESUMEN

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.

Asunto(s)

Ciclo Celular/genética , Daño del ADN , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Respuesta al Choque Térmico , Ribonucleótido Reductasas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Genes Fúngicos , Mitosis , ARN de Hongos , Transducción de Señal

RESUMEN

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.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulación Fúngica de la Expresión Génica , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Transducción de Señal , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Serina-Treonina Quinasas/genética , Schizosaccharomyces/genética

RESUMEN

Cyclophilins are peptidyl-prolyl cis-trans isomerases (PPIases) which have been implicated in intracellular protein folding, transport and assembly. Cyclophilins are also known as the intracellular receptors for the immunosuppressive drug cyclosporin A (CsA). The most common type of cyclophilins are the 18 kDa cytosolic proteins containing only the highly conserved core domain for PPIase and CsA binding activities. The wis2+ gene of the fission yeast Schizosaccharomyces pombe was isolated as a multicopy suppressor of wee1-50 cdc25-22 win1-1, a triple mutant strain which exhibits a cell cycle defect phenotype. Sequence analysis of wis2+ reveals that it encodes a 40 kDa cyclophilin-like protein, homologous to the mammalian cyclophilin 40. The 18 kDa cyclophilin domain (CyP-18) of wis2 is followed by a C-terminal region of 188 amino acids. The C-terminal region of wis2 is essential for suppression of the triple mutant defect. Furthermore this region of the protein is able to confer suppression activity on the 18 kDa S.pombe cyclophilin, cyp1, since a hybrid protein consisting of an 18 kDa S.pombe cyclophilin (cyp1) fused to the C-terminus of wis2 shows suppression activity. We also demonstrate that the level of wis2+ mRNA increases 10- to 20-fold upon heat shock of S.pombe cells suggesting a role for wis2+ in the heat-shock response.

Asunto(s)

Isomerasas de Aminoácido/genética , Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Supresores , Schizosaccharomyces/genética , Isomerasas de Aminoácido/biosíntesis , Isomerasas de Aminoácido/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Ciclo Celular/genética , ADN de Hongos/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Calor , Humanos , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Isomerasa de Peptidilprolil , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo Restrictivo , Schizosaccharomyces/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

We identified the phh1+ 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.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Diferenciación Sexual , Transducción de Señal/fisiología , Estrés Fisiológico , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , División Celular , Medios de Cultivo/química , Fertilidad/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Calor , Quinasas Quinasa Quinasa PAM/genética , Datos de Secuencia Molecular , Nitrógeno/química , Nitrógeno/metabolismo , Presión Osmótica , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Esporas

RESUMEN

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.

Asunto(s)

Ciclo Celular/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Schizosaccharomyces/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Proteínas Fúngicas/genética , Fase G1/genética , Fase G2/genética , Genes Fúngicos/fisiología , Genes Recesivos , Datos de Secuencia Molecular , Mutación/genética , Schizosaccharomyces/citología , Schizosaccharomyces/fisiología , Temperatura , Factores de Transcripción/genética

RESUMEN

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

Asunto(s)

Meiosis/fisiología , ARN de Hongos/fisiología , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Precursores del ARN/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Schizosaccharomyces/genética

RESUMEN

Fission yeast temperature-sensitive cut5 (cell untimely torn) mutants are defective in initiation and/or elongation of DNA replication but allow mitosis and cell division at a restrictive temperature. We show that the cut5 protein (identical to rad4) (i) is an essential component of the replication checkpoint system but not the DNA damage checkpoint, and (ii) negatively regulates the activation of M phase kinase at mitotic entry. Even if the replication checkpoint has been activated previously, cut5 mutations allow mitosis and cell division after shift to 36 degrees C. Transcription of cut5+ is not under the control of the START gene cdc10+. The cut5 protein is enriched in the nucleus, consisting of repeating domains. An essential domain which resembles the proto-oncoprotein Ect2 has a strong negative effect on the entry into mitosis when overexpressed. Expression of the cut5 mutant phenotype requires the function of the M phase regulator genes cdc2+, cdc25+ and cdc13+. The cut5 protein forms a novel, essential link between DNA synthesis and M phase activation in the replication checkpoint control pathway.

Asunto(s)

Cromatina/metabolismo , Cromosomas Fúngicos/metabolismo , Replicación del ADN , ADN de Hongos/biosíntesis , Proteínas de Unión al ADN , Proteínas Fúngicas/fisiología , Metafase , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Transglutaminasas , Secuencia de Aminoácidos , Cromátides/metabolismo , Secuencia de Consenso , ADN de Hongos/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/química , Schizosaccharomyces/citología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética

RESUMEN

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.

Asunto(s)

Replicación del ADN/genética , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Mitosis/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Transglutaminasas , Ciclo Celular/genética , Reparación del ADN/genética , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo

RESUMEN

The cdc22+ gene of Schizosaccharomyces pombe is required early in the cell cycle, and its transcript varies in concentration in step with the cell cycle, with a peak level at the G1-S boundary. The sequences of the cdc22+ gene and of a multicopy suppressor of cdc22ts mutations, suc22+, have been determined. The cdc22+ open reading frame, which is interrupted in the genome by a single intron very close to its 5' end, encodes a protein of 811 amino acids, which has an amino acid sequence highly similar to that of the large subunit of ribonucleotide reductase from several species. The suc22+ gene contains an uninterrupted open reading frame of 391 amino acids, very similar to the sequence of the small subunit of ribonucleotide reductase. Disruption of either gene is lethal. Upstream of the cdc22+ coding region are seven short sequence elements similar to the recognition sequence for MluI, which are involved in regulating periodic transcription of the gene. Inhibition of DNA synthesis by hydroxyurea results in a several-fold increase in the level of the cdc22+ transcript. In contrast, hydroxyurea does not induce the 1.5 kb transcript of suc22+, but results in the induction of a 1.9 kb mRNA which hybridises to suc22+ DNA.

Asunto(s)

Proteínas de Ciclo Celular , Ciclo Celular/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Ribonucleótido Reductasas/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Genes Supresores , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Plásmidos , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Homología de Secuencia de Aminoácido , Transcripción Genética