Tyrosine phosphorylation of cdc2 is required for the replication checkpoint in Schizosaccharomyces pombe

The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea. In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15. On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism. We have undertaken biochemical and genetic studies to resolve this controversy. We report that the DNA replication checkpoint in S. pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis. These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggests that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint.     

The Spg1p GTPase is an essential, dosage-dependent inducer of septum formation in Schizosaccharomyces pombe

The spg1 gene (septum-promoting GTPase) was cloned as a multicopy suppressor of a dominant-negative mutant of the Cdc7p kinase. It encodes a small GTPase of the Ras superfamily. spg1 is an essential gene. Null or heat-sensitive alleles do not make a division septum, but growth, S-phase, and mitosis continue in the absence of cell division, producing elongated, multinucleate cells. Increased expression of Spg1p induces septum formation in G2, S-phase, and pre-Start G1-arrested cells. This requires the activity of Cdc7p kinase, but not p34(cdc2). Increased expression of Cdc7p bypasses the requirement for Spg1p. Spg1p and Cdc7p can be coimmunoprecipitated from cell extracts, and interact in the two-hybrid system. These data indicate that Spg1p is a key element in controlling the onset of septum formation in Schizosaccharomyces pombe, and that it acts through the Cdc7p kinase.     

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint

In fission yeast both DNA polymerase alpha (pol alpha) and delta (pol delta) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol epsilon) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol epsilon (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29-39]. We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol epsilon does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol alpha rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol alpha, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.     

cut11(+): A gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe

The "cut" mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11(+) suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11(+) encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.     

The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe

BACKGROUND: The small GTP-binding protein Rho has been shown to regulate the formation of the actin cytoskeleton in animal cells. We have previously isolated two rho genes, rho1+ and rho2+, from the fission yeast Schizosaccharomyces pombe in order to investigate the function of Rho using genetic techniques. In this paper, we report the cellular function of Rho1. RESULTS: We found that Rho1 is essential for cell viability and cell polarity using gene disruption and by exogenous expression of botulinum C3 ADP-ribosyltransferase. In cells expressing either a constitutively active Rho1 or a dominant-negative Rho1, actin patches were delocalized. Both the cell wall and secondary septum were thick and stratified in cells expressing the constitutively active Rho1, while the cell wall of cells expressing the dominant-negative Rho1 seemed to be loosely organized. Furthermore, inactivation of Rho1 is apparently required for the separation of daughter cells. Cell fractionation studies suggested that Rho1 is predominantly membrane-bound. Moreover, we observed that Rho1 is localized to the cell periphery and to the septum. CONCLUSIONS: Rho1 is involved in actin patch localization, the control of cell polarity, the regulation of septation, and cell wall synthesis.     

Cdm1, the smallest subunit of DNA polymerase d in the fission yeast Schizosaccharomyces pombe, is non-essential for growth and division

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the most abundant protein kinases in the brain and has a broad substrate specificity [M.K. Bennett, N.E. Erondu, M.B. Kennedy, Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem. 258 (1983) 12735-12744 [1]; J.R. Goldenring, B. Gonzalez, J.S. McGuire, Jr., R.J. DeLorenzo, Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins, J. Biol. Chem. 258 (1983) 12632-12640 [4]; M.B. Kennedy, P. Greengard, Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1293-1297 [10]; T. Yamauchi, H. Fujisawa, Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain, FEBS Lett. 116 (1980) 141-144 [20]; T. Yamauchi, H. Fujisawa, Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase, Eur. J. Biochem. 132 (1983) 15-21 [21]]. The alpha and beta isoforms of CaM kinase II are known to be expressed almost exclusively in the brain [P.I. Hanson, H. Schulman, Ca2+/calmodulin-dependent protein kinases, Annu. Rev. Biochem. 61 (1992) 559-601 [7]]. To elucidate the cellular function of CaM kinase II, we introduced cDNA of wild-type CaM kinase II alpha- or beta-isoform, and of mutant alpha-isoform (Ala-286 kinase) into two different types of neuroblastoma, Neuro2a (Nb2a) and NG108-15, thus generating cell lines stably producing elevated levels of these kinases. The mutant alpha-isoform is markedly suppressed in its autophosphorylation by replacement of Thr-286 with Ala [Y.-L. Fong, W.L. Taylor, A.R. Means, T.R. Soderling, Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate, J. Biol. Chem. 264 (1989) 16759-16763 [3]; P.I. Hanson, M.S. Kapiloff, L.L. Lou, M.G. Rosenfeld, H. Schulman, Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation, Neuron 3 (1989) 59-70 [6]; S. Ohsako, H. Nakazawa, S. Sekihara, A. Ikai, T. Yamauchi, Role of Threonine-286 as autophosphorylation site for appearance of Ca2+-independent activity of calmodulin-dependent protein kinase II alpha subunit, J. Biochem. 109 (1991) 137-143 [15]]. We provided evidence that CaM kinase II played a role in regulating neurite outgrowth and growth cone motility in these cells, and that the autophosphorylation is essential for the kinase to sufficiently exert its cellular function in vivo [Y. Goshima, S. Ohsako, T. Yamauchi, Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility, J. Neurosci. 13 (1993) 559-567 [5]]. Neurite outgrowth was further stimulated by treatment with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or chelerythrine, inhibitors of protein kinase C [T. Nomura, K. Kumatoriya, Y. Yoshimura, T. Yamauchi, Overexpression of alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II in neuroblastoma cells-H-7 promotes neurite outgrowth, Brain Res. 766 (1997) 129-141 [14]]. The morphological change stimulated with protein kinase inhibitors was rapid and was greater in the beta than alpha cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing the kinase stimulated with H-7. These results suggest that CaM kinase II and protein kinase C play an important role in the control of cell change. (c) 1998 Elsevier Science B.V. All rights reserved.     

A novel fission yeast gene, kms1+, is required for the formation of meiotic prophase-specific nuclear architecture

From archival records, we assessed outcomes achieved by preschoolers with both severe mental retardation and autistic features: (a) an experimental group (n = 11), which received intensive behavioral treatment, and (b) a comparison group (n = 10), which received minimal treatment. At intake (mean CA = 3.08 years), the groups did not differ significantly on any variable. At follow-up children in the experimental group obtained a higher mean IQ and evinced more expressive speech than did those in the comparison group. Behavior problems diminished in both groups. Results indicate that intensively treated children achieved clinically meaningful gains relative to the comparison group but remained quite delayed.     

The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA

Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression of GCN4 mRNA. The phenotypes of gcd10 mutants were suppressed by high-copy-number IMT genes, encoding initiator methionyl tRNA (tRNAiMet), or LHP1, encoding the yeast homolog of the human La autoantigen. The gcd10-504 mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet. Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNAiMet is the essential function of Gcd10p. A gcd14-2 mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMet processing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMet maturation. The chromatographic behavior of elongator and initiator tRNAMet on a RPC-5 column indicated that both species are altered structurally in gcd10Delta cells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable in gcd10Delta tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.     

The UDP-Glc:Glycoprotein glucosyltransferase is essential for Schizosaccharomyces pombe viability under conditions of extreme endoplasmic reticulum stress

Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions. We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides. The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT). Both single mutants grew normally at 28 degreesC. On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28 degreesC and did not grow at 37 degreesC. The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation. It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37 degreesC and had, when grown at 28 degreesC, a phenotype of growth and morphology almost identical to that of wild-type cells. These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation-deglucosylation catalyzed by GT and GII.     

The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised

Premature initiation of cytokinesis can lead to loss of chromosomes, and 'cutting' of the nucleus. Therefore, the proper spatial and temporal co-ordination of mitosis and cytokinesis is essential for maintaining the integrity of the genome. The fission yeast cdc16 gene is implicated both in the spindle assembly checkpoint and control of septum formation. To identify other proteins involved in these controls, we have isolated multicopy suppressors of the cdc16-116 mutation, and the characterization of one of these, dma1 (defective in mitotic arrest), is presented here. dma1 is not an essential gene, but in a dma1 null background (dma1-D1) the function of the spindle assembly checkpoint is compromised. If assembly of the spindle is prevented, dma1-D1 cells do not arrest, the activity of cdc2 kinase decays and cells form a division septum without completing a normal mitosis. dma1-D1 cells also show an increased rate of chromosome loss during exponential growth. Upon ectopic expression from an inducible promoter, dma1p delays progress through mitosis and inhibits septum formation, giving rise to elongated, multinucleate cells. We propose that dma1 is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is impaired.     

Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe

Gastric effects of subchronic treatment with the cholecystokinin-B (CCK-B)/gastrin receptor antagonist CI-988 were investigated in cynomolgus monkeys. In preliminary range-finding studies, CI-988 was given orally to 1 monkey per sex for 14 days at doses of 50, 100, 200, and 500 mg/kg/day. Subchronic studies of CI-988 were subsequently conducted using 5 monkeys per sex at doses of 0, 5, 25, and 75 mg/kg for 4 or 13 wk. High-dose monkeys were dosed initially at 100 mg/kg, but the dose was not well tolerated and was decreased to 75 mg/kg after 8 days of treatment. One male monkey at 75 mg/kg was euthanatized in extremis on day 23. In the range-finding study, minimal to moderate, multifocal to diffuse degeneration of gastric glands, primarily in the fundic region, was observed at 100 mg/kg and above, with frank gastric mucosal atrophy occurring at 200 and 500 mg/kg. Minimal to mild gastric gland degeneration was also observed in the subchronic study after 4 wk at 25 and 75 mg/kg, but histopathologic gastric changes were remarkably absent after 13 wk. Mucosal height in the stomach fundus was decreased 19.8% in 75-mg/kg males at week 4, and although gastric mucosa appeared histologically normal after 13 wk, mucosal height remained 28.6% less than that of controls. In females at 75 mg/kg, fundic mucosal height was decreased 7% and 5% at weeks 4 and 13, respectively, but decreases were not statistically significant. Mean serum gastrin concentrations were increased 10-fold in males only after 4 wk at 75 mg/kg, but were comparable to controls during week 13. CI-988-induced gastric gland degeneration is consistent with antagonism of gastrin's trophic activity toward gastric mucosa. Notwithstanding decrements in gastric mucosal height, disappearance of mild histopathologic findings despite continued treatment with the ligand suggests some degree of adaptation to subchronic CCK-B/gastrin inhibition, although the mechanism of accommodation has yet to be delineated.     

The structural H19 gene is required for transgene imprinting

The product of the H19 gene is an untranslated RNA that is expressed exclusively from the maternal chromosome during mammalian development. The H19 gene and its 5'-flanking sequence are required for the genomic imprinting of two paternally expressed genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), that lie 90 and 115 kb 5' to the H19 gene, respectively. In this report, the role of the H19 gene in its own imprinting is investigated by introducing a Mus spretus H19 gene into heterologous locations in the mouse genome. Multiple copies of the transgene were sufficient for its paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene resulted in loss of imprinting of the transgene. That is, high expression and low levels of DNA methylation were observed upon both paternal and maternal inheritance. The removal of 701 bp at the 5' end of the structural gene resulted in a similar loss of paternal-specific DNA methylation, arguing that those sequences are required for both the establishment and maintenance of the sperm-specific gametic mark. The M. spretus H19 transgene could not rescue the loss of Igf-2 imprinting in trans in H19 deletion mice, implying a cis requirement for the H19 gene. In contrast to a previous report in which overexpression of a marked H19 gene was a prenatal lethal, expression of the M. spretus transgene had no deleterious effect, leading to the conclusion that the 20-base insertion in the marked gene created a neomorphic mutation.     

The cdr2(+) gene encodes a regulator of G2/M progression and cytokinesis in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G2/M cell size control. The G2/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p. Cdc2p activation is regulated both positively and negatively. cdr2(+) was identified in a screen for regulators of mitotic control during nutrient deprivation. We have cloned cdr2(+) and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S. pombe Cdr1p/Nim1p. cdr2(+) is not essential for viability, but cells lacking cdr2(+) are elongated relative to wild-type cells, spending a longer period of time in G2. Because of this property, upon nitrogen deprivation cdr2(+) mutants do not arrest in G1, but rather undergo another round of S phase and arrest in G2 from which they are able to enter a state of quiescence. Genetic evidence suggests that cdr2(+) acts as a mitotic inducer, functioning through wee1(+), and is also important for the completion of cytokinesis at 36 degrees C. Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1(+), suggesting that cdr2(+) encodes a second activity involved in cytokinesis.     

The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila

BACKGROUND: Drosophila axis formation requires a series of inductive interactions between the oocyte and the somatic follicle cells. Early in oogenesis, Gurken protein, a member of the transforming growth factor alpha family, is produced by the oocyte to induce the adiacent follicle cells to adopt a posterior cell fate. These cells subsequently send an unidentified signal back to the oocyte to induce the formation of a polarised microtubule array that defines the anterior-posterior axis. The polarised microtubules also direct the movement of the nucleus and gurken mRNA from the posterior to the anterior of the oocyte, where Gurken signals a second time to induce the dorsal follicle cells, thereby polarising the dorsal-ventral axis. RESULTS: In addition to its previously described role in the localisation of oskar mRNA, the mago nashi gene is required in the germ line for the transduction of the polarising signal from the posterior follicle cells. Using a new in vivo marker for microtubules, we show that mago nashi mutant oocytes develop a symmetric microtubule cytoskeleton that leads to the transient localisation of bicoid mRNA to both poles. Furthermore, the oocyte nucleus often fails to migrate to the anterior, causing the second Gurken signal to be sent in the same direction as the first. This results in a novel phenotype in which the anterior of the egg is ventralised and the posterior dorsalised, demonstrating that the migration of the oocyte nucleus determines the relative orientation of the two principal axes of Drosophila. The mago nashi gene is highly conserved from plants to animals, and encodes a protein that is predominantly localised to nuclei. CONCLUSIONS: The mago nashi gene plays two essential roles in Drosophila axis formation: it is required downstream of the signal from the posterior follicle cells for the polarisation of the oocyte microtubule cytoskeleton, and has a second, independent role in the localisation of oskar mRNA to the posterior of the oocyte.     

DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo.     

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.