Schizosaccharomyces

Biochim Biophys Acta, 2003 Apr 1, 1611(1-2), 217 - 22
Effects of hexavalent chromium on the plasma membranes of sensitive and tolerant mutants of Schizosaccharomyces pombe . An EPR study; Farkas N et al.; The interactions of chromium(VI) with the plasma membranes of chromium-sensitive (chr-51S) and chromium-tolerant (chr1-66T) mutants and their parental strain (6chr(+)) of a Schizosaccharomyces pombe strain were studied by electron paramagnetic resonance (EPR) spectroscopy . 5-doxylstearic acid (5-SASL) and 3-doxylbutyric acid (HO-185) spin probes were used to label the membranes . The order parameter S from the EPR spectra was calculated at different temperatures (0-25 degrees C) in order to characterize the internal dynamics of the membranes . In control experiments, both mutants exhibited differences in structural transitions in the both 5-SASL- and the HO-185-labeled membranes in comparison with their parental strain, suggesting differences in the membrane composition and/or rotational dynamics of these mutants . Addition of K(2)Cr(2)O(7) (225 microM) induced small decreases in the phase transition temperatures of the 5-SASL-labeled membranes of the parental and chromium-sensitive strains . More pronounced effects of the chromium compound on the HO-185-labeled membranes were detected as evidence that the membrane perturbations are mostly localized in the environment of the lipid-water interface.

Gene, 2003 Mar 13, 306, 13 - 25
The Plasmodium falciparum family of Rab GTPases; Quevillon E et al.; Rab GTPases are key regulators of vesicular traffic in eukaryotic cells . Here we sought a global characterization and description of the Plasmodium falciparum family of Rab GTPases . We used a combination of bioinformatic analyses, experimental testing of predictions, structure modelling and phylogenetics . These analyses led to the identification of seven new parasite Rabs . Accordingly we estimate that the P . falciparum family is made up of 11 genes . We show that ten members of this family are transcribed in infected erythrocytes . Concerning the various members of the family, a series of specific as well as global conclusions can be drawn . Rabs predicted to be compartment-specific show different subcellular distributions . This is demonstrated for PfRab1A and PfRab11A, with the generation of specific antisera . The sequence analyses reveal several peculiarities, with possible functional implications . One of the transcribed genes, Pfrab5b, does not encode a classical C-terminus, suggestive of a novel regulatory role for this GTPase . Another, Pfrab5a, previously identified as a rab gene located on chromosome 2, possesses a 30-amino-acid insertion in its GTP-binding domain . Structural considerations suggest that this insertion could represent a novel interaction interface . We used conserved RabF and RabSF motifs to discriminate between specific parasite Rabs, and followed their predicted change in position on the structure of PfRab6, as GTP is hydrolysed to GDP . This allowed us to propose their involvement in potential interaction surfaces, that we extended to human Rab6 and the motifs known to mediate Rabkinesine-6 binding . Finally, we compared the P . falciparum Rab family to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe and found that parasite Rabs segregate into possible functional clads . Such grouping into clads may give clues to parasite Rab function, and may shed light on P . falciparum secretory/endocytic pathways.

J Cell Biol, 2003 Mar 31, 160(7), 1083 - 92 Epub 2003 Mar 24.
Mid2p stabilizes septin rings during cytokinesis in fission yeast; Berlin A et al.; Septins are filament-forming proteins with a conserved role in cytokinesis . In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell-cell separation, a late stage in cytokinesis . Here, we identified a protein Mid2p on the basis of its sequence similarity to S . pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p . Like septin mutants, mid2delta mutants had delays in cell-cell separation . mid2delta mutants were defective in septin organization but not contractile ring closure or septum formation . In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract . In mid2delta cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure . FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2delta cells . Mid2p colocalized with septins and required septins for its localization . A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function . No genetic interactions were found between mid2 and the related gene mid1 . Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.

Genes Cells, 2003 Apr, 8(4), 357 - 70
The small GTPase Rho4 is involved in controlling cell morphology and septation in fission yeast; Nakano K et al.; BACKGROUND: Rho family small GTPases have been shown to be involved in various cellular activities, including the organization of actin cytoskeleton in eukaryotic cells . There are six rho genes in the fission yeast Schizosaccharomyces pombe . Cdc42 is known to control the polarity of the cell . Rho1, Rho2 and Rho3 play important roles in controlling cell shape and septation . On the other hand, Rho4 and Rho5 have not yet been characterized . Here we report the function of rho4+ in fission yeast . RESULTS: Gene disruption revealed that rho4+ is not essential for cell growth . However, rho4-null cells were abnormally elongated and had multiple septa of irregular shape at 37 degrees C . In these cells, F-actin patches were randomly localized all over the cell periphery, and cytoplasmic microtubules (MTs) were misoriented . On the other hand, the exogenous expression of a constitutively active Rho4-G23V or Rho4-Q74L in wild-type cells induced depolarization of F-actin patches and cytoplasmic MTs . Rho4 was localized to the cell periphery during interphase and septum during mitosis . Both the binding of GTP and isoprenylation of its C-terminus were necessary for the localization . Furthermore, the localization of Rho4 was likely to be controlled by Rho GAP and Rho GDI . CONCLUSION: Rho4 may control cell morphogenesis and septation by regulating both the actin cytoskeleton and cytoplasmic MTs.

Genes Cells, 2003 Apr, 8(4), 341 - 55
A brute force postgenome approach to identify temperature-sensitive mutations that negatively interact with separase and securin plasmids; Matsumura T et al.; BACKGROUND: The fission yeast Schizosaccharomyces pombe separase/Cut1 and securin/Cut2 are required for anaphase-specific activation of proteolysis that leads to proper sister chromatid separation . We intended to identify ts (temperature sensitive) strains whose growth was inhibited by multicopy plasmid pCUT1 or pCUT2 at the permissive temperature . RESULTS: After a one-by-one transformation of 1015 randomly isolated ts strains, 18 transformants that retarded in colony formation at the permissive or semipermissive temperature were isolated . Six of them, in the absence of pCUT1 or pCUT2, produced mitotic phenotypes with condensed chromosomes at the restrictive temperature . Gene cloning established that these mutants were defective in either the subunits (Cut9, Cut23, Cut20 or Apc10) of APC (anaphase promoting complex)/cyclosome or Cut8, a regulator for 26S proteasome localization . The inhibitory effect of separase against APC/cyclosome mutations was abolished when the catalytic site mutation C1730A was introduced and overproduced, indicating that inhibition needs an active separase . Securin/Cut2 overproduction also caused a negative effect on these mutants . Surprisingly, the phenotypes of cut9 and cut23 in the presence of pCUT1 or pCUT2 were not the mitotic arrest, and they were strikingly different depending on pCUT1 or pCUT2 . CONCLUSIONS: This study shows the functional link between separase/Cut1 and APC/cyclosome in a separase activity-dependent manner . The negative effect of active separase overproduction on APC/cyclosome mutations is possibly due to the direct inhibition of APC/cyclosome . In addition, the manner of the inhibition by high copy securin and separase plasmids were quite different each other and did not result in the mitotic block.

Curr Genet, 1990 Mar, 17(3), 191 - 4
Thiamin regulates agglutination and zygote formation in Schizosaccharomyces pombe; Schweingruber ME et al.; Nutritional conditions regulate mating of the fission yeast S . pombe . To investigate how nutritional signals are monitored by the cell and translated into appropriate mating behaviour, effects of unique and specific growth factors would be desirable . We show that thiamin can inhibit sexual agglutination and zygote formation in S . pombe . A concentration of 50 nM thiamin in the culture medium is required for full growth of a thiamin auxotrophic strain . At this concentration thiamin starts to inhibit mating of wild-type cells of opposite heterothallic mating type and at a 1 microM concentration zygote formation is inhibited by more than 95% . Growth conditions modulate the inhibitory effect of thiamin . Thiamin acts only for a restricted period of time and seems to inhibit commitment to zygote formation rather than the cell aggregation and fusion process itself . Pyrithiamin, a thiamin antagonist, inhibits growth as well as mating.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Mar 25, 786(1-2), 81 - 91
Identification and characterisation of Schizosaccharomyces pombe cyclophilin 3, a cyclosporin A insensitive orthologue of human USA-CyP; Pemberton TJ et al.; We have identified nine cyclophilins encoded in the genome of the fission yeast Schizosaccharomyces pombe (Sp) . Cyclophilin 3 is an orthologue of hUSA-CyP, which is associated with Prp4/Prp3 in the {U4/U6.U5} snRNP complex and Prp18, both of which are components of the pre-mRNA splicing machinery . PPIase assays have shown SpCyp3 and hUSA-CyP to have comparable activity and substrate specificity, but SpCyp3 has a reduced sensitivity to CsA correlating with a difference in the catalytic site . Prp3, Prp4 and Prp18 proteins exist in S . pombe and nuclear localisation of SpCyp3 has been shown, indicating conservation of function between hUSA-CyP and SpCyp3.

J Biol Chem, 2003 Jun 6, 278(23), 20540 - 6 Epub 2003 Mar 20.
The UDP-glucose:glycoprotein glucosyltransferase is organized in at least two tightly bound domains from yeast to mammals; Guerin M et al.; The endoplasmic reticulum UDP-Glc:glycoprotein glucosyltransferase (GT) exclusively glucosylates nonnative glycoprotein conformers . GT sequence analysis suggests that it is composed of at least two domains: the N-terminal domain, which composes 80% of the molecule, has no significant similarity to other known proteins and was proposed to be involved in the recognition of non-native conformers and the C-terminal or catalytic domain, which displays a similar size and significant similarity to members of glycosyltransferase family 8 . Here, we show that N- and C-terminal domains from Rattus norvegicus and Schizosaccharomyces pombe GTs remained tightly but not covalently bound upon a mild proteolytic treatment and could not be separated without loss of enzymatic activity . The notion of a two-domain protein was reinforced by the synthesis of an active enzyme upon transfection of S . pombe GT null mutants with two expression vectors, each of them encoding one of both domains . Transfection with the C-terminal domain-encoding vector alone yielded an inactive, rapidly degraded protein, thus indicating that the N-terminal domain is required for proper folding of the C-terminal catalytic portion . If, indeed, the N-terminal domain is, as proposed, also involved in glycoprotein conformation recognition, the tight association between N- and C-terminal domains may explain why only N-glycans in close proximity to protein structural perturbations are glucosylated by the enzyme . Although S . pombe and Drosophila melanogaster GT N-terminal domains display an extremely poor similarity (16.3%), chimeras containing either yeast N-terminal and fly C-terminal domains or the inverse construction were enzymatically and functionally active in vivo, thus indicating that the N-terminal domains of both GTs shared three-dimensional features.

J Biol Chem, 2003 Jul 4, 278(27), 25256 - 63 Epub 2003 Mar 19.
Control of cell polarity in fission yeast by association of Orb6p kinase with the highly conserved protein methyltransferase Skb1p; Wiley DJ et al.; In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control . Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis . To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p . Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response . We found that Skb1p associates with Orb6p in S . pombe cells and that the two proteins interact directly in vitro . Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S . pombe cells . Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips . Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress . Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression . Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.

Nat Cell Biol, 2003 Apr, 5(4), 341 - 5
Human CENP-I specifies localization of CENP-F, MAD1 and MAD2 to kinetochores and is essential for mitosis; Liu ST et al.; The kinetochore, a macromolecular complex located at the centromere of chromosomes, provides essential functions for accurate chromosome segregation . Kinetochores contain checkpoint proteins that monitor attachments between the kinetochore and microtubules to ensure that cells do not exit mitosis in the presence of unaligned chromosomes . Here we report that human CENP-I, a constitutive protein of the kinetochore that shares limited similarity with Mis6 of Schizosaccharomyces pombe, is required for the localization of CENP-F and the checkpoint proteins MAD1 and MAD2 to kinetochores . Depletion of CENP-I from kinetochores causes the cell cycle to delay in G2 . Although monopolar chromosomes in CENP-I-depleted cells fail to establish bipolar connections, the cells are unable to arrest in mitosis . These cells are transiently delayed in mitosis in a MAD2-dependent manner, even though their kinetochores are depleted of MAD2 . The delay is extended considerably when the number of unattached kinetochores is increased . This suggests that no single unattached kinetochore in CENP-I-depleted cells can arrest mitosis . The collective output from many unattached kinetochores is required to reach a threshold signal of 'wait for anaphase' to sustain a prolonged mitotic arrest.

J Biol Chem, 2003 May 23, 278(21), 18895 - 901 Epub 2003 Mar 13.
Physical and functional interaction of the yeast corepressor Tup1 with mRNA 5'-triphosphatase; Mukai Y et al.; The Tup1-Ssn6 complex is an important corepressor in Saccharomyces cerevisiae that inhibits transcription through interactions with the basal transcription machinery and by remodeling chromatin . In a two-hybrid screen for factors that interact with the Schizosaccharomyces pombe Tup1 ortholog, Tup11, we isolated the pct1+ cDNA . The pct1+ gene encodes an mRNA 5'-triphosphatase, which catalyzes the first step of mRNA capping reactions . Pct1 did not interact with the S . pombe Ssn6 ortholog . In vitro glutathione S-transferase pull-down experiments revealed that Pct1 binds to the WD repeat regions of Tup11 and the functionally redundant Tup12 protein . Similarly, the S . cerevisiae Tup1 protein associates with the mRNA 5'-triphosphatase encoded by the CET1 gene . The highly conserved C-terminal domain of Cet1 interacts with Tup1 in vitro, and Tup1-Ssn6 complexes co-purify with the Cet1 protein, indicating that in vivo interactions also occur between these proteins . Over-expression of CET1 compromised repression of an MFA2-lacZ reporter gene that is subject to Tup1-Ssn6 repression . These genetic and biochemical interactions between Tup1-Ssn6 and Cet1 indicate that the capping enzyme associated with RNA polymerase II is a target of the corepressor complex.

FEBS Lett, 2003 Mar 13, 538(1-3), 192 - 6
The dihydroceramide desaturase is not essential for cell viability in Schizosaccharomyces pombe; Garton S et al.; Recent studies have identified a new family of desaturase-like polypeptide sequences in many higher eukaryotes . Functional characterisation of one member of this family, from Schizosaccharomyces pombe, revealed the enzyme to be a sphingolipid desaturase . This S . pombe gene designated SDCB3b8.07c was identified as the dihydroceramide Delta(4)-desaturase, responsible for the synthesis of sphingosine . Homologous recombination was used to disrupt the endogenous S . pombe dihydroceramide Delta(4)-desaturase . Surprisingly, this had no effect on cell viability, indicating that sphingosine may not be crucial for normal S . pombe functions . This observation has implications for our understanding of the role of sphingosine and its phosphorylated metabolite sphingosine-1-phosphate in lower eukaryotes.

Mol Biol Cell, 2003 Mar, 14(3), 1109 - 24
The fission yeast spo14+ gene encoding a functional homologue of budding yeast Sec12 is required for the development of forespore membranes; Nakamura-Kubo M et al.; The Schizosaccharomyces pombe spo14-B221 mutant was originally isolated as a sporulation-deficient mutant . However, the spo14(+) gene is essential for cell viability and growth . spo14(+) is identical to the previously characterized stl1(+) gene encoding a putative homologue of Saccharomyces cerevisiae Sec12, which is essential for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus . In the spo14 mutant cells, ER-like membranes were accumulated beneath the plasma membrane and the ER/Golgi shuttling protein Rer1 remained in the ER . Sec12 is a guanine nucleotide exchange factor for the Sar1 GTPase . Overproduction of psr1(+) coding for an S . pombe Sar1 homologue suppressed both the sporulation defect of spo14-B221 and cold-sensitive growth of newly isolated spo14-6 and spo14-7 mutants . These results indicate that Spo14 is involved in early steps of the protein secretory pathway . The spo14-B221 allele carries a single nucleotide change in the branch point consensus of the fifth intron, which reduces the abundance of the spo14 mRNA . During meiosis II, the forespore membrane was initiated near spindle pole bodies; however, subsequent extension of the membrane was arrested before its closure into a sac . We conclude that Spo14 is responsible for the assembly of the forespore membrane by supplying membrane vesicles.

EMBO J, 2003 Mar 17, 22(6), 1431 - 41
Protein kinase Cdelta is responsible for constitutive and DNA damage-induced phosphorylation of Rad9; Yoshida K et al.; The mammalian homolog of the Schizosaccharomyces pombe Rad9 is involved in checkpoint signaling and the induction of apoptosis . While the mechanisms responsible for the regulation of human Rad9 (hRad9) are not known, hRad9 is subject to hyperphosphorylation in the response of cells to DNA damage . The present results demonstrate that protein kinase Cdelta (PKCdelta) associates with Rad9 and that DNA damage induces this interaction . PKCdelta phosphorylates hRad9 in vitro and in cells exposed to genotoxic agents . The functional significance of the interaction between hRad9 and PKCdelta is supported by the finding that activation of PKCdelta is necessary for formation of the Rad9-Hus1-Rad1 complex . We also show that PKCdelta is required for binding of hRad9 to Bcl-2 . In concert with these results, inhibition of PKCdelta attenuates Rad9-mediated apoptosis . These findings demonstrate that PKCdelta is responsible for the regulation of Rad9 in the Hus1-Rad1 complex and in the apoptotic response to DNA damage.

EMBO J, 2003 Mar 17, 22(6), 1419 - 30
Pathway utilization in response to a site-specific DNA double-strand break in fission yeast; Prudden J et al.; We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe . Tech nology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch(16), using the Saccharomyces cerevisiae HO-endonuclease and its target site, MATa . DSB repair in this context was predominantly through interchromosomal gene conversion . We found that the homologous recombination (HR) genes rhp51(+), rad22A(+), rad32(+) and the nucleotide excision repair gene rad16(+) were required for efficient interchromosomal gene conversion . Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3(+) . Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55Delta background involving ku70(+) and rhp51(+) . Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70Delta and lig4Delta non-homologous end joining (NHEJ) mutant backgrounds compared with wild type . Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion . These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways.

Glycobiology, 2003 Feb, 13(2), 87 - 95 Epub 2002 Nov 26.
In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir; Abe H et al.; A glycosyltransferase was fused to the yeast cell wall protein Pir, which forms the Pir1-4 protein family and is incorporated into the cell wall by an unknown linkage to be displayed at the yeast cell surface . We first expressed the PIR1-HA-gma12+ fusion, in which gma12+ encodes alpha-1,2-galactosyltransferase from the fission yeast Schizosaccharomyces pombe under the Saccharomyces cerevisiae GAPDH promoter . The alpha-1,2-galactosyltransferase activity was detected at the surface of the intact cells that produce Pir1-HA-Gma12 fusion . To further demonstrate sequential oligosaccharide synthesis, two plasmids containing PIR1-HA-KRE2 and PIR2-FLAG-MNN1 fusion genes were constructed in which KRE2 and MNN1 encode alpha-1,2-mannosyltransferase and alpha-1,3-mannosyltransferase from S . cerevisiae, respectively . The intact yeast cells transformed with these two plasmids added mannoses initially with an alpha-1,2 linkage and subsequently with an alpha-1,3 linkage to the alpha-1,2-mannobiose acceptor in the presence of a GDP-mannose donor, demonstrating that Pir1 and Pir2 can be used as anchors to simultaneously immobilize several glycosyltransferases at the yeast cell surface . Based on the high acceptor specificity of glycosyltransferases, we propose a simple in vitro method for oligosaccharide synthesis using the yeast intact cell as a biocatalyst.

Appl Environ Microbiol, 2003 Mar, 69(3), 1861 - 5
Heterologous expression of the Saccharomyces cerevisiae PGU1 gene in Schizosaccharomyces pombe yields an enzyme with more desirable properties for the food industry; Sieiro C et al.; The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe . The optimum pH and temperature for the recombinant enzyme were 5 and 40 degrees C, respectively, these being around 0.5 U higher and 5 degrees C lower than those shown by the native enzyme . The K(m) value was about fourfold higher than that of the S . cerevisiae enzyme . The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.

Genome Res, 2003 Mar, 13(3), 399 - 406
Schizosaccharomyces pombe essential genes: a pilot study; Decottignies A et al.; After completion of the Schizosaccharomyces pombe genome sequence, we have carried out a pilot gene deletion project to assess the feasibility of a genome-wide deletion project and to estimate the percentage of essential genes . Using a PCR-based gene deletion procedure, we investigated 100 genes within a 253-kb region of chromosome II . Eight of nine genes located within a region of 18 kb could not be deleted, suggesting that systematic deletion of all fission yeast genes may be difficult to achieve using this PCR approach . The percentage of essential genes was found to be 17.5% . Further deletion of selected S . pombe genes revealed that whether a gene is essential or not is correlated with the timing of its appearance on the tree of life and its conservation within all branches of the tree . None of the investigated ancient genes in fission yeast that have been lost in the Saccharomyces cerevisiae lineage are essential . In agreement with S . cerevisiae and Caenorhabditis elegans genome analyses, our data suggest that natural selection has preferentially kept the genes required for vital functions . We propose that many of the essential eukaryotic genes appeared with the first eukaryotic cell and have remained conserved in all species.

Biochem J, 2003 Jun 1, 372(Pt 2), 651 - 60
Mutagenesis of the HMGB (high-mobility group B) protein Cmb1 (cytosine-mismatch binding 1) of Schizosaccharomyces pombe: effects on recognition of DNA mismatches and damage; Kunz C et al.; Cmb1 (cytosine-mismatch binding 1) is a high-mobility group (HMG) protein of Schizosaccharomyces pombe, which consists of 223 amino acids and has a single HMG domain at the C-terminal end . We have created several mutant and deletion forms of the Cmb1 protein and studied the effects on general DNA binding and specific binding to DNA mismatches and damaged DNA . Cmb1Delta41 (i.e . Cmb1 from which the 41 N-terminal amino acids have been deleted) bound specifically to cytosine-containing mismatches, to the cisplatin-induced intrastrand cross-links cis -GG and cis -AG and to an O (6)-methylguanine lesion . DNA binding was not affected when the 45 N-terminal amino acids were deleted, but was abolished in the absence of the 50 N-terminal amino acids, and was reduced when Cmb1 was truncated by between five and eleven C-terminal amino acids . Cmb1, both with and without the C-terminal truncations, retained its DNA binding affinity after heating at 95 degrees C . The cmb1 gene was induced when S . pombe cells were treated with cisplatin . Mitotic mutation rates were increased in a S . pombe cmb1 null mutant and in a cmb1-(1-212) mutant, which encodes a Cmb1 protein lacking the 11 C-terminal amino acids . We conclude that mutation avoidance by Cmb1 is distinct from Msh2-dependent mismatch repair, but related to nucleotide excision repair.

Mol Biochem Parasitol, 2003 Mar, 127(1), 37 - 46
An early ancestor in the evolution of splicing: a Trypanosoma cruzi serine-arginine-rich protein (TcSR) is functional in cis-splicing; Portal D et al.; A novel serine-arginine-rich protein designated TcSR was identified in Trypanosoma cruzi . The deduced amino acid sequence reveals that TcSR is a member of the SR protein family of splicing factors that contains two RNA-binding domains at the N-terminal side and several serine-arginine repeats at the COOH-terminus . Over expression of either TcSR or the human SR-protein associated splicing factor/splicing factor 2 (ASF/SF2) in wild-type Schizosaccharomyces pombe, provoked an elongated phenotype similar to that of fission yeast over expressing the SR-containing splicing factor Prp2, a U2AF(65) orthologue . When a double mutant strain lacking two SR protein-specific protein kinases was used, expression of TcSR or human SR ASF/SF2 splicing factor reverted the mutant to a wild-type phenotype . Transient expression of TcSR in HeLa cells stimulated the inclusion of the EDI exon of human fibronectin in an in vivo functional alternative cis-splicing assay . Inclusion was dependent on a splicing enhancer sequence present in the EDI exon . In addition, TcSR and peptides carrying TcSR-RS domain sequences were phosphorylated by a human SR protein kinase . These results indicate that TcSR is a member of the SR splicing network and that some components common to the trans- and cis-splicing machineries evolved from the early origins of the eukaryotic lineage.

Mol Biochem Parasitol, 2003 Mar, 127(1), 9 - 21
Trypanosoma cruzi TcSRPK, the first protozoan member of the SRPK family, is biochemically and functionally conserved with metazoan SR protein-specific kinases; Portal D et al.; A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism . The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family . In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs . TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark . Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs . In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene . When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2 . This inhibition was similar to that observed with human SRPK1 . This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism . The implications in the origins and control of splicing are discussed.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2334 - 9 Epub 2003 Feb 25.
The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1 kinase; Lee JK et al.; Previous studies in Saccharomyces cerevisiae have defined an essential role for the Dbf4-Cdc7 kinase complex in the initiation of DNA replication presumably by phosphorylation of target proteins, such as the minichromosome maintenance (Mcm) complex . We have examined the phosphorylation of the Mcm complex by the Dfp1-Hsk1 kinase, the Schizosaccharomyces pombe homologue of Dbf4-Cdc7 . In vitro, the purified Dfp1-Hsk1 kinase efficiently phosphorylated Mcm2p . In contrast, Mcm2p, present in the six-subunit Mcm complex, was a poor substrate of this kinase and required Cdc23p (homologue of Mcm10p) for efficient phosphorylation . In the presence of Cdc23p, Dfp1-Hsk1 phosphorylated the Mcm2p and Mcm4p subunits of the Mcm complex . Cdc23p interacted with both the Mcm complex and Dfp1-Hsk1 by selectively binding to the Mcm467 subunits and Dfp1p, respectively . The N terminus of Cdc23p was found to interact directly with Dfp1-Hsk1 and was essential for phosphorylation of the Mcm complex . Truncated derivatives of Cdc23p that complemented the temperature-sensitive phenotype of cdc23 mutant cells also stimulated the phosphorylation of Mcm complex, implying that this activity might be a critical role of Cdc23p in vivo . These results suggest that Cdc23p participates in the activation of prereplicative complex by recruiting the Dfp1-Hsk1 kinase and stimulating the phosphorylation of the Mcm complex.

Trends Plant Sci, 2003 Feb, 8(2), 53 - 5
DNA-RNA-protein gang together in silence; Stokes T; Two recent reports demonstrate interdependence between DNA and histone methylation in Arabidopsis . ddm1 (decrease in DNA methylation 1) mutants switch histone methylation from a form associated with inactive chromatin to a form connected to actively transcribed genomic regions . The loss of DNA methylation and shift in histone methylation cause transcriptional derepression of heterochromatic regions . In a related report, small RNAs in Schizosaccharomyces pombe mark histone methylation to form heterochromatin, suggesting that methylation systems work alongside RNA metabolism.

Biochem J, 2003 May 15, 372(Pt 1), 97 - 104
Characterization of SUMO-conjugating enzyme mutants in Schizosaccharomyces pombe identifies a dominant-negative allele that severely reduces SUMO conjugation; Ho JC et al.; The phenotypes of mutants defective in the Schizosaccharomyces pombe SUMO (small, ubiquitin-like modifier)-conjugating enzyme Hus5 (the homologue of Ubc9) show that it is required for recovery from S-phase arrest . Unlike the case with ubiquitination, where ligases are required, SUMO-conjugating enzymes are sufficient for substrate recognition and conjugation of SUMO on to target proteins, at least in vitro . Thus SUMO-conjugating enzymes are likely to be important regulators of sumoylation . Here, we report on the characterization of two hus5 alleles . Although hus5.17 and hus5.62 respond in a similar manner to UV and ionizing radiation, they have different responses to the DNA-synthesis inhibitor, hydroxyurea . In addition, SUMO (Pmt3) is mislocalized in hus5.17 cells, but not in hus5.62 mutant cells . The mutations in hus5.62 and hus5.17 map to Ala(129) and the 5' splice site of intron 2 respectively . We have characterized the Hus5.62 protein and shown, in vitro, that it still interacts with SUMO and at least one protein, Rad22, which is a SUMO-modified target . The Hus5.62 protein is also capable of forming a thioester link with SUMO, but it does not function in sumoylation assays, either in the modification of Rad22 or in SUMO chain formation . When overexpressed in wild-type S . pombe cells, the Hus5.62 protein has a dominant-negative effect on sumoylation.

DNA Res, 2002 Dec 31, 9(6), 209 - 15
Abundant poly(A)-bearing RNAs that lack open reading frames in Schizosaccharomyces pombe; Watanabe T et al.; We report here that 6.9% (68/987) of randomly selected cDNA clones from an S . pombe cDNA library lack apparently long open reading frames which we denote prl . One of them, prl1, was examined further because multiple bands were observed when it was used as a probe in northern blot analysis . These multiple bands appear to be derived from overlapping transcripts from both DNA strands, including non-coding RNAs and antisense RNAs in addition to mRNA . Such mechanisms may increase the transcriptional variation in S . pombe cells.

Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2663 - 72
His-to-Asp phosphorelay circuitry for regulation of sexual development in Schizosaccharomyces pombe; Nakamichi N et al.; The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), and two response regulators (Mcs4 and Prr1) . The results of recent extensive studies on the S . pombe His-to-Asp phosphorelay circuitry suggested that it is involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals . The functions of these histidine kinases have not yet been fully characterized . Here we characterize a homothallic (h90) mutant lacking the genes for all the histidine kinases, with special reference to sexual development . Homothallic phk1/2/3delta cells underwent mating precociously in a nitrogen-deficient medium . Surprisingly, the mutant cells underwent mating even in a nitrogen-sufficient medium, under which conditions wild-type cells did so rarely if at all . Under anaerobic (or microaerobic) growth conditions, wild-type cells did not undergo sexual development even in a nitrogen-deficient medium, but the homothallic phk1/2/3delta cells mated efficiently . Oxidative reagents such as H2O2 induced sexual development in wild-type cells grown anaerobically . On the basis of these results, we propose the novel view that the S . pombe His-to-Asp phosphorelay, initiated by the Phk histidine kinases, is crucial for regulation of sexual development . This Phk-mediated signaling pathway is linked to the well-documented canonical pathway for induction of the sexual development, in that both converge at the initiation of meiosis through activation of ste11+, mam2+, and mei2+ transcription.

J Mol Biol, 2003 Mar 7, 326(5), 1463 - 73
Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold; Bauer S et al.; The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase . Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase . The enzyme represents a novel family of phosphoryl transferring enzymes . It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape . The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN . Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form . The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix . In contrast, no significant changes are observed upon binding of riboflavin . The ribityl side-chain might be covered by a rather flexible flap II . The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45 . This may explain the preference for zinc observed in vitro.

J Mol Biol, 2003 Feb 28, 326(4), 1081 - 94
The fission yeast spSet1p is a histone H3-K4 methyltransferase that functions in telomere maintenance and DNA repair in an ATM kinase Rad3-dependent pathway; Kanoh J et al.; We have characterized spSet1p, the Schizosaccharomyces pombe ortholog of the budding yeast histone H3 methyltransferase Set1p . SpSet1p catalyzes methylation of H3 at K4, in vivo and in vitro . Deleting spset1 partially affects telomeric and centromeric silencing . Strikingly, lack of spSet1p causes elongation of telomeres in wild-type cells and in most DNA damage checkpoint rad mutant cells, but not in cells lacking the ATM kinase Rad3 or its associated protein Rad26 . Interestingly, spset1 deletion specifically causes a reduction in sensitivity to ultraviolet radiation of the PCNA-like checkpoint mutants hus1 and rad1, but not of cells devoid of Rad3 . This partial suppression was not due to restoration of checkpoint function or to transcriptional induction of DNA repair genes . Moreover, spset1 allows recovery specifically of the crb2 checkpoint mutant upon treatment with the replication inhibitor hydroxyurea but not upon UV irradiation . Nevertheless, the pathway induced in spset1 cells cannot substitute for the Mus81/Rqh1 DNA damage tolerance pathway . Our results suggest that SpSet1p and the ATM kinase Rad3 function in a common genetic pathway linking chromatin to telomere length regulation and DNA repair.

Curr Genet, 2003 Feb, 42(5), 252 - 9 Epub 2002 Dec 14.
Heterologous expression and characterization of Schizosaccharomyces pombe vacuolar carboxypeptidase Y in Saccharomyces cerevisiae; Takegawa K et al.; To investigate the intracellular transport mechanism of the vacuolar carboxypeptidase of Schizosaccharomyces pombe (SpCPY), SpCPY was expressed in Saccharomyces cerevisiae and its biosynthesis and sorting were examined . When Sac . cerevisiae prc1Delta, devoid of intrinsic (Sc) CPY activity, was transformed with a plasmid carrying the Sch . pombe cpy1(+) gene, CPY activity was restored . Pulse-chase experiments revealed that SpCPY is initially synthesized in a pro-precursor form and then converted to a heterodimer, the mature form, in Sac . cerevisiae cells . SpCPY was not processed into intermediate or mature forms in pep4 mutant cells, indicating that SpCPY was proteolytically cleaved in a PEP4-dependent manner in Sac . cerevisiae . Several vps mutants, which are defective in vacuolar protein-sorting, exhibited a defect in the maturation of SpCPY . Moreover, the maturation of SpCPY was severely inhibited in a vps10 strain, although the pro- segment of SpCPY does not contain a QRPL-like sequence, which is the putative targeting signal of ScCPY . When SpCPY was expressed in a wild-type strain, more than 90% of ScCPY was normally sorted to the vacuole, indicating that SpCPY does not compete with ScCPY for vacuolar sorting . In contrast, expression of SpCPY resulted in a missorting of a ScCPY-invertase fusion protein to the cell surface . These results suggested that there are two different binding sites for SpCPY and ScCPY on Vps10p and that the binding of SpCPY to Vps10p interferes with the binding of a ScCPY-invertase fusion protein.

Curr Genet, 2003 Feb, 42(5), 241 - 51 Epub 2002 Dec 13.
Pre-mRNA splicing in Schizosaccharomyces pombe: regulatory role of a kinase conserved from fission yeast to mammals; Kuhn AN et al.; Most primary messenger RNA transcripts (pre-mRNAs) in eukaryotes contain intervening sequences that must be precisely removed to generate a functional mRNA . The excision of the intervening sequences, the introns, from a pre-mRNA and the concomitant joining of the flanking sequences, the exons, is called pre-mRNA splicing . Pre-mRNA splicing takes place in large ribonucleoprotein machinery, the spliceosome . Although the function and components of this machinery appear to be highly conserved between organisms, many distinct differences between budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe, have been found, emphasizing their evolutionary distance . Most interestingly, fission yeast appears to reflect the more conservative evolutionary development regarding pre-mRNA splicing . Many spliceosomal components, including the five small nuclear RNAs, which most likely form the catalytic core of the spliceosome, show a higher degree of similarity with the components of the splicing machinery found in mammals . In addition, several regulatory components of the spliceosome detected in mammals are absent in Sac . cerevisiae, but present in Sch . pombe . Here, we review recent progress made in our understanding of the control of pre-mRNA splicing in Sch . pombe . The focus is on Prp4p kinase, first discovered in fission yeast and also present in mammals, but absent in Sac . cerevisiae . Results from both mammals and Sch . pombe suggest that Prp4p plays a key role in regulating pre-mRNA splicing and in connecting this process with the cell cycle.

Mol Genet Genomics, 2003 Feb, 268(5), 684 - 91 Epub 2003 Jan 15.
Isolation and characterisation of a calnexin homologue, clxA, from Aspergillus niger; Wang H et al.; We describe the isolation of a gene (clxA) encoding calnexin from laboratory and industrial strains of Aspergillus niger . Calnexin is a chaperone, which specifically recognises monoglucosylated glycoproteins in the endoplasmic reticulum, and is thus an essential component of the process that assesses the folded state of nascent secreted glycoproteins . Manipulation of chaperones has previously been adopted in attempts to overcome some of the problems associated with the secretion of heterologous proteins from filamentous fungi . The A . niger clxA gene encodes a 562-residue protein with strong homology to the calnexin of Schizosaccharomyces pombe . The clxAgene product complements a S . pombe cnx1 mutant . Motifs associated with genes controlled via the Unfolded Protein Response (UPR) were identified by sequence homology in the promoter of clxA . Steady-state levels of clxA mRNA were elevated in a strain expressing bovine prochymosin fused to the catalytic domain of glucoamylase . The ORF is punctuated by four introns, and contains two sets of four repeated peptide motifs that are characteristic of the calnexin family, together with a putative membrane-spanning domain . Deletion studies indicate that clxA is not an essential gene in A . niger.

Mol Genet Genomics, 2003 Feb, 268(5), 585 - 97 Epub 2003 Jan 15.
Mkp1 and Mkp2, two MAPKAP-kinase homologues in Schizosaccharomyces pombe, interact with the MAP kinase Sty1; Asp E et al.; Mkp1 ( MAPKAP kinase Schizosaccharomyces pombe 1) and Mkp2 are two members from fission yeast of the sub-class of putative MAPK-activated protein kinases in yeasts, the other known members being Rck1 and Rck2 from Saccharomyces cerevisiae . The Mkp1 protein is readily co-immunoprecipitated with Sty1 from S . pombe extracts; Mkp2 shows a weaker interaction with Sty1 . In mkp1 mutants, conjugation and meiosis proceed more readily and rapidly than in wild-type cells, in analogy to what was previously found for S . cerevisiae rck1 mutants . Conversely, overexpression of mkp1(+) delays meiosis . Mkp1 is phosphorylated in vivo in a sty1(+)-dependent manner; this modification is removed when cells are starved for nitrogen, a condition that is conducive to entry into stationary phase and meiosis . Overexpression of mkp1(+), like a sty1 mutation, also causes vegetative cells to elongate . The level of Mkp1 phosphorylation drops as cells enter mitosis . We have localised Mkp1 to the cytoplasm, excluded from the nucleus, in vegetative cells . The Mkp1 protein accumulates in zygotic asci and is concentrated within spores . The mkp2(+) gene has no noticeable impact on meiosis . Mkp2 is excluded from the nucleus in vegetative cells, and is concentrated at the septa of dividing cells . Mkp2 does not accumulate in meiotic cells.

Eukaryot Cell, 2003 Feb, 2(1), 159 - 69
Bgs3p, a putative 1,3-beta-glucan synthase subunit, is required for cell wall assembly in Schizosaccharomyces pombe; Martin V et al.; beta-Glucans are the main components of the fungal cell wall . Fission yeast possesses a family of beta-glucan synthase-related genes . We describe here the cloning and characterization of bgs3(+), a new member of this family . bgs3(+) was cloned as a suppressor of a mutant hypersensitive to Echinocandin and Calcofluor White, drugs that interfere with cell wall biosynthesis . Disruption of the gene is lethal, and a decrease in Bgs3p levels leads to rounded cells with thicker walls, slightly reduces the amount of the beta-glucan, and raises the amount of alpha-glucan polymer . These cells finally died . bgs3(+) is expressed in vegetative cells grown in different conditions and during mating and germination and is not enhanced by stress situations . Consistent with the observed expression pattern, Bgs3-green fluorescence protein (GFP-Bgs3p) was found at the growing tips during interphase and at the septum prior to cytokinesis, always localized to growth areas . We also found GFP-Bgs3p in mating projections, during the early stages of zygote formation, and at the growing pole during ascospore germination . We conclude that Bgs3p localization is restricted to growth areas and that Bgs3p is a glucan synthase homologue required for cell wall biosynthesis and cell elongation in the fission yeast life cycle.

Genes Cells, 2003 Feb, 8(2), 163 - 78
Overproduction of a conserved domain of fission yeast and mammalian translation initiation factor eIF4G causes aberrant cell morphology and results in disruption of the localization of F-actin and the organization of microtubules; Hashemzadeh-Bonehi L et al.; BACKGROUND: The recruitment of mRNA for translation involves the assembly at the 5'cap of a complex of three initiation factors: the cap binding protein eIF4E, the ATP-dependent RNA helicase eIF4A and the scaffold protein eIF4G . eIF4G mediates the binding of this mRNA-protein complex to the 43S ribosomal preinitiation complex . There is growing recognition that the components of the translational apparatus interact functionally with cytoskeletal components . Here we report specific effects of the over-expression of human and fission yeast eIF4G domains on cell morphology in Schizosaccharomyces pombe . RESULTS: A single gene encoding fission yeast eIF4G was identified and demonstrated to be essential . We have over-expressed fragments corresponding to the conserved functional domains of eIF4G . At expression levels that did not disrupt rates of overall translation or protein accumulation, a fragment of S . pombe eIF4G, 4G-NOB, corresponding to the minimal region of human eIF4G required to support cap-independent mRNA recruitment, was found to impair cell proliferation in fission yeast . This resulted from defects in cytokinesis, and was associated with the disruption of both microtubules and actin microfilaments . The over-expressed fragment was itself localized to the cell ends, the nuclear periphery and the septum . CONCLUSIONS: This is the first demonstration of a link between a translation initiation factor and mechanisms controlling cell morphology . The data suggest a direct or indirect interaction between the functional domains of eIF4G and cellular structures involved in cytokinesis.

J Biol Chem, 2003 Apr 18, 278(16), 14565 - 77 Epub 2003 Feb 10.
The Schizosaccharomyces pombe Cuf1 is composed of functional modules from two distinct classes of copper metalloregulatory transcription factors; Beaudoin J et al.; In fission yeast, the genes encoding proteins that are components of the copper transporter family are controlled at the transcriptional level by the Cuf1 transcription factor . Under low copper availability, Cuf1 induces expression of the copper transporter genes . In contrast, sufficient levels of copper inactivate Cuf1 and expression of its target genes . Our study reveals that Cuf1 harbors a putative copper-binding motif, Cys-X-Cys-X(3)-Cys-X-Cys-X(2)-Cys-X(2)-His, within its carboxyl-terminal region to sense changing environmental copper levels . Binding studies reveal that the amino-terminal 174-residue segment of Cuf1 expressed as a fusion protein in Escherichia coli specifically interacts with the cis-acting copper transporter promoter element CuSE (copper-signaling element) . Within this region, the first 61 amino acids of Cuf1 exhibit more overall homology to the Saccharomyces cerevisiae Ace1 copper-detoxifying factor (from residues 1 to 63) than to Mac1, its functional ortholog . Consistently, we demonstrate that a chimeric Cuf1 protein bearing the amino-terminal 63-residue segment of Ace1 complements cuf1 Delta null phenotypes . Furthermore, we show that Schizosaccharomyces pombe cuf1Delta mutant cells expressing the full-length S . cerevisiae Ace1 protein are hypersensitive to copper ions, with a concomitant up-regulation of CuSE-mediated gene expression in fission yeast . Taken together, these studies reveal that S . cerevisiae Ace1 1-63 is functionally exchangeable with S . pombe Cuf1 1-61, and the nature of the amino acids located downstream of this amino-terminal conserved region may be crucial in dictating the type of regulatory response required to establish and maintain copper homeostasis.

J Cell Sci, 2003 Mar 1, 116(Pt 5), 867 - 74
Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe; Wachtler V et al.; Sterol-rich membrane domains exist in unicellular and multicellular eukaryotes . They are thought to provide a structural framework for interactions among a subset of proteins by selectively incorporating some proteins while excluding others . Although most studies have focused on the biophysical and biochemical properties of sterol-rich membrane domains and incorporated proteins, relatively little is known about their intracellular distribution . Using a cytological approach we show here that in the fission yeast Schizosaccharomyces pombe, sterols are enriched in the plasma membrane at the growing cell tips and at the site of cytokinesis . The distribution of sterols is regulated in a cell-cycle-dependent manner and requires a functional secretory pathway . By manipulating the integrity of sterol-rich membrane domains using sterol sequestering agents and genetic means, we show that these domains are important for multiple processes regulating cytokinesis . In these cells, defects in proper maintenance of the actomyosin ring and/or its attachment to the overlying plasma membrane were observed . Furthermore, the stability of a plasma membrane protein that colocalises with sterol-rich membrane domains was compromised . Taken together, our studies establish S . pombe as a genetically tractable model organism in which to study the role(s) of sterol-rich membrane domains in cell polarity and cytokinesis.

Oncogene, 2003 Feb 6, 22(5), 637 - 48
hob1+, the fission yeast homolog of Bin1, is dispensable for endocytosis or actin organization, but required for the response to starvation or genotoxic stress; Routhier EL et al.; BAR (Bin/Amphiphysin/Rvs) adapter proteins have been suggested to regulate endocytosis, actin organization, apoptosis, and transcription, but their precise roles are obscure . There are at least five mammalian genes that encode BAR adapter proteins, including the evolutionarily conserved and ubiquitously expressed Bin1/Amphiphysin-II and Bin3 genes . Bin1 holds special interest as certain splice isoforms localize to the nucleus, interact with the c-Abl and c-Myc oncoproteins, and display tumor suppressor properties . To obtain functional insights, we embarked upon a genetic analysis of the two BAR adapter proteins expressed in the fission yeast Schizosaccharomyces pombe . In a previous work, a role in actin organization and cytokinesis was identified for the Bin3 homolog hob3+ . In this study, a role in stress signaling was defined for the Bin1 homolog, hob1+ . Notably, hob1+ was dispensable for endocytosis, actin organization, or osmotic sensitivity . Instead, mutation of hob1+ led to slight cell elongation and faulty cell cycle arrest upon nutrient starvation . These defects were complemented by Bin1, but not by Amphiphysin-I, arguing that these genes have distinct functions despite their structural similarity . hob1 delta mutant cells were also hypersensitive to genotoxic stress . This was not related to a faulty checkpoint response, but mutation in the checkpoint gene rad3(+) further exacerbated the sensitivity of hob1 delta mutant cells . Interestingly, mutation of the cell cycle regulator wee1+ partially relieved the sensitivity defect, suggesting that hob1+ may influence the efficiency of DNA repair or checkpoint release after DNA damage . Genetic and biochemical evidence indicated that hob3+ is epistatic to hob1+ in the response to genotoxic stress . Our findings indicate that the Bin1 homolog hob1+ participates in DNA damage signaling and they suggest a novel role for BAR adapter proteins in stress response processes.

Genes Dev, 2003 Feb 1, 17(3), 330 - 5
Early-replicating heterochromatin; Kim SM et al.; Euchromatin, which has an open structure and is frequently transcribed, tends to replicate in early S phase . Heterochromatin, which is more condensed and rarely transcribed, usually replicates in late S phase . Here, we report significant deviation from this correlation in the fission yeast, Schizosaccharomyces pombe . We found that heterochromatic centromeres and silent mating-type cassettes replicate in early S phase . Only heterochromatic telomeres replicate in late S phase . Research in other laboratories has shown that occasionally other organisms also replicate some of their heterochromatin in early S phase . Thus, late replication is not an obligatory feature of heterochromatin.

Gene, 2003 Jan 30, 304, 133 - 41
A simple Cre-loxP method for chromosomal N-terminal tagging of essential and non-essential Schizosaccharomyces pombe genes; Werler PJ et al.; To facilitate the N-terminal tagging of essential genes at their genomic locus and under control of their own promoters we have developed a series of novel polymerase chain reaction templates . Initially, a 1.8 kb DNA fragment is integrated upstream of the ATG of the gene of interest . This fragment encodes the tag, a loxP site, a selectable marker, an exogenous nmt1 promoter and a second loxP site . In a single homologous integration event, the gene of interest is placed under control of the thiamine regulated nmt1 promoter, allowing identification of potential integrants on the basis of phenotype . Subsequently, this integrant strain is transformed with a plasmid expressing the Cre recombinase . This results in excision of the marker and nmt1 promoter and leaves sequences encoding an in-frame tag at the N-terminus of the gene of interest under the control of its native promoter . We have created TAP-cdc22, TAP-suc22 and TAP-rad50 strains using this N-tagging system, and developed a range of vectors for introducing TAP-, (His)10HA-, (His)6Myc- and EGFP.

Biochem Biophys Res Commun, 2003 Feb 14, 301(3), 641 - 5
Fission yeast synaptobrevin is involved in cytokinesis and cell elongation; Edamatsu M et al.; Synaptobrevin is a vesicle-associated membrane protein playing an essential role in regulated vesicle transport . In this study, we characterized Syb1, synaptobrevin of Schizosaccharomyces pombe . Syb1 was located on various sizes of vesicle-like structures in the cytoplasm and enriched in the medial region and cell ends . Transport of Syb1 to the medial region was mainly dependent on F-actin and Myo52/Myo4 . Syb1 is essential for cell viability and most of the syb1-null cells showed a round or short cylindrical form . These results suggest that Syb1 is involved in membrane trafficking of cytokinesis and cell elongation.

Exp Cell Res, 2003 Feb 1, 283(1), 101 - 15
The kic1 kinase of schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell-cell separation; Tang Z et al.; The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation . Some of the kinases in the family are shown to phosphorylate serine-arginine-rich splicing factors and to regulate pre-mRNA splicing . However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear . Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe, and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis . The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade . In addition, kic1(+) together with dsk1(+), which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth . The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals . Furthermore, since cell division and cell-cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1(+) revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes .

Nat Biotechnol, 2003 Mar, 21(3), 321 - 4 Epub 2003 Feb 03.
Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase; Belteki G et al.; Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast . Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites . Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange . The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision . To stabilize the trans event, functional mutant recognition sites had to be identified . None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange . Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells . This enzyme recombines between two heterotypic sites: attB and attP . The product sites of the recombination event (attL and attR) are not substrates for the integrase . Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.

J Biol Chem, 2003 Apr 11, 278(15), 12826 - 33 Epub 2003 Jan 31.
Ure2, a prion precursor with homology to glutathione S-transferase, protects Saccharomyces cerevisiae cells from heavy metal ion and oxidant toxicity; Rai R et al.; Ure2, the protein that negatively regulates GATA factor (Gln3, Gat1)-mediated transcription in Saccharomyces cerevisiae, possesses prion-like characteristics . Identification of metabolic and environmental factors that influence prion formation as well as any activities that prions or prion precursors may possess are important to understanding them and developing treatment strategies for the diseases in which they participate . Ure2 exhibits primary sequence and three-dimensional homologies to known glutathione S-transferases . However, multiple attempts over nearly 2 decades to demonstrate Ure2-mediated S-transferase activity have been unsuccessful, leading to the possibility that Ure2 may well not participate in glutathionation reactions . Here we show that Ure2 is required for detoxification of glutathione S-transferase substrates and cellular oxidants . ure2 Delta mutants are hypersensitive to cadmium and nickel ions and hydrogen peroxide . They are only slightly hypersensitive to diamide, which is nitrogen source-dependent, and minimally if at all hypersensitive to 1-chloro-2,4-dinitrobenzene, the most commonly used substrate for glutathione S-transferase enzyme assays . Therefore, Ure2 shares not only structural homology with various glutathione S-transferases, but ure2 mutations possess the same phenotypes as mutations in known S . cerevisiae and Schizosaccharomyces pombe glutathione S-transferase genes . These findings are consistent with Ure2 serving as a glutathione S-transferase in S . cerevisiae.

Yeast, 2003 Feb, 20(3), 221 - 31
Isolation and characterization of the plasma membrane biotin transporter from Schizosaccharomyces pombe; Stolz J; The fission yeast Schizosaccharomyces pombe is auxotrophic for biotin (vitamin H) and growth depends on biotin uptake over the plasma membrane . Here a biotin transport mutant of Saccharomyces cerevisiae is used to identify the vht1(+) gene encoding the Schizosaccharomyces pombe plasma membrane transport protein for biotin . SpVht1p belongs to the family of allantoate transporters and has only little sequence homology to the S . cerevisiae biotin transporter . Although having dissimilar primary structures, the biotin transporters in Sz . pombe and S . cerevisiae share similar biochemical properties and regulation . Like in S . cerevisiae, biotin uptake in Sz . pombe is a high-affinity process, is optimal at acidic pH values and inhibited by protonophores, indicating that SpVht1p acts as a proton-biotin symporter . Desthiobiotin, the metabolic precursor of biotin, is also imported by SpVht1p . Deletion of vht1(+) abolishes growth on low external concentrations of the vitamin, showing that vht1(+) encodes the only protein that mediates biotin uptake in Sz . pombe . Expression of vht1(+) is maximal at low external biotin concentrations, indicating that Sz . pombe can adjust the rate of biotin uptake to meet the requirement for the vitamin .

Yeast, 2003 Feb, 20(3), 193 - 206
Role of phosphatidylinositol 3-phosphate in formation of forespore membrane in Schizosaccharomyces pombe; Onishi M et al.; Phosphatidylinositol (PI) 3-kinase (encoded by the pik3(+) gene) in Schizosaccharomyces pombe has been identified as a homologue of VPS34p, a protein required for proper vesicular protein sorting . The clone defective in this protein carries enlarged vacuoles and exhibits sensitivity to high temperature or high ion concentration . The effect of disruption of pik3(+) on sporulation of Sz . pombe was examined . The diploid cells underwent G(1) arrest and meiosis . However, the spores formed by the deltapik3 cells were not viable . Electron-microscopic analysis revealed that the growth of the forespore membrane of deltapik3 cells was not correctly orientated, failing to engulf the nucleus or forming extremely small spores, as was confirmed by the use of Spo3p-GFP and GFP-Psy1p, which are markers of the forespore membrane . The coating materials found along the forespore membrane of the wild-type were greatly reduced in these cells . PI 3-P, the product of Pik3p, was detected on the forespore membrane, suggesting that PI 3-P-dependent vesicle transport may take place in formation of the forespore membrane . Misshaped forespore membrane, accumulation of vesicles, formation of small non-viable spores, and suppression by over expression of Psy1p were the phenotypes commonly seen in deltapik3 and deltaspo3 cells, suggesting a relationship between the functions of Pik3p and Spo3p in formation of the forespore membrane in Sz . pombe .

J Biol Chem, 2003 Apr 18, 278(16), 13627 - 32 Epub 2003 Jan 28.
Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1; Hausmann S et al.; Fcp1 is an essential protein serine phosphatase that dephosphorylates the C-terminal domain (CTD) of RNA polymerase II . By testing the effects of serial N- and C-terminal deletions of the 723-amino acid Schizosaccharomyces pombe Fcp1, we defined a minimal phosphatase domain spanning amino acids 156-580 . We employed site-directed mutagenesis (introducing 24 mutations at 14 conserved positions) to locate candidate catalytic residues . We found that alanine substitutions for Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298) abrogated the phosphatase activity with either p-nitrophenyl phosphate or CTD-PO(4) as substrates . Structure-activity relationships were determined by introducing conservative substitutions at each essential position . Our results, together with previous mutational studies, highlight a constellation of seven amino acids (Asp(170), Asp(172), Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298)) that are conserved in all Fcp1 orthologs and likely comprise the active site . Five of these residues (Asp(170), Asp(172), Lys(280), Asp(297), and Asp(298)) are conserved at the active site of T4 polynucleotide 3'-phosphatase, suggesting that Fcp1 and T4 phosphatase are structurally and mechanistically related members of the DXD phosphotransferase superfamily.

Mol Cell Biol, 2003 Feb, 23(4), 1441 - 52
Direct kinase-to-kinase signaling mediated by the FHA phosphoprotein recognition domain of the Dun1 DNA damage checkpoint kinase; Bashkirov VI et al.; The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae . It belongs to the Chk2 family of checkpoint kinases, which includes S . cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2 . Dun1 is required for DNA damage-induced transcription of certain target genes, transient G(2)/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55 . Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo . trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases . The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G(2)/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1 . As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea . We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

J Exp Bot, 2003 Feb, 54(383), 699 - 706
Genes encoding two essential DNA replication activation proteins, Cdc6 and Mcm3, exhibit very different patterns of expression in the tobacco BY-2 cell cycle; Dambrauskas G et al.; Very little is known about the expression patterns in plants of genes that encode proteins involved in the initiation of DNA replication . Partial cDNA sequences that encode Cdc6 and Mcm3 in tobacco have been isolated . The sequences were used as probes in northern blots which suggested that, in the cell cycle of synchronized tobacco BY-2 cells, expression of CDC6 is confined to late G(1) and S-phase whereas the expression of MCM3 is not confined to any particular cell cycle phase . These data were confirmed and extended by real-time PCR measurements of mRNA abundance through the cell cycle . CDC6 exhibits a very clear peak of expression in S-phase whereas MCM3, expressed at a much lower level than CDC6, is not cell-cycle-regulated . These patterns of cell cycle gene expression resemble those found in the fission yeast Schizosaccharomyces pombe rather than those in budding yeast or mammalian cells.

Cell, 2003 Jan 24, 112(2), 207 - 17
Schizosaccharomyces pombe Int6 and Ras homologs regulate cell division and mitotic fidelity via the proteasome; Yen HC et al.; Yin6 is a yeast homolog of Int6, which is implicated in tumorigenesis . We show that Yin6 binds to and regulates proteasome activity . Overexpression of Yin6 strengthens proteasome function while inactivation weakens and causes the accumulation of polyubiquitinated proteins including securin/Cut2 and cyclin/Cdc13 . Yin6 regulates the proteasome by preferentially interacting with Rpn5, a conserved proteasome subunit, and affecting its localization/assembly . We showed previously that Yin6 cooperates with Ras1 to mediate chromosome segregation; here, we demonstrate that Ras1 similarly regulates the proteasome via Rpn5 . In yeast, human Int6 binds Rpn5 and regulates its localization . We propose that human Int6, either alone or cooperatively with Ras, influences proteasome activities via Rpn5 . Inactivating Int6 can lead to accumulation of mitotic regulators affecting cell division and mitotic fidelity.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 592 - 7
{Mapping the interaction site of Rpb2 and Rpb3 subunit of fission yeast RNA polymerase II}; Qu Z et al.; To map the interacting site of subunit Rpb2 to subunit Rpb3 of RNA polymerase II in fission yeast Schizosaccharomyces pombe, the yeast two-hybrid system was employed in this paper to screen the interacting clones between Rpb2 and Rpb3.4 fragments of Rpb2 cDNA were cloned into the Ga14 BD vector pAS2 . The 4 clones were named as pAS2 Rpb2-1, 2-2, 2-3 and 2-4, respectively . The complete cDNA of Rpb3 was cloned into the Gal 4 AD vector pGADGH . The clone was named as pGADGH Rpb3 . The two-hybrid plasmids pGADGH Rpb3 and pAS2Rpb2-1, 2-2, 2-3 or 2-4 respectively were cotransformed into host cell yeast Y190 . The interaction positive cotransformants were identified by beta-gal activity assay . The beta-gal positive cotransformants were selected from pGADGH Rpb3 and pAS2Rpb2-4 two-hybrid system . DNA sequencing and alignment results showed that the interacting site of Rpb2 to Rpb3 located within the fragment from base 2701 to 2966 of Rpb2 cDNA, or within the C-termini polypeptide from amino acid 902 to 989 of Rpb2 protein.

Shi Yan Sheng Wu Xue Bao, 2000 Jun, 33(2), 141 - 9
{The different effects of CaM inhibitors of phenothiazines on the proliferation of Saccharomyces cerevisiae and Schizosaccharomyces pombe}; Lu L et al.; Low concentration of phenothiazines apparently stimulated the proliferation of S . pombe, the cell density incubated for 54 hours by preincubating the cells with 20 mumol/L trifluoperazine (TFP) in the EMM-Ca medium was two times more than the control . The stimulation was more obvious with lowing the concentration of calcium in the culture medium, TFP cooperated and complemented with calcium in stimulating the proliferation of S . pombe . When the original inoculated cell density was 5 x 10(6) cells/ml or during the logarithm period of growth curve, the proliferation of S . pombe wasn't affected by the low concentration of TFP . While when the concentration of TFP was increased to 100 mumol/L, the promotion effect of TFP on proliferation of S . pombe declined obviously and the proliferation of S . pombe was inhibited completely when TFP up to 200 mumol/L . The cell proliferation also could be inhibited by CaM antagonist W7 and W7-agarose, the inhibition was increased with increasing the concentration of antagonist . On the other hand, 20 mumol/L TFP used by the same method as above arrested the cell division cycle of Saccharomyces cerevisiae at a single G2 + M nuclei stage, the cells was penetrated easily by TFP, the fluorescence in cells was very obvious when TFP was 20 mumol/L, but it was difficult to penetrat by TFP in the cells of S . pombe and the Ca2+ influx of S . pombe could be induced rapidly by 20 mumol/L TFP . In this article, the cause of different effects of TFP on cell proliferation of S . pombe and S . cerevisiae was discussed, it was due to the difference of penetration of TFP and stimulation by calcium in the two kinds of cells.

Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 198 - 203
{Continuous ethanol fermentation using tapioca starch by immobilized yeast cell without carrier}; Ma Y et al.; A widespread interest has been noted in continuous power ethanol fermentation utilizing systems with immobilized yeast cell through self-flocculating . We studied continuous ethanol fermentation by a naturally flocculent strain of Schizosaccharomyces pombe and the performance of two-stage continuous ethanol fermentation system using suspended-bioreactors(total effective volume 1.5 L) has been established: the high biomass levels achieved allow efficient ethanol conversion(72.7 g/L, average), residual glucose(3.74 g/L) and high productivity(9.7 g/L.h) for 150 g/L glucose concentration in feed(tapioca syrup) with average 90% total conversion of substrate . The system was run steadily during a month research.

Shi Yan Sheng Wu Xue Bao, 1999 Mar, 32(1), 39 - 45
{Comparative study of dependence of the cell proliferation of Saccharomyces cerevisiae and Schizosaccharomyces pombe on Ca2+}; Yuan S et al.; Under the same experimental conditions, exogenous Ca2+ had no effect on the proliferation of S . cerevisiae, but it could obviously stimulate the proliferation of S . pombe . Ca2+ chelator EGTA had no inhibition effect on the proliferation of S . cerevisiae, but it apparently inhibited the proliferation of S . pombe and the inhibition could be effectively overcome by adding Ca2+ . Non-special ion chelator EDTA could inhibit the proliferation of both S . cerevisiae and S . pombe, but the inhibition could not be overcome by adding Ca2+ . The results above directly showed that the dependence of the proliferation of the two kinds of yeast on exogeneous Ca2+ was different . The growth rate of S . cerevisiae was about 3 times that of S . pombe and the proliferation of S . cerevisiae was independent on the exogenous Ca2+, which was similar to transformed cells . Therefore, in order to understand the relationship between the disorder of cell cycle and cell transformation, it was very important to study the mechanism of different effects of exogenous Ca2+ on the proliferation of the two kinds of yeast.

Parasitol Int, 2003 Mar, 52(1), 41 - 52
Characterisation of a sexual stage-specific gene encoding ORC1 homologue in the human malaria parasite Plasmodium falciparum; Li JL et al.; The origin recognition complex (ORC) is a multisubunit protein composed of six polypeptides that binds to replication origins and is essential for the initiation of chromosomal DNA replication . Using the Vectorette technique, we have isolated a novel gene encoding an ORC1-like protein from the human malaria parasite Plasmodium falciparum . The gene has no introns and encodes a protein (PfORC1) of 1189 amino acid residues with a predicted molecular mass of 139 kDa . PfORC1 contains all conserved sequences in the ORC1/Cdc6/Cdc18 family and displays the highest homology to the Schizosaccharomyces pombe ORC1 . However, PfORC1 possesses an extensive N-terminal segment with several interesting features including multiple potential phosphorylation sites, a large proportion of charged amino acids, four copies of a heptamer repeat, two nuclear localisation signals, and a leucine zipper motif . Southern blot analyses show that the Pforc1 gene is present as a single copy per haploid genome and is located on chromosome 12 . A 5600 nucleotide transcript of this gene is expressed predominantly in the sexual erythrocytic stage, indicating that PfORC1 may be involved in gametogenesis during which DNA is quickly replicated .

Genome Biol . 2002;3(12):REVIEWS1035 . Epub 2002 Nov 28.
RNAi hushes heterochromatin; Bailis JM et al.; Repeated DNA elements and region-specific protein modifications combine within chromosomes to form a transcriptionally silent chromatin structure called heterochromatin . Recent work in the fission yeast Schizosaccharomyces pombe reveals that RNA is also an integral component of silent heterochromatin, providing a new perspective on how heterochromatin is organized and maintained in eukaryotic cells.

Mol Microbiol, 2003 Feb, 47(3), 781 - 92
Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins; Ladds G et al.; G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems . They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response . The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins . Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways . A number of systems have been developed for the analysis of GPCRs . All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening . Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host . To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins . These enable GPCRs to be coupled to the Sz . pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene . We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors . We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems . Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor . In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors . Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant . This is the first time that human GPCRs have been coupled to the signalling pathway in Sz . pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.

DNA Repair (Amst), 2002 Nov 3, 1(11), 869 - 80
Structural and functional conservation of error-free DNA postreplication repair in Schizosaccharomyces pombe; Brown M et al.; DNA postreplication repair (PRR) is a cellular process by which cells survive replication-blocking lesions without removing the lesion . In the budding yeast Saccharomyces cerevisiae, MMS2 plays a key role in the error-free PRR pathway: the mms2 null mutant displays an increased spontaneous mutation rate and sensitivity to a variety of DNA damaging agents . In contrast, its human homologs appear to play a different role . In order to address whether the MMS2-mediated PRR pathway is conserved in eukaryotes, we isolated a Schizosaccharomyces pombe cDNA homologous to MMS2, which we named spm2(+) . Using spm2(+) as a bait in a yeast two-hybrid screen, we identified a fission yeast cDNA homologous to UBC13 from various species and named it spu13(+) . Two-hybrid analysis confirmed physical interaction between Spm2 and Spu13, and between Spm2 and budding yeast Ubc13 . Genetic analysis shows that both spm2(+) and spu13(+) are able to functionally complement the corresponding budding yeast mutants . Furthermore, deletion of either spm2(+), spu13(+) or both genes from fission yeast results in an increased sensitivity to DNA damaging agents, suggesting that spm2(+) and spu13(+) indeed function in PRR . The fact that the spm2(-) spu13(-) double mutant showed sensitivity similar to that of the single mutant indicates that these two gene products act at the same step . Hence, our data strongly support the hypothesis that the PRR function mediated by UBC13-MMS2 is conserved throughout eukaryotes.

Mol Biol Cell, 2003 Jan, 14(1), 313 - 23
Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe; Coll PM et al.; Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton . Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S . pombe . We have identified a new GEF, named Gef1p, specifically regulating Cdc42p . Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays . Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro . Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele . Gef1p localizes to the septum . gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis . The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators . However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions . Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p . In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.

Mol Biol Cell, 2003 Jan, 14(1), 214 - 29
Global transcriptional responses of fission yeast to environmental stress; Chen D et al.; We explored transcriptional responses of the fission yeast Schizosaccharomyces pombe to various environmental stresses . DNA microarrays were used to characterize changes in expression profiles of all known and predicted genes in response to five stress conditions: oxidative stress caused by hydrogen peroxide, heavy metal stress caused by cadmium, heat shock caused by temperature increase to 39 degrees C, osmotic stress caused by sorbitol, and DNA damage caused by the alkylating agent methylmethane sulfonate . We define a core environmental stress response (CESR) common to all, or most, stresses . There was a substantial overlap between CESR genes of fission yeast and the genes of budding yeast that are stereotypically regulated during stress . CESR genes were controlled primarily by the stress-activated mitogen-activated protein kinase Sty1p and the transcription factor Atf1p . S . pombe also activated gene expression programs more specialized for a given stress or a subset of stresses . In general, these "stress-specific" responses were less dependent on the Sty1p mitogen-activated protein kinase pathway and may involve specific regulatory factors . Promoter motifs associated with some of the groups of coregulated genes were identified . We compare and contrast global regulation of stress genes in fission and budding yeasts and discuss evolutionary implications.

Mol Cell Biol, 2003 Feb, 23(3), 791 - 803
Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint; Weiss RS et al.; Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein . In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress . Unlike hus1(-) fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G(2)/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment . However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect . Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent . However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses . Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway . Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

Nucleic Acids Res, 2003 Jan 15, 31(2), 759 - 68
A comparison of three fission yeast mitochondrial genomes; Bullerwell CE et al.; The fission yeasts are members of the fungal order Schizosaccharomycetales, a candidate deep-diverging group within Ascomycota . Although a great deal of molecular information is available from Schizosaccharomyces pombe, a model eukaryote, very little is available from other members of this group . In order to better characterize mitochondrial genome evolution in this fungal lineage, the mitochondrial DNA (mtDNA) of two additional fission yeasts, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus var . japonicus, was sequenced . Whereas the mtDNA of S.pombe is only 19 431 bp, the mtDNA of S.octosporus is 44 227 bp, and that of S.japonicus var . japonicus is over 80 kb . The size variation of these mtDNAs is due largely to non-coding regions . The gene content in the latter two mtDNAs is almost identical to that of the completely sequenced S.pombe mtDNA, which encodes 25 tRNA species, the large and small mitochondrial ribosomal RNAs (rnl and rns), the RNA component of mitochondrial RNaseP (rnpB), mitochondrial small subunit ribosomal protein 3 (rps3), cytochrome oxidase subunits 1, 2 and 3 (cox1, cox2 and cox3) and ATP-synthase subunits 6, 8 and 9 (atp6, atp8 and atp9) . However, trnI2(cau) (C modified to lysidine) is absent in the S.octosporus mtDNA, as are corresponding ATA codons in its protein-coding genes, and rps3 and rnpB are not found in the mtDNA of S.japonicus var . japonicus . The mtDNA of S.octosporus contains five double hairpin elements, the first report of these elements in an ascomycete . This study provides further evidence in favor of the mobility of these elements, and supports their role in mitochondrial genome rearrangement . The results of our phylogenetic analysis support the monophyly of the Schizosaccharomycetales, but question their grouping within the Archiascomycota.

Mol Cells, 2002 Dec 31, 14(3), 444 - 8
The Pap1-independent induction by metal ions of a third gene encoding glutathione S-transferase gene from the fission yeast; Sa JH et al.; A third gene that encodes glutathione S-transferase (GSTIII) was previously cloned from the fission yeast Schizosaccharomyces pombe . Using the GSTIII-lacZ fusion plasmid pGDA-19, its expression was shown to be enhanced by various metal ions . In the present study, four additional fusion plasmids, pGDA-29, pGDA-39, PGDA-49, and pGDA-59, were designed to carry 998, 378, 276, and 115 bp upstream regions from the translational initiation point, respectively . The major activation region was located between -998 and -378 bp upstream of the GSTIII gene . Regulatory sequences that are responsible for the induction by metal ions reside between -998 and -378 bp and between -276 and -115 bp upstream of the gene . The overexpressed Pap1 exerts a repression effect on the GSTIII expression via -998 to approximately -378 bp region, whereas it exerts an activation effect on the GSTIII expression via -270 to approximately -115 bp region . However, the induction of the GSTIII gene by metal ions occurs independent of Pap1.

Mol Cells, 2002 Dec 31, 14(3), 437 - 43
Post-transcriptional regulation of ura4+ gene expression by glucose in Schizosaccharomyces pombe; Kim MJ et al.; Glucose-inducible gene expression is a fundamental cellular response for optimal cell growth, but identities of glucose-inducible genes and its regulatory mechanism remain largely elusive in Schizosaccharomyces pombe . Here we report that ura4+, encoding orotidine monophosphate decarboxylase (OMPdecase), shows glucose-inducible expression regulated at post-transcriptional level . The ura4+ mRNA level was rapidly decreased by approximately 50% within 20 min after glucose depletion and it was readily recovered upon glucose-readdition within 1 h . Glucose at above 2% similarly raised the transcript level of ura4+, while low concentration (0.1%) was not effective . Interestingly, control of mRNA turnover would be the main regulatory step of the glucose-dependent expression of ura4+ . Moreover, stress-activated MAPK (SAPK) pathway was partially responsible for the glucose-regulated expression of ura4+ and rrg1+, another example of glucose-dependent mRNA stability control in S . pombe . These results suggest that the SAPK pathway might participate in the glucose-dependent regulation of ura4+ and rrg1+ mRNA stabilities.

Mol Cells, 2002 Dec 31, 14(3), 431 - 6
Pap1-dependent regulation of the GSTII gene from the fission yeast; Lim CJ et al.; The genomic DNA encoding a second glutathione S-transferase (GSTII) was previously isolated from the fission yeast Schizosaccharomyces pombe . Its expression was shown to be induced by menadione, mercuric chloride, o-dinitrobenzene, and NO-generating S-nitroso-N-acetylpenicillamine using the GSTII-lacZ fusion harboring the 910 bp upstream region from the translational initiation point . In this study, the additional fusion plasmids pGST50-590 and pGST50-6R-590 were constructed to carry the 590 bp upstream region in the vectors YEp357 and YEp367R, respectively . The synthesis of beta-galactosidase from the fusion plasmid pGST50-590 was about 3-fold higher than that from the fusion plasmid pGST50-F, indicating the presence of negatively activating sequence in the -910 to approximately -590 region . It was also enhanced by the same agents, which induced the synthesis of beta-galactosidase from the fusion plasmid pGST50-F . The synthesis of beta-galactosidase from both fusion plasmids pGST50-F and pGST50-590 was enhanced by the overexpressed Pap1 protein . The synthesis of beta-galactosidase from the two YEp367R derivatives pGST50-6R-F and pGST50-6R-590 was greatly decreased in the Pap1-negative strain TP108-3C . These results propose the Pap1-dependent regulation of the GSTII gene from the fission yeast.

Mol Cells, 2002 Dec 31, 14(3), 425 - 30
Regulation of septation and cytokinesis during resumption of cell division requires uvi31+, a UV-inducible gene of fission yeast; Kim MJ et al.; uvi31+ is a sequence homolog of Escherichia coli bolA gene in Schizosaccharomyces pombe, identified as a UV-inducible gene . Here, the cellular function of uvi31+ was investigated by null mutant analysis . Deletion of uvi31+ led to a delayed germination of spore and defects in subsequent cell division . However, the uvi31 mutant cell proliferated faster with smaller cell size than the wild-type cell during vegetative growth . In addition, the uvi31 mutant was sensitive to UV-light . It showed a normal cell cycle delay after UV-irradiation but displayed aberrant septum formation and defective cytokinesis when released from the UV damage checkpoint . These results suggest that uvi31+ may be involved in control of cell division, especially during the resumption from cell cycle arrest.

J Biol Chem, 2003 Mar 14, 278(11), 9318 - 21 Epub 2003 Jan 07.
Exposure of single-stranded telomeric DNA causes G2/M cell cycle arrest in Saccharomyces cerevisiae; Pang TL et al.; In Saccharomyces cerevisiae, Cdc13p is a single-stranded TG(1-3) DNA binding protein that protects telomeres and maintains telomere length . A mutant allele of CDC13, cdc13-1, causes accumulation of single-stranded TG(1-3) DNA near telomeres along with a G(2)/M cell cycle arrest at non-permissive temperatures . We report here that when the single-stranded TG(1-3) DNA is masked by its binding proteins, such as S . cerevisiae Gbp2p or Schizosaccharomyces pombe Tcg1, the growth arrest phenotype of cdc13-1 is rescued . Mutations on Gbp2p that disrupt its binding to the single-stranded TG(1-3) DNA render the protein unable to complement the defects of cdc13-1 . These results indicate that the presence of a single-stranded TG(1-3) tail in cdc13-1 cells serves as the signal for the cell cycle checkpoint . Moreover, the binding activity of Gbp2p to single-stranded TG(1-3) DNA appears to be associated with its ability to restore the telomere-lengthening phenotype in cdc13-1 cells . These results indicate that Gbp2p is involved in modulating telomere length.

Mol Microbiol, 2003 Jan, 47(2), 507 - 18
Rga5p is a specific Rho1p GTPase-activating protein that regulates cell integrity in Schizosaccharomyces pombe; Calonge TM et al.; Schizosaccharomyces pombe Rho1p regulates (1,3)beta-d-glucan synthesis and is required for cell integrity maintenance and actin cytoskeleton organization, but nothing is known about the regulation of this protein . At least nine different S . pombe genes code for proteins predicted to act as Rho GTPase-activating proteins (GAPs) . The results shown in this paper demonstrate that the protein encoded by the gene named rga5+ is a GAP specific for Rho1p . rga5+ overexpression is lethal and causes morphological alterations similar to those reported for Rho1p inactivation . rga5+ deletion is not lethal and causes a mild general increase in cell wall biosynthesis and morphological alterations when cells are grown at 37 degrees C . Upon mild overexpression, Rga5p localizes to growth areas and possesses both in vivo and in vitro GAP activity specific for Rho1p . Overexpression of rho1+ in rga5Delta cells is lethal, with a morphological phenotype resembling that of the overexpression of the constitutively active allele rho1G15V . In addition (1,3)beta-d-glucan synthase activity, regulated by Rho1p, is increased in rga5Delta cells and decreased in rga5-overexpressing cells . Moreover, the increase in (1,3)beta-d-glucan synthase activity caused by rho1+ overexpression is considerably higher in rga5Delta than in wild-type cells . Genetic interactions suggest that Rga5p is also important for the regulation of the other known Rho1p effectors, Pck1p and Pck2p.

J Biol Chem, 2003 Mar 14, 278(11), 9185 - 94 Epub 2003 Jan 02.
Identification of Uhp1, a ubiquitinated histone-like protein, as a target/mediator of Rhp6 in mating-type silencing in fission yeast; Naresh A et al.; Mating-type silencing in Schizosaccharomyces pombe is brought about by cooperative interactions between cis-acting DNA sequences flanking mat2P and mat3M and the trans-acting factors, namely Swi6, Clr1-Clr4, Clr6, and Rik1 . In addition, DNA repair gene rhp6, which plays a role in post-replication DNA repair and ubiquitination of proteins including histones, is also involved in silencing, albeit in a unique way; its effect on silencing and chromatin structure of the donor loci is dependent on their switching competence . Earlier, we hypothesized the existence of a mediator of Rhp6 that plays a role in reestablishment of the chromatin structure coincidentally with DNA replication associated with mating-type switching . Here we report the identification of a 22-kDa protein as an in vivo target and mediator of Rhp6 in mating-type silencing . The level of this protein is greatly elevated in sng1-1/rhp6(-) mutant and rhp6Delta as compared with wild type strain . Both the deletion and overexpression of the gene encoding this protein elicit switching-dependent loss of silencing . Furthermore, the 22-kDa protein undergoes Rhp6-dependent multiubiquitination and associates with mat2 locus during S phase in wild type cells . Interestingly, it contains a histone-fold motif similar to that of histone H2A, and like histone H2A, it interacts strongly with histone H2B in vitro . These results indicate that the 22-kDa protein, renamed as the ubiquitinated histone-like protein Uhp1, is an in vivo target/mediator of Rhp6 in silencing . Thus, regulation of association of Uhp1 with chromatin and ubiquitination followed by degradation may play a role in reestablishment of inactive chromatin structure at the silent mating-type loci.

DNA Repair (Amst), 2002 Jun 21, 1(6), 463 - 82
Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants; Oakley TJ et al.; The Saccharomyces cerevisiae TOP3 gene encodes the type IA topoisomerase (Top3p) that is highly conserved in evolution . Deletion of TOP3 leads to a reduction in cell viability, hyper-recombination between repetitive DNA sequences, and abnormalities in both cell cycle progression and responses to DNA damaging agents . Deletion of SGS1, encoding the sole RecQ family helicase in S . cerevisiae, strongly suppresses the phenotypic effects of loss of TOP3 function . Here, we show that many of the adverse phenotypic effects of TOP3 deletion can also be partially alleviated by disruption of homologous recombination (HR) functions . This genetic interaction is seen both in strains deleted for TOP3 and in wild-type strains over-expressing a dominant-negative Top3p mutant form that confers a top3-like phenotype . Moreover, we show that this genetic interaction is conserved in the distantly-related fission yeast, Schizosaccharomyces pombe . Our results implicate topoisomerase III enzymes in recombination repair events required for cellular protection against DNA damaging agents and DNA replication inhibitors.

Biochim Biophys Acta, 2003 Jan 10, 1609(1), 71 - 9
Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H(+)-ATPase reconstituted into vesicles; Holoubek A et al.; Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Delta(psi)) generated by the Schizosaccharomyces pombe plasma membrane H(+)-ATPase reconstituted into vesicles . Oxonol VI was used for quantitative measurements of the Delta(psi) because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V . However, oxonol V has a superior sensitivity to Delta(psi) at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution . Oxonol VI was found to be a good emission-ratiometric probe . We have shown that the reconstituted H(+)-ATPase generates Delta(psi) of about 160 mV on the vesicle membrane . The generated Delta(psi) was stable at least over tens of minutes . An influence of the H(+) membrane permeability on the Delta(psi) buildup was demonstrated by manipulating the H(+) permeability with the protonophore CCCP . Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H(+)-ATPase-mediated Delta(psi) buildup.

J Gen Appl Microbiol, 1997 Jun, 43(3), 169 - 174
Induction of sexual co-flocculation of heterothallic fission yeast (Schizosaccharomyces pombe) cells by mating pheromones; Miyata M et al.; Heterothallic fission yeast (Schizosaccharomyces pombe) cells preincubated with sex pheromone, P- or M-factor of the obverse mating-type cells, in mannose synthetic medium (MSM) results in remarkably increased sexual co-flocculation with obverse mating-type cells almost without time lag, i.e., within 10 min . By contrast, comparable flocculation requires over 1 h if untreated control cells are mixed with obverse mating-type cells . The agglutinin of P cells is more inducible than that of M cells . These pheromonal inductions of sexual co-flocculation are inhibited by the addition of cycloheximide or tunicamycin during preincubation but not by chloramphenicol or hydroxyurea . These results demonstrate that, in addition to (a) the repression of cell division (G1 arrest) and (b) the activation of cell wall autolytic processes (mating-specific elongation of cells: formation of their conjugation tubes), mating pheromones of fission yeast have another important role; (c) to induce sexual co-flocculation (agglutinability) . Using our experimental system of preincubation with sexual pheromones, we show that M-agglutinin is heat-stable and its induction is inhibited by tunicamycin, but that P-agglutinin is heat-labile and its induction is only partially inhibited by tunicamycin.

J Gen Appl Microbiol, 1997 Aug, 43(4), 209 - 215
Characterization of multicopy suppressor genes that complement a defect in the Wis1-Sty1 MAP kinase cascade involved in stress responses in Schizosaccharomyces pombe; Yamada H et al.; The Wis1-Sty1 mitogen-activated protein (MAP) kinase cascade is one of the major signaling systems involved in a wide range of stress responses in Schizosaccharomyces pombe . It is known that Deltawis1 and Deltasty1 mutants exhibit highly pleiotropic phenotypes, including a phenotype of temperature sensitivity for growth . In this study, we screened multicopy suppressor genes that allow both the Deltawis1 and Deltasty1 mutants to grow simultaneously at a non-permissive temperature, 37 degrees C . Two such multicopy suppressors were cloned and characterized as sds23(+) and hxk2(+) genes . The former is known to specify a protein that functions as a multicopy suppressor for mutations of the PP1 protein phosphatase and the 20S cyclosome/anaphase-promoting complex (APC), and the latter encodes hexokinase 2 . It was revealed that the multicopy sds231 gene restored a defect in the mating efficiency caused by the Deltawis1 and Deltasty1 mutations, whereas the multicopy hxk2(+) gene suppressed a phenotype of heat-shock sensitivity for growth of these mutant cells . These findings are discussed with special reference to the Wis1-Sty1 MAP kinase signaling pathway in S . pombe.

Biochim Biophys Acta, 2003 Jan 2, 1619(1), 89 - 97
Characterisation of the gptA gene, encoding UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase, from the filamentous fungus, Aspergillus niger; Sorensen TK et al.; The production of asparagine (N)-linked oligosaccharides is of vital importance in the formation of glycosylated proteins in eukaryotes and is mediated by the dolichol pathway . As part of studies to allow manipulation of this pathway, the gene coding for the production of the enzyme UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase (GPT), catalysing the first step in the assembly of dolichol-linked oligosaccharides, was cloned from the filamentous fungus Aspergillus niger . Degenerate-PCR was used to amplify a 470-bp fragment of the gene, which was labelled as a probe to obtain a full-length clone from a genomic library of A . niger . This contained a 1557-bp open reading frame encoding a highly hydrophobic protein of 468 amino acids with a predicted molecular weight of 51.4 kDa . The gene contained two intron sequences and putative dolichol recognition sites (PDRSs) were present in the deduced amino acid sequence . Comparison with other eukaryotic GPTs revealed the A . niger GPT to share 45-47% identity with yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and 41-42% identity with mammals (mouse, hamster, human) . Nested-PCR of a cDNA library was used to confirm the position of an intron . A complete cDNA clone of A . niger gpt was obtained by employing a recombinant PCR approach . This was used to rescue a conditional lethal mutant of S . cerevisiae carrying a dysfunctional gpt gene by heterologous expression, confirming that the gpt genes from A . niger and S . cerevisiae are functionally equivalent.

Nucleic Acids Res, 2002 Dec 15, 30(24), 5347 - 59
Characterization of the fission yeast ribosomal DNA binding factor: components share homology with Upstream Activating Factor and with SWI/SNF subunits; Liu M et al.; A ribosomal DNA (rDNA) binding activity was previously characterized in fission yeast that recognized the upstream ribosomal RNA (rRNA) gene promoter in a sequence specific manner and which stimulated rRNA synthesis . It was found to share characteristics with Saccharomyces cerevisiae's Upstream Activating Factor (UAF), an RNA polymerase I (pol I) specific transcription stimulatory factor . Putative fission yeast homologs of the S.cerevisiae UAF subunits, Rrn5p and Rrn10p, were identified . The Schizosaccharomyces pombe rDNA binding activity/transcriptional stimulatory activity was found to co-fractionate with both SpRrn5h and SpRrn10h . Analysis of polypeptides interacting with SpRrn10h uncovered a 27 kDa polypeptide (Spp27) homologous to a SWI/SNF component (now known to be homologous to Uaf30p) . The contributions of the S.pombe and S.cerevisiae upstream rDNA promoter domains were assessed in cross-species transcriptional assays . Furthermore, comparative genomic analysis revealed putative Rrn5p, Rrn10p, Rrn9p and p27 homologs in multiple non-vertebrates . The S.pombe rDNA binding activity is proposed to be an RNA pol I specific SWI/SNF type factor.

Yeast, 2003 Jan 15, 20(1), 69 - 78
Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae; Eksteen JM et al.; Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e . alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known . Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L . kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe . Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L . kononenkoae . LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides . Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da . The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz . pombe . When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S . cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host . In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S . cerevisiae resulted in the production of biologically active alpha-amylase . The LKA2-encoded alpha-amylase produced by S . cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages . The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C . Besides LKA1, LKA2 is only the second L . kononenkoae gene ever cloned and expressed in S . cerevisiae . The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S . cerevisiae for the efficient bioconversion of starch into commercially important commodities .

J Biol Chem, 2003 Mar 7, 278(10), 8487 - 93 Epub 2002 Dec 17.
High conservation of the Set1/Rad6 axis of histone 3 lysine 4 methylation in budding and fission yeasts; Roguev A et al.; Histone 3 lysine 4 (H3 Lys(4)) methylation in Saccharomyces cerevisiae is mediated by the Set1 complex (Set1C) and is dependent upon ubiquitinylation of H2B by Rad6 . Mutually exclusive methylation of H3 at Lys(4) or Lys(9) is central to chromatin regulation; however, S . cerevisiae lacks Lys(9) methylation . Furthermore, a different H3 Lys(4) methylase, Set 7/9, has been identified in mammals, thereby questioning the relevance of the S . cerevisiae findings for eukaryotes in general . We report that the majority of Lys(4) methylation in Schizosaccharomyces pombe, like in S . cerevisiae, is mediated by Set1C and is Rad6-dependent . S . pombe Set1C mediates H3 Lys(4) methylation in vitro and contains the same eight subunits found in S . cerevisiae, including the homologue of the Drosophila trithorax Group protein, Ash2 . Three additional features of S . pombe Set1C each involve PHD fingers . Notably, the Spp1 subunit is dispensable for H3 Lys(4) methylation in budding yeast but required in fission yeast, and Sp_Set1C has a novel proteomic hyperlink to a new complex that includes the homologue of another trithorax Group protein, Lid (little imaginal discs) . Thus, we infer that Set1C is highly conserved in eukaryotes but observe that its links to the proteome are not.

Dev Cell, 2002 Dec, 3(6), 779 - 90
Dma1 prevents mitotic exit and cytokinesis by inhibiting the septation initiation network (SIN); Guertin DA et al.; In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) triggers cytokinesis after mitosis . We investigated the relationship between Dma1p, a spindle checkpoint protein and cytokinesis inhibitor, and the SIN . Deletion of dma1 inactivates the spindle checkpoint and allows precocious SIN activation, while overexpressing Dma1p reduces SIN signaling . Dma1p seems to function by inhibiting the SIN activator, Plo1p kinase, since dma1 overexpression and deletion phenotypes suggest that Dma1p antagonizes Plo1p localization . Furthermore, failure to maintain high cyclin-dependent kinase (CDK) activity during spindle checkpoint activation in dma1 deletion cells requires Plo1p . Dma1p itself localizes to spindle pole bodies through interaction with Sid4p . Our observations suggest that Dma1p functions to prevent mitotic exit and cytokinesis during spindle checkpoint arrest by inhibiting SIN signaling.

Yeast, 2002 Dec, 19(16), 1437 - 45
Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species; Kimura Y et al.; We recently discovered, on the chromosome of Saccharomyces cerevisiae sigma 1278b, novel MPR1 and MPR2 genes required for resistance to a toxic analogue of L-proline, L-azetidine-2-carboxylic acid . The MPR genes, which were absent in the S . cerevisiae genome project strain S288C, encoded a novel acetyltransferase of 229 amino acids that detoxifies the analogue by acetylating it . The MPR1 gene homologue found in Schizosaccharomyces pombe was also shown to encode a similar acetyltransferase . To further analyse the origin and the physiological role of the yeast novel gene, we report here the comparative analysis of the MPR1 gene in the S . cerevisiae complex spp . which belong to the Saccharomyces sensu stricto group . Only the type strain of S . paradoxus exhibited resistance and acetyltransferase activity to L-azetidine-2-carboxylic acid . PCR was then used to isolate the new MPR1 homologue (Spa MPR1) from S . paradoxus with the primers based on the sequence of the MPR1 gene . Gene expression and enzymatic analysis showed that the cloned Spa MPR1 gene encodes an L-azetidine-2-carboxylic acid acetyltransferase of 231 amino acids, which has 87% identity to the MPR1 protein . We also found in the protein databases that S . bayanus contains a DNA fragment that is partly homologous to the MPR1 gene . However, the gene product was considered to lose the enzymatic activity, possibly due to the gene truncation or the base substitution(s) at the important region for catalysis . Further, genomic PCR analysis showed that most of the S . cerevisiae complex spp . have the sequence highly homologous to the MPR1 gene .

Yeast, 2002 Dec, 19(16), 1381 - 98
Helicase activity is only partially required for Schizosaccharomyces pombe Rqh1p function; Ahmad F et al.; The RecQ-related family of DNA helicases is required for the maintenance of genomic stability in organisms ranging from bacteria to humans . In humans, mutation of three RecQ-related helicases, BLM, WRN and RecQL4, cause the cancer-prone and premature ageing diseases of Bloom syndrome, Werner's syndrome and Rothmund-Thompson syndrome, respectively . In the fission yeast Schizosaccharomyces pombe, disruption of the rqh1(+) gene, which encodes the single Sz . pombe RecQ-related helicase, causes cells to display reduced viability and elevated levels of chromosome loss . After S-phase arrest or DNA damage, cells lacking rqh1(+) function display elevated levels of homologous recombination and defective chromosome segregation . Here we show that, like other RecQ family members, the Rqh1p protein displays 3' to 5' DNA helicase activity . Interestingly, however, unlike other RecQ family members, the helicase activity of Rqh1p is only partially required for its function in recovery from S-phase arrest or DNA damage . We also report that high cellular levels of Rqh1p result in lethal chromosome segregation defects, while more moderate levels of Rqh1p cause significantly elevated rates of chromosome loss . This suggests that careful regulation of RecQ-like protein levels in eukaryotic cells is vital for maintaining genome stability .

Curr Genet, 2002 Nov, 42(2), 73 - 84 Epub 2002 Oct 22.
Prospects for functional genomics in Schizosaccharomyces pombe; Sunnerhagen P; Schizosaccharomyces pombe is well established as an experimental organism for basic research, with well developed technologies for molecular biology, genetics and cell biology . Its full genome sequence has recently been published . Here, the prerequisites for functional genomics studies in Sch . pombe are examined and compared with those of some established prominent functional genomics model organisms, especially Saccharomyces cerevisiae . It is argued that functional genomics studies in certain areas of cellular and molecular biology could potentially be more efficiently performed in Sch . pombe than in most other experimental organisms.

Curr Biol, 2002 Dec 10, 12(23), 2048 - 54
Proteomics analysis identifies new components of the fission and budding yeast anaphase-promoting complexes; Yoon HJ et al.; The anaphase-promoting complex (APC) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators . Components of the APC have been identified through genetic screens in both Schizosaccharomyces pombe and Saccharomyces cerevisiae as well as through biochemical purification coupled with mass spectrometric protein identification . With these approaches, 11 subunits of the core S . cerevisiae APC have been identified . Here, we have applied a tandem affinity purification approach coupled with direct analysis of the purified complexes by mass spectrometry (DALPC) to reveal additional subunits of both the S . pombe and S . cerevisiae APCs . Our data increase the total number of identified APC subunits to 13 in both yeasts and indicate that previous approaches were biased against the identification of small subunits . These results underscore the power of direct analysis of protein complexes by mass spectrometry and set the foundation for further functional and structural studies of the APC.

J Biol Chem, 2003 Feb 28, 278(9), 7180 - 8 Epub 2002 Dec 09.
Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control; Pei Y et al.; RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5 . Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO(4), and Spt5 . Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9 . The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk domain . We show that the Cdk domain interacts with S . pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S . pombe Spt5 . We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb . Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo . Deleting the C-terminal domain of SpCdk9 causes a severe growth defect . We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest . This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.

Mol Genet Genomics, 2002 Dec, 268(4), 553 - 62 Epub 2002 Nov 07.
The Schizosaccharomyces pombe genes sep10 and sep11 encode putative general transcriptional regulators involved in multiple cellular processes; Szilagyi Z et al.; We have previously described the genetic analysis of eleven complementation groups ( sep6- sep16) defined by Schizosaccharomyces pombe mutants that are defective in cell separation and sexual differentiation . Here we report on the cloning and characterisation of two members of this set, sep10 and sep11 . Sequencing of the full-length sep10 revealed a continuous ORF that encodes a conserved protein with possible functions in general transcriptional regulation . The coding region of sep11 is interrupted by introns and the putative s ep11 protein shows no sequence similarity with known proteins of other species . Disruption of each gene causes temperature sensitivity . Simultaneous disruption of both genes is lethal, demonstrating that sep10 and sep11 perform related, overlapping functions . Overexpression of aff1/ste11, a pivotal regulator of sexual development, suppresses the sterility of sep10 (-) cells, which suggests that sep10 is needed for the activity of aff1/ste11.

Mol Genet Genomics, 2002 Dec, 268(4), 525 - 34 Epub 2002 Nov 12.
Activation of the urease of Schizosaccharomyces pombe by the UreF accessory protein from soybean; Bacanamwo M et al.; Plant orthologs of the bacterial urease accessory genes ureD and ureF, which are required for the insertion of the nickel ion at the active site, have been isolated from soybean ( Glycine max L . Merr.), tomato ( Lycopersicon esculentum) and Arabidopsis thaliana . The functionality of soybean UreD and UreF was tested by measuring their ability to complement urease-negative mutants of Schizosaccharomyces pombe, a eukaryote which produces a "plant-like" urease of ~90 kDa . The S . pombe ure4 mutant was complemented by a 12-kb fragment of S . pombe genomic DNA, which was shown by PCR to contain a putative ureD gene . However, ure4 was not complemented by a UreD cDNA soybean, expressed under the control of a strong promoter . In contrast, an S . pombe ure3 mutation was complemented by both a 10-kb fragment of S . pombe DNA containing ureF and the UreF cDNA from soybean . Soybean Eu2 is a candidate urease accessory gene; its product cooperates with the Eu3 protein in activating apourease in vitro . However, the sequences of UreD and UreF transcripts from two eu2/eu2 mutants, recovered as RT-PCR products, revealed no mutational alteration, suggesting that Eu2 encodes neither UreD nor UreF.

Nucleic Acids Res, 2002 Dec 1, 30(23), 5036 - 55
Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes; Pandey R et al.; Sequence similarity and profile searching tools were used to analyze the genome sequences of Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster for genes encoding three families of histone deacetylase (HDAC) proteins and three families of histone acetyltransferase (HAT) proteins . Plants, animals and fungi were found to have a single member of each of three subfamilies of the GNAT family of HATs, suggesting conservation of these functions . However, major differences were found with respect to sizes of gene families and multi-domain protein structures within other families of HATs and HDACs, indicating substantial evolutionary diversification . Phylogenetic analysis identified a new class of HDACs within the RPD3/HDA1 family that is represented only in plants and animals . A similar analysis of the plant-specific HD2 family of HDACs suggests a duplication event early in dicot evolution, followed by further diversification in the lineage leading to Arabidopsis . Of three major classes of SIR2-type HDACs that are found in animals, fungi have representatives only in one class, whereas plants have representatives only in the other two . Plants possess five CREB-binding protein (CBP)-type HATs compared with one to two in animals and none in fungi . Domain and phylogenetic analyses of the CBP family proteins showed that this family has evolved three distinct types of CBPs in plants . The domain architecture of CBP and TAF(II)250 families of HATs show significant differences between plants and animals, most notably with respect to bromodomain occurrence and their number . Bromodomain-containing proteins in Arabidopsis differ strikingly from animal bromodomain proteins with respect to the numbers of bromodomains and the other types of domains that are present . The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.

Biochemistry, 2002 Dec 10, 41(49), 14560 - 8
Cooperative binding of single-stranded telomeric DNA by the Pot1 protein of Schizosaccharomyces pombe; Lei M et al.; The fission yeast Pot1 (protection of telomeres) protein is a single-stranded telomeric DNA-binding protein and is required to protect the ends of chromosomes . Its N-terminal DNA-binding domain, Pot1pN, shows sequence similarity to the first OB fold of the telomere-binding protein alpha subunit of Oxytricha nova . The minimal-length telomeric ssDNA required to bind Pot1pN was determined to consist of six nucleotides, GGTTAC, by gel filtration chromatography and filter-binding assay (K(D) = 83 nM) . Pot1pN is a monomer, and each monomer binds one hexanucleotide . Experiments with nucleotide substitutions demonstrated that the central four nucleotides are crucial for binding . The dependence of Pot1pN-ssDNA binding on salt concentration was consistent with a single ionic contact between the protein and the ssDNA phosphate backbone, such that at physiological salt condition 83% of the free energy of binding is nonelectrostatic . Subsequent binding experiments with longer ssDNAs indicated that Pot1pN binds to telomeric ssDNA with 3' end preference and in a highly cooperative manner that mainly results from DNA-induced protein-protein interactions . Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.

Eukaryot Cell, 2002 Aug, 1(4), 613 - 25
The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region; Nielsen IS et al.; Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe . Silencing is mediated by a number of gene products and cis-acting elements . We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing . Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby . Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing . Rhp18 is the S . pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p . Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced . Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing . However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination . We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.

Eukaryot Cell, 2002 Aug, 1(4), 558 - 67
Schizosaccharomyces pombe Git7p, a member of the Saccharomyces cerevisiae Sgtlp family, is required for glucose and cyclic AMP signaling, cell wall integrity, and septation; Schadick K et al.; The Schizosaccharomyces pombe fbp1 gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose . Mutations that confer constitutive fbp1 transcription identify git (glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation . Four of these genes encode the three subunits of a heterotrimeric G protein (gpa2, git5, and git11) and a G protein-coupled receptor (git3) . Three additional genes, git1, git7, and git10, act in parallel to or downstream from the G protein genes . Here, we describe the cloning and characterization of the git7 gene . The Git7p protein is a member of the Saccharomyces cerevisiae Sgtlp protein family . In budding yeast, Sgtlp associates with Skplp and plays an essential role in kinetochore assembly, while in Arabidopsis, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1 . Like S . cerevisiae Sgtlp, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as S . pombe cells lacking either adenylate cyclase or protein kinase A are viable . In addition, git7 mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect . Two alleles of git7 that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus . The S . cerevisiae and human SGT1 genes both suppress git7-93 but not git7-235 for glucose repression of fbp1 transcription and benomyl sensitivity . This allele-specific suppression indicates that the Git7p/Sgtlp proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets . Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in S . pombe, S . cerevisiae, and humans.

Eukaryot Cell, 2002 Jun, 1(3), 448 - 57
Divergent subunit interactions among fungal mRNA 5'-capping machineries; Takagi T et al.; The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5'-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase) . The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription . Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1) . For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans . Surprisingly, the S . pombe capping enzyme subunits do not interact with each other . Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding . The S . pombe RTPase gene (pct1+) is essential for viability . Pct1 can replace the S . cerevisiae RTPase when GTase activity is supplied by the S . pombe or mouse enzymes but not by the S . cerevisiae GTase . The C . albicans capping enzyme subunits do interact with each other . However, this interaction is not essential in vivo . Our results reveal an unexpected diversity among the fungal capping machineries.

Eukaryot Cell, 2002 Feb, 1(1), 44 - 55
A long terminal repeat retrotransposon of fission yeast has strong preferences for specific sites of insertion; Singleton TL et al.; The successful dispersal of transposons depends on the critical balance between the fitness of the host and the ability of the transposon to insert into the host genome . One method transposons may use to avoid the disruption of coding sequences is to target integration into safe havens . We explored the interaction between the long terminal repeat retrotransposon Tf1 and the genome of the yeast Schizosaccharomyces pombe . Using techniques that were specifically designed to detect integration of Tf1 throughout the genome and to avoid bias in this detection, we generated 51 insertion events . Although 60.2% of the genome of S . pombe is coding sequence, all but one of the insertions occurred in intergenic regions . We also found that Tf1 was significantly more likely to insert into intergenic regions that included polymerase II promoters than into regions between convergent gene pairs . Interestingly, 8 of the 51 insertion sites were isolated multiple times from genetically independent cultures . This result suggests that specific sites in intergenic regions are targeted by Tf1 . Perhaps the most surprising observation was that per kilobase of nonrepetitive sequence, Tf1 was significantly more likely to insert into chromosome 3 than into one of the other two chromosomes . This preference was found not to be due to differences in the distribution or composition of intergenic sequences within the three chromosomes.

Eukaryot Cell, 2002 Oct, 1(5), 787 - 98
A forkhead transcription factor is important for true hyphal as well as yeast morphogenesis in Candida albicans; Bensen ES et al.; Candida albicans is an important pathogen of immunocompromised patients which grows with true hyphal, pseudohyphal, and yeast morphologies . The dynamics of cell cycle progression are markedly different in true hyphal relative to pseudohyphal and yeast cells, including nuclear movement and septin ring positioning . In Saccharomyces cerevisiae, two forkhead transcription factors (ScFKH1 and ScFKH2) regulate the expression of B-cyclin genes . In both S . cerevisiae and Schizosaccharomyces pombe, forkhead transcription factors also influence morphogenesis . To explore the molecular mechanisms that connect C . albicans morphogenesis with cell cycle progression, we analyzed CaFKH2, the single homolog of S . cerevisiae FKH1/FKH2 . C . albicans cells lacking CaFkh2p formed constitutive pseudohyphae under all yeast and hyphal growth conditions tested . Under hyphal growth conditions levels of hyphae-specific mRNAs were reduced, and under yeast growth conditions levels of several genes encoding proteins likely to be important for cell wall separation were reduced . Together these results imply that Fkh2p is required for the morphogenesis of true hyphal as well as yeast cells . Efglp and Cphlp, two transcription factors that contribute to C . albicans hyphal growth, were not required for the pseudohyphal morphology of fkh2 mutants, implying that Fkh2p acts in pathways downstream of and/or parallel to Efglp and Cphlp . In addition, cells lacking Fkh2p were unable to damage human epithelial or endothelial cells in vitro, suggesting that Fkh2p contributes to C . albicans virulence.

Eukaryot Cell, 2002 Oct, 1(5), 758 - 73
mcl1+, the Schizosaccharomyces pombe homologue of CTF4, is important for chromosome replication, cohesion, and segregation; Williams DR et al.; The fission yeast minichromosome loss mutant mcl1-1 was identified in a screen for mutants defective in chromosome segregation . Missegregation of the chromosomes in mcl1-1 mutant cells results from decreased centromeric cohesion between sister chromatids . mcl1+ encodes a beta-transducin-like protein with similarity to a family of eukaryotic proteins that includes Ctf4p from Saccharomyces cerevisiae, sepB from Aspergillus nidulans, and AND-1 from humans . The previously identified fungal members of this protein family also have chromosome segregation defects, but they primarily affect DNA metabolism . Chromosomes from mcl1-1 cells were heterogeneous in size or structure on pulsed-field electrophoresis gels and had elongated heterogeneous telomeres . mcl1-1 was lethal in combination with the DNA checkpoint mutations rad3delta and rad26delta, demonstrating that loss of Mcl1p function leads to DNA damage . mcl1-1 showed an acute sensitivity to DNA damage that affects S-phase progression . It interacts genetically with replication components and causes an S-phase delay when overexpressed . We propose that Mcl1p, like Ctf4p, has a role in regulating DNA replication complexes.

Int J Med Microbiol, 2002 Oct, 292(5-6), 391 - 404
Pneumocystis; Stringer JR; Pneumocystis organisms can cause pneumonia in mammals that lack a strong immune defense . The genus Pneumocystis contains many different organisms that can be distinguished by DNA sequence analysis . These different organisms are different species of yeast-like fungi that are most closely related to the ascomycete, Schizosaccharomyces pombe . Each species of Pneumocystis appears to be specific for the mammal in which it is found . The species that infects humans is Pneumocystis jiroveci . P . jiroveci has not been found in any other mammal and the species of Pneumocystis found in other mammals have not been seen in humans . Genetic variation among P . jiroveci samples is common, suggesting that there are many strains . Strain analysis shows that adults can be infected by more than one strain, and suggests that pneumonia can be the result of infection occurring proximal to the time of disease, rather than to reactivation of dormant organisms acquired in early childhood . Nevertheless, long-term colonisation may be occurring . A large fraction of normal children and animals show evidence of infection . A Pneumocystis species that grows in rats has been shown to possess a complex genetic system for surface antigen variation, a strategy employed by other microbes that dwell in immunocompetent hosts . These findings, together with strong host specificity, suggest that Pneumocystis species may be obligate parasites . The source of infection is not clear . Pneumocystis DNA is detectable in the air, but is scarce except in environments occupied by individuals with Pneumocystis pneumonia . In a few cases, there is direct evidence of person to person transmission . In general, however, patients and their contacts have been found to have different strains of P . jiroveci.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2224 - 7
Involvement of a CCAAT-binding complex in the expression of a nitrogen-starvation-specific gene, isp6+, in Schizosaccharomyces pombe; Nakashima A et al.; The fission yeast gene isp6+ is needed in nitrogen-starvation response but its transcriptional regulation has been unclear . isp6+ was repressed under nutrient conditions, in which cAMP-dependent protein kinase A, the stress-activated protein kinase cascade, and the CCAAT-binding complex were concerned . The CCAAT-binding complex also was involved in the induction of isp6+ during nitrogen starvation.

Mikrobiologiia, 2002 Sep-Oct, 71(5), 611 - 8
{Involvement of microbial alkyl hydroxybenzenes in the regulation of autolytic degradation of yeast cells.}; Karpekina TA et al.; A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d2 (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis) . Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d1, which belong to alkylhydroxybenzenes and fulfil functions of chemical chaperons . The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.

Mol Biol Evol, 2002 Dec, 19(12), 2039 - 50
Comparative genomics of the RBR family, including the Parkinson's disease-related gene parkin and the genes of the ariadne subfamily; Marin I et al.; Genes of the RBR family are characterized by the RBR signature (two RING finger domains separated by an IBR/DRIL domain) . The RBR family is widespread in eukaryotes, with numerous members in animals (mammals, Drosophila, Caenorhabditis) and plants (Arabidopsis) . But yeasts, such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, contain only two RBR genes . We determined the phylogenetic relationships and the most likely orthologs in different species of several family members for which functional data are available . These include: (1) parkin, whose mutations are involved in forms of familial Parkinson's disease; (2) the ariadne genes, recently characterized in Drosophila and mammals; (3) XYbp and Dorfin, two mammalian genes whose products interact with the centrosome; (4) XAP3, RBCK1, and UIP28, mammalian genes encoding Protein Kinase-C-binding proteins; and (5) ARA54, an androgen receptor coactivator . Because several of these genes are involved in ubiquitination, we used phylogenetic and structural analyses to explore the hypothesis that all RBR proteins might play a role in ubiquitination . We show that the involvement of RBR proteins in ubiquitination predates the animals-plants-fungi divergence . On the basis of the evidence provided by cases of gene fusion, we suggest that Ariadne proteins interact with cullin domain-containing proteins to form complexes with ubiquitin-ligase activity.

Mol Cell Biol, 2002 Dec, 22(24), 8491 - 505
The Ran GTPase system in fission yeast affects microtubules and cytokinesis in cells that are competent for nucleocytoplasmic protein transport; Salus SS et al.; Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport . These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells . The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum . Overexpression of Imp2p, which specifically destabilizes these structures, restores viability . (iii) Ran-dependent processes differ in their requirements for active Ran in vivo . Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust . Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.

J Mol Biol, 2002 Nov 29, 324(3), 399 - 407
The constraints protein-protein interactions place on sequence divergence; Teichmann SA; Structural analyses on a small number of protein families have shown that residues in protein interfaces are more conserved than average amino acid residues . This is also true of other ligand-binding and active site residues . This raises the question whether protein interactions place additional constraints on sequence divergence beyond this general background of functional restrictions on all different types of proteins . In order to investigate this, the sequence identities of Saccharomyces cerevisiae (SC) proteins to their Schizosaccharomyces pombe (SP) orthologues were used as a measure of sequence divergence . The SC proteins were divided into those in stable complexes, those that participate in transient interactions and the remaining proteins . All types of proteins can undergo extensive divergence: all three sequence identity distributions range from less than 20 to over 90% . However, overall, protein interactions do place additional constraints on sequence divergence and the distributions differ significantly: proteins not known to be involved in interactions have an average sequence identity of 38% while this value is 46% for proteins in stable complexes . Proteins that have transient interactions are intermediate between the two, with an average sequence identity of 41% . This trend is independent of whether the proteins are involved in informational functions (transcription, translation and replication) or not and of protein dispensability.

Mol Cells, 2002 Oct 31, 14(2), 312 - 7
A glucose-inducible gene in Schizosaccharomyces pombe, rrg1+, is involved in negative regulation of G2/M progression; Kim MJ et al.; A glucose-inducible gene in S . pombe is rrg1+ . Its mRNA level is rapidly decreased and increased by glucose-depletion and readdition, respectively . The previous study revealed that the rrg1+ expression was regulated by glucose-dependent mRNA stability control . To understand the significance of the glucose-dependent expression of rrg1+, the cellular function of rrg1+ was explored . Deletion of the rrg1+ gene from the haploid chromosome of S . pombe cells did not lead to cell lethality but brought about cell size reduction, which was accompanied by fast cell proliferation . In accordance with this result, the overexpression of the Rrgl protein under the control of the nmt1 promoter produced elongated cells of G2 delay, and consequently resulted in the slowing-down of cell proliferation . In addition, the rrg1+ mRNA level showed cell-cycle dependent changes, peaking at G2/M . These results demonstrate that Rrg1 might be involved in the negative regulation of cell proliferation and G2/M progression for cell size control.

Mol Cells, 2002 Oct 31, 14(2), 300 - 4
Regulation of the manganese-containing superoxide dismutase gene from fission yeast; Jung HI et al.; The manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates a potentially toxic superoxide radical into hydrogen peroxide and dioxygen . To study the regulation of the Schizosaccharomyces pombe MnSOD gene, the 943 bp upstream region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357, which resulted in the fusion plasmid pMS14 . Restriction mapping and nucleotide sequencing confirmed its construction . The synthesis of beta-galactosidase from the fusion plasmid was induced by aluminum chloride, menadione, cadmium chloride, manganese chloride, and hydrogen peroxide . It was also induced by NO-generating S-nitroso-N-acetylpenicillamine (SNAP) . However, cupric chloride and zinc chloride did not affect the synthesis of beta-galactosidase from the fusion plasmid . The beta-galactosidase synthesis appeared to be independent of the Pap1 protein . These results suggest that some metals, oxidative stress, and nitric oxide regulate the S . pombe MnSOD gene.

J Basic Microbiol, 2002, 42(6), 408 - 19
Chromate sensitivity in fission yeast is caused by increased glutathione reductase activity and peroxide overproduction; Pesti M et al.; The Cr(VI)-sensitive mutant chr-51S of the Schizosaccharomyces pombe accumulated chromate (CrO(4) (2-)) and reduced Cr(V) to much greater extent, than did its parental strain 6 chr(+) . Sublethal doses of K(2)Cr(2)O(7) did not induce any adaptive stress response, while H(2)O(2) or menadione pretreatment proved protective against the cell injuries caused by Cr(VI) . The intracellular GSH concentration in chr-51S cells was approximately half of that for the 6 chr(+) . Moreover, the glutathione disulfide reducing capacity of chr-51S was characterized by significantly increased glutathione reductase (GR) and glucose-6-phosphate dehydrogenase activities . These data strongly suggested that, instead of GSH, the NADPH/GR system was the major one-electron Cr(VI) reductant in vivo . The increased Cr(V) reduction in chr-51S mutant was accompanied by high intracellular superoxide and peroxide concentrations, required for formation of the hydroxyl radical ((*)OH) . The decreased intracellular GSH levels and the Cr(VI)-sensitive phenotype of the chr-51S cells indicates that GSH might act effectively against chromate by scavenging (*)OH.

Cell Chromosome . 2002 Sep 19;1(1):1.
Distinct functions of S . pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II; Sharif WD et al.; BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction . We report that these functions are separable in the fission yeast Schizosaccharomyces pombe . RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination . rec12-117, rec12-D15 (null), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products . Since S . pombe contains only three chromosome pairs, many of those aneuploid products were viable . The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products . The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II . Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors . CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I . Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I . In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

Biochem J, 2003 Feb 15, 370(Pt 1), 265 - 73
Degradation of human thymidine kinase is dependent on serine-13 phosphorylation: involvement of the SCF-mediated pathway; Ke PY et al.; The expression level of human thymidine kinase (hTK) is regulated in a cell-cycle-dependent manner . One of the mechanisms responsible for the fluctuation of TK expression in the cell cycle can be attributed to protein degradation during mitosis . Given the facts that cell-cycle-dependent proteolysis is highly conserved in all eukaryotes and yeast cells are an excellent model system for protein-degradation study, here we report on the use of Saccharomyces cerevisiae and Schizosaccharomyces pombe to investigate the degradation signal and mechanism required for hTK degradation . We found that the stability of hTK is significantly reduced in mitotic yeasts . Previously, we have observed that Ser-13 is the site of mitotic phosphorylation of hTK in HeLa cells {Chang, Huang and Chi (1998) J . Biol . Chem . 273, 12095-12100} . Here, we further provide evidence that the replacement of Ser-13 by Ala (S13A) renders hTK stable in S . pombe and S . cerevisiae . Most interestingly, we demonstrated that degradation of hTK is impaired in S . cerevisiae carrying a temperature-sensitive mutation in the proteasomal gene pre1-1 or the Skp1-Cullin-1/CDC53-F-box (SCF) complex gene cdc34 or cdc53, suggesting the contribution of the SCF-mediated pathway in hTK degradation . As phosphorylation is a prerequisite signal for SCF recognition, our results implied that phosphorylation of Ser-13 probably contributes to the degradation signal for hTK via the SCF-mediated proteolytic pathway.

Plant Physiol, 2002 Nov, 130(3), 1230 - 40
Molecular characterization of plant ubiquitin-conjugating enzymes belonging to the UbcP4/E2-C/UBCx/UbcH10 gene family; Criqui MC et al.; The anaphase promoting complex or cyclosome is the ubiquitin-ligase that targets destruction box-containing proteins for proteolysis during the cell cycle . Anaphase promoting complex or cyclosome and its activator (the fizzy and fizzy-related) proteins work together with ubiquitin-conjugating enzymes (UBCs) (E2s) . One class of E2s (called E2-C) seems specifically involved in cyclin B1 degradation . Although it has recently been shown that mammalian E2-C is regulated at the protein level during the cell cycle, not much is known concerning the expression of these genes . Arabidopsis encodes two genes belonging to the E2-C gene family (called UBC19 and UBC20) . We found that UBC19 is able to complement fission yeast (Schizosaccharomyces pombe) UbcP4-140 mutant, indicating that the plant protein can functionally replace its yeast ortholog for protein degradation during mitosis . In situ hybridization experiments were performed to study the expression of the E2-C genes in various tissues of plants . Their transcripts were always, but not exclusively, found in tissues active for cell division . Thus, the UBC19/20 E2s may have a key function during cell cycle, but may also be involved in ubiquitylation reactions occurring during differentiation and/or in differentiated cells . Finally, we showed that a translational fusion protein between UBC19 and green fluorescent protein localized both in the cytosol and the nucleus in stable transformed tobacco (Nicotiana tabacum cv Bright Yellow 2) cells.

J Biol Chem, 2003 Jan 10, 278(2), 991 - 7 Epub 2002 Nov 08.
Nak1, an essential germinal center (GC) kinase regulates cell morphology and growth in Schizosaccharomyces pombe; Huang TY et al.; We have identified and characterized Nak1, a 652- amino acid NH(2)-terminal kinase belonging to the group II germinal center kinase (GCK) family, in Schizosaccharomyces pombe . We found that nak1 is essential for cell proliferation . Furthermore, partial repression of nak1, under regulation of an integrated nmt1 promoter, resulted in an aberrant round cellular morphology, actin and microtubule mislocalization, slow growth, and cell division defects . Overexpression of either a kinase-inactive mutant (Nak1(K39R)) or the non-catalytic domain resulted in similar phenotypes, suggesting dominant-negative effects . By deletion analysis, we mapped the region responsible for this dominant-negative effect to the COOH-terminal 99 residues . Furthermore, we found that deletion of the COOH-terminal 99 residues inhibited Nak1 autophosphorylation, and expression of a partially inactive (Nak1(T171A)) or truncated (Nak1(1-562)) protein only weakly suppressed morphological and growth phenotypes, indicating that both kinase and COOH-terminal regions are important for Nak1 function . GFP-Nak1 localized uniformly throughout the cytoplasm, unlike many other proteins which influence cell polarity that preferentially localize to cell ends . Together, our results implicate Nak1 in the regulation of cell polarity, growth, and division and suggest that the COOH-terminal end plays an important role in the regulation of this kinase.

Folia Microbiol (Praha), 2002, 47(4), 401 - 6
Fluctuations during growth of the plasma membrane H(+)-ATPase activity of Saccharomyces cerevisiae and Schizosaccharomyces pombe; Nso E et al.; The plasma membrane H(+)-ATPase activity was determined under various growth conditions using the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . Under early batch-growth conditions in a rich medium, the budding yeast S . cerevisiae ATPase specific activity increased 2- to 3-fold during exponential growth . During late exponential growth, a peak of ATPase activity, followed by a sudden decrease, was observed and termed "growth-arrest control" . The growth arrest phenomenon of S . cerevisiae could not be related to the acidification of the culture medium or to glucose exhaustion in the medium or to variation of glucose activation of the H(+)-ATPase . Addition of ammonium to a proline minimum medium also stimulated transiently the ATPase activity of S . cerevisiae . Specific activity of the fission yeast S . pombe ATPase did not show a similar profile and steadily increased to reach a plateau in stationary growth . Under synchronous mitotic growth conditions, the ATPase activity of S . cerevisiae increased during the cell division cycle according to the "peak" type cycle, while that of S . pombe was of the "step" type.

Mol Microbiol, 2002 Nov, 46(4), 1095 - 105
The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host; Weiler F et al.; Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function . The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z . bailii virus M) that stably persists within the yeast cell cytosol . In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system . Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment . Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications . Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype . Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z . bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.

Mol Cell, 2002 Oct, 10(4), 907 - 16
Increased recombination intermediates and homologous integration hot spots at DNA replication origins; Segurado M et al.; We have studied the relationship between DNA replication and recombination in Schizosaccharomyces pombe using two-dimensional gel electrophoresis and functional analysis . Our results indicate that the activation of replication origins (ORIs) during the mitotic cell cycle is associated with the generation of joint DNA molecules between sister chromatids . The frequency of integration by homologous recombination was up to 50-fold higher than the genomic average within a narrow window overlapping the ars1 replication initiation site . The S . pombe rad22Delta, rhp51Delta, and rhp54Delta mutants, deficient in mitotic recombination, activate ORIs very inefficiently and accumulate abnormal replication intermediates . These results focus on the general link between replication and recombination previously found in several systems and suggest a role for recombination in the initiation of eukaryotic DNA replication.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Nov, 34(6), 737 - 42
Cloning and expression of the AtGRP9 gene related to salt stress tolerance in Arabidopsis thaliana; Tang YX et al.; The fission yeast Schizosaccharomyces pombe is a single cell eukaryotic organism . Taking advantage of the genetic simplicity of S.pombe and of the functional conservation of some salt tolerance-related proteins, this organism was used as a simple system to identify Arabidopsis thaliana salt-tolerance proteins by screening for cDNA clones that confer salt resistance when overexpressed . By this "cross-phylogenetic" screen, an A.thaliana gene was isolated encoding a glycine-rich protein, which was named AtGRP9 . Overexpression of AtGRP9 in yeast cells led to significant increase in salt tolerance of the yeast strain . Northern blotting analysis revealed that AtGRP9 was expressed specifically in the roots of A.thaliana and was induced by NaCl . The growth of some transgenic plants was easily controlled by using the medium with different NaCl concentrations . Taken together, these results show that AtGRP9 may be involved in the salt stress response in A.thaliana.

Mol Cell Biol, 2002 Dec, 22(23), 8366 - 74
Two ubiquitin-conjugating enzymes, Rhp6 and UbcX, regulate heterochromatin silencing in Schizosaccharomyces pombe; Choi ES et al.; Methylation of histone H3 has been linked to the assembly of higher-order chromatin structures . Very recently, several examples, including the Schizosaccharomyces pombe mating-type region, chicken beta-globin locus, and inactive X-chromosome, revealed that H3-Lys9-methyl (Me) is associated with silent chromatin while H3-Lys4-Me is prominent in active chromatin . Surprisingly, it was shown that homologs of Drosophila Su(var)3-9 specifically methylate the Lys9 residue of histone H3 . Here, to identify putative enzymes responsible for destabilization of heterochromatin, we screened genes whose overexpressions disrupt silencing at the silent mat3 locus in fission yeast . Interestingly, we identified two genes, rhp6(+) and ubcX(+) (ubiquitin-conjugating enzyme participating in silencing), both of which encode ubiquitin-conjugating enzymes . Their overexpression disrupted silencing at centromeres and telomeres as well as at mat3 . Additionally, the overexpression interfered with centromeric function, as confirmed by elevated minichromosome loss and antimicrotubule drug sensitivity . On the contrary, deletion of rhp6(+) or ubcX(+) enhanced silencing at all heterochromatic regions tested, indicating that they are negative regulators of silencing . More importantly, chromatin immunoprecipitation showed that their overexpression alleviated the level of H3-Lys9-Me while enhancing the level of H3-Lys4-Me at the silent regions . On the contrary, their deletions enhanced the level of H3-Lys9-Me while alleviating that of H3-Lys4-Me . Taken together, the data suggest that two ubiquitin-conjugating enzymes, Rhp6 and UbcX, affect methylation of histone H3 at silent chromatin, which then reconfigures silencing.

J Cell Sci, 2002 Dec 1, 115(Pt 23), 4629 - 39
The small GTPase Rho3 and the diaphanous/formin For3 function in polarized cell growth in fission yeast; Nakano K et al.; We identified a novel Rho gene rho3(+) and studied its interaction with diaphanous/formin for3(+) in the fission yeast Schizosaccharomyces pombe . Both rho3 null cells and for3 null cells showed defects in organization of not only actin cytoskeleton but also cytoplasmic microtubules (MTs) . rho3 for3 double null cells had defects that were more severe than each single null cell: polarized growth was deficient in the double null cells . Function of For3 needed the highly conserved FH1 and FH2 domains, an N-terminal region containing a Rho-binding domain, and the C-terminal region . For3 bound to active forms of both Rho3 and Cdc42 but not to that of Rho1 . For3 was localized as dots to the ends of interphase cells and to the mid-region in dividing cells . This localization was probably dependent on its interaction with Rho proteins . Overexpression of For3 produced huge swollen cells containing depolarized F-actin patches and thick cytoplasmic MT bundles . In addition, overexpression of a constitutively active Rho3Q71L induced a strong defect in cytokinesis . In conclusion, we propose that the Rho3-For3 signaling system functions in the polarized cell growth of fission yeast by controlling both actin cytoskeleton and MTs.

J Cell Sci, 2002 Dec 1, 115(Pt 23), 4555 - 64
Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest; Capasso H et al.; In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage . Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents . We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog . Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response . We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345 . Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage . These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome . These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.

Nucleic Acids Res, 2002 Nov 1, 30(21), 4781 - 92
The severe slow growth of Deltasrs2 Deltarqh1 in Schizosaccharomyces pombe is suppressed by loss of recombination and checkpoint genes; Maftahi M et al.; Our interest in the Schizosaccharomyces pombe RecQ helicase, rqh1+, led us to investigate the function of a related putative DNA helicase, srs2+ . We identified the srs2+ homolog in S.pombe, and found that srs2+ is not essential for cell viability . A Deltasrs2 Deltarqh1 double mutant grows extremely slowly with aberrant shaped cells and low viability . This slow growth does not appear to be related to stalled replication, as Deltasrs2 Deltarqh1 cells showed higher survival rates, compared with Deltarqh1, when stalled forks were increased by UV irradiation or hydroxy urea treatment . Consistent with this result, we found that Deltasrs2 Deltarqh1 cells progress through S-phase with a slight delay, but undergo a checkpoint-dependent arrest presumably at G2/M . Further, we found that Deltasrs2 Deltarqh1 slow growth is related to recombination, as loss of either the rhp51+ or rhp57+ recombination genes improves cell growth in the double mutant . Deltasrs2 is also synthetic lethal with Deltarhp54, another homologous recombination gene . This lethality is suppressed in a Deltarhp51 background . Together, these results demonstrate a clear genetic interaction between rqh1+, srs2+ and the genes of the homologous recombination pathway.

Nucleic Acids Res, 2002 Nov 1, 30(21), 4728 - 39
The fission yeast pfh1(+) gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase; Tanaka H et al.; The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease-helicase protein required for Okazaki fragment processing . To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele . One of the complementation groups of such suppressors defined a novel gene, pfh1(+), encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins . The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5' to 3' DNA helicase activity in vitro . Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo . Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature . This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the completion of DNA replication . Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.

Yeast, 2002 Nov, 19(15), 1335 - 50
Molecular and structural characterization of the spindle pole bodies in the fission yeast Schizosaccharomyces japonicus var japonicus; Horio T et al.; The structure and localization of the microtubule organization centres (MTOCs) of the fission yeast Schizosaccharomyces japonicus var . japonicus were examined by fluorescence microscopy and electron microscopy . Spindle pole bodies (SPBs), which are the fungal equivalent of centrosomes, of Sz . japonicus were visualized by immunofluorescent staining using a monoclonal anti-gamma-tubulin antibody . The behaviour of the SPBs during the cell cycle mostly coincided with previous reports on the most widely used fission yeast Schizosaccharomyces pombe . We cloned the gamma-tubulin gene from Sz . japonicus by PCR using redundant sets of primers corresponding to conserved regions of known gamma-tubulins . The predicted amino acid sequence of Sz . japonicus gamma-tubulin was most similar to the Sz . pombe gamma-tubulin . Under the electron microscope, the SPBs of Sz . japonicus were detected as electron-dense multilayered structures located just outside the nuclear envelope . The SPBs of Sz . japonicus were composed of three electron-dense layers and were surrounded by fuzzy material . Each layer showed structural changes according to the progression of the cell cycle . In mitotic cells, the SPBs were located on the fenestrae of the nuclear envelopes through which the mitotic spindle microtubules ran into the nucleoplasm . Our results show that Sz . japonicus is a very potent and attractive organism for the investigation of the microtubule nucleation system and morphogenesis in yeasts . The Accession No . for the nucleotide sequence of the Sz . japonicus gtb1(+) gene is AF159163 .

Genetics, 2002 Oct, 162(2), 689 - 703
The sal3(+) gene encodes an importin-beta implicated in the nuclear import of Cdc25 in Schizosaccharomyces pombe; Chua G et al.; In Schizosaccharomyces pombe, the nuclear accumulation of Cdc25 peaks in G2 and is necessary for the proper timing of mitotic entry . Here, we identify the sal3(+) gene product as an importin-beta homolog that participates in the nuclear import of Cdc25 . Loss of sal3(+) results in a cell cycle delay, failure to undergo G1 arrest under nitrogen-starvation conditions, and mislocalization of Cdc25 to the cytosol . Fusion of an exogenous classical nuclear localization sequence (cNLS) to Cdc25 restores its nuclear accumulation in a sal3 disruptant and suppresses the sal3 mutant phenotypes . In addition, we show that enhanced nuclear localization of Cdc25 at endogenous levels of expression advances the onset of mitosis . These results demonstrate that the nuclear translocation of Cdc25 is important for the timing of mitotic entry and that Sal3 plays an important role in this process.

Genetics, 2002 Oct, 162(2), 591 - 602
The 2.1-kb inverted repeat DNA sequences flank the mat2,3 silent region in two species of Schizosaccharomyces and are involved in epigenetic silencing in Schizosaccharomyces pombe; Singh G et al.; The mat2,3 region of the fission yeast Schizosaccharomyces pombe exhibits a phenomenon of transcriptional silencing . This region is flanked by two identical DNA sequence elements, 2.1 kb in length, present in inverted orientation: IRL on the left and IRR on the right of the silent region . The repeats do not encode any ORF . The inverted repeat DNA region is also present in a newly identified related species, which we named S . kambucha . Interestingly, the left and right repeats share perfect identity within a species, but show approximately 2% bases interspecies variation . Deletion of IRL results in variegated expression of markers inserted in the silent region, while deletion of the IRR causes their derepression . When deletions of these repeats were genetically combined with mutations in different trans-acting genes previously shown to cause a partial defect in silencing, only mutations in clr1 and clr3 showed additive defects in silencing with the deletion of IRL . The rate of mat1 switching is also affected by deletion of repeats . The IRL or IRR deletion did not cause significant derepression of the mat2 or mat3 loci . These results implicate repeats for maintaining full repression of the mat2,3 region, for efficient mat1 switching, and further support the notion that multiple pathways cooperate to silence the mat2,3 domain.

Microbiol Res, 2002, 157(3), 197 - 200
Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe; Massardo DR et al.; In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast . Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants . For one of them (mut-1) we show that the mutation is chromosomally encoded . The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.

Biochim Biophys Acta, 2002 Oct 11, 1578(1-3), 21 - 8
Regulation of ornithine decarboxylase by antizymes and antizyme inhibitor in zebrafish (Danio rerio); Hascilowicz T et al.; Mammalian polyamine synthesis is regulated by a unique feedback mechanism . When cellular polyamine levels increase, antizyme, an ornithine decarboxylase (ODC) inhibitory protein, is induced by polyamine-dependent translational frameshifting . Antizyme not only inhibits ODC, a key enzyme in polyamine synthesis, it also targets the enzyme degradation by the 26S proteasome . Furthermore, it suppresses cellular uptake of polyamines . Previously, we isolated two zebrafish antizymes with different expressions and activities . This suggested that a common feedback mechanism of polyamine metabolism might operate in mammals and zebrafish (Danio rerio) . In the present study, cDNAs of zebrafish ODC and antizyme inhibitor, another regulatory protein that inhibits antizyme action, were cloned . The presence of ODC and antizyme inhibitor mRNAs was confirmed by Northern blotting in embryos and adult fish, as well as in a zebrafish-derived cell line (BRF41) . The activity of the ODC cDNA expression product was inhibited by short and long zebrafish antizymes, and recombinant zebrafish antizyme inhibitor reversed this inhibition . In the BRF41 cells, the ODC half-life was considerably longer than that of mammalian ODC but shorter than that of Schizosaccharomyces pombe . Spermidine elicited a rapid decay of ODC activity and ODC protein in a protein synthesis-dependent manner.

Nat Struct Biol, 2002 Nov, 9(11), 828 - 32
Structure of the SET domain histone lysine methyltransferase Clr4; Min J et al.; Methylation of histone H3 lysine 9 is an important component of the 'histone code' for heterochromatic gene silencing . The SET domain-containing Clr4 protein, a close relative of Su(var)3-9 proteins in higher eukaryotes, specifically methylates lysine 9 of histone H3 and is essential for silencing in Schizosaccharomyces pombe . Here we report the 2.3 A resolution crystal structure of the catalytic domain of Clr4 . The structure reveals an overall fold rich in beta-strands, a potential active site consisting of a SAM-binding pocket, and a connected groove that could accommodate the binding of the N-terminal tail of histone H3 . The pre-SET motif contains a triangular zinc cluster coordinated by nine cysteines distant from the active site, whereas the post-SET region is largely flexible but proximal to the active site . The structure provides insights into the architecture of SET domain histone methyltransferases and establishes a paradigm for further characterization of the Clr4 family of epigenetic regulators.

Biochem Biophys Res Commun, 2002 Oct 25, 298(2), 185 - 92
High-affinity Src-SH2 ligands which do not activate Tyr(527)-phosphorylated Src in an experimental in vivo system; Mandine E et al.; The Src-SH2 domain has been determined to play a key role in many signaling pathways, especially in osteoclast-mediated bone resorption . Therefore, non-peptidic small molecules, mimicking the natural pYEEI peptide ligand, have been designed, to inhibit SH2-mediated protein-protein interactions and provide therapeutic treatment of certain diseases such as osteoporosis . However it has been shown in vitro that phosphopeptidic ligands of the SH2 domain are able to increase Src kinase activity by disrupting the intramolecular interactions between the Tyr(521)-phosphorylated C-terminal tail and the SH2 domain, thereby inducing a change from a "closed" inactive to an "open" active conformation of Src . Thus it was not clear whether non-peptidic ligands would limit their action to the inhibition of the signaling cascade by interfering with the intermolecular SH2 binding, or would activate the enzyme as do phosphopeptides . To address this question we have investigated the effects of a series of both peptidic and non-peptidic ligands of the SH2 domain on Src kinase activation, both in vitro in an ELISA based assay and in vivo using csk and src double transformed Schizosaccharomyces pombe . We found that, in the peptide series, the extent of c-Src activation is directly correlated to the respective binding affinity for Src-SH2 . By contrast such correlation is not valid for non-peptidic ligands, some high-affinity SH2 binders showing no detectable Src activation in vivo . These results have significant implications for the design of SH2 binders, as they allow a way to inhibit Src-SH2-mediated signal transduction in target cells, without activating Src in non-target cells, thereby reducing the possibility of side effects.

Biochem J, 2003 Feb 1, 369(Pt 3), 519 - 28
Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5',5"'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations; Ingram SW et al.; Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (Ap(n)A, n =5 or 6) and diphosphoinositol polyphosphates {diphosphoinositol pentakisphosphate (PP-InsP(5)) and bisdiphosphoinositol tetrakisphosphate ({PP}(2)-InsP(4))} in vitro . The in vivo substrates of Aps1 are unknown . We report here the identification of Ap(5)A, PP-InsP(5), {PP}(2)-InsP(4) and a novel diphosphoinositol polyphosphate ({PP}(x)-InsP(x)) in S . pombe using HPLC methods . Ap(5)A was present at 0.06 pmol/mg of protein (approx . 4 nM) . PP-InsP(5), {PP}(x)-InsP(x) and {PP}(2)-InsP(4) were present at 15 pmol/mg (approx . 1.1 microM), 15 pmol/mg (approx . 1.1 microM) and 30 pmol/mg (approx . 2.2 microM) respectively, while the intracellular concentration of InsP(6) was 0.5 nmol/mg of protein (approx . 36 microM) . Disruption of aps1 resulted in a 52% decrease in Ap(6)A hydrolase activity in vitro, no detectable change in the intracellular Ap(5)A concentration, and 3-fold increased intracellular concentrations of PP-Ins P(5) and {PP}(x)-InsP(x) . Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30 degrees C . Overexpression of aps1 via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity in vitro caused no detectable changes in the intracellular concentrations of {PP}(2)-InsP(4), {PP}(x)-InsP(x) or PP-InsP(5), but paradoxical increases of approx . 2.5- and 55-fold respectively in the intracellular Ap(5)A concentration . Overexpression of aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells . No phenotypic changes or changes in intracellular Ap(5)A occurred upon overexpression of aps1 E93Q, which encodes a mutated Aps1 lacking significant enzymic activity . We conclude that Aps1 degrades PP-InsP(5) and {PP}(x)-InsP(x) in vivo.

Eur J Biochem, 2002 Oct, 269(20), 5056 - 65
Cold induces stress-activated protein kinase-mediated response in the fission yeast Schizosaccharomyces pombe; Soto T et al.; In the fission yeast Schizosaccharomyces pombe the Wak1p/Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK) pathway relays environmental signals to the transcriptional machinery and modulates gene expression via a cascade of protein phosphorylation . Cells of S . pombe subjected to cold shock (transfer from 28 degrees C to 15 degrees C) transiently activated the Sty1p mitogen-activated protein kinase (MAPK) by phosphorylation . Induction of this response was completely abolished in cells disrupted in the upstream response regulator Mcs4p . The cold-triggered Sty1p activation was partially dependent on Wak1p MAPKKK and fully dependent on Wis1p MAPKK suggesting that the signal transmission follows a branched pathway, with the redundant MAPKKK Win1p as alternative transducer to Wis1p, which subsequently activates the effector Sty1p MAPK . Also, the bZIP transcription factor Atf1p became phosphorylated in a Sty1p-dependent way during the cold shock and this phosphorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+ and ntp1+ genes . Strains deleted in transcription factors Atf1p or Pcr1p were unable to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to do so . These data reveal that S . pombe responds to cold by inducing the SAPK pathway . However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth at low temperature by an as yet undefined mechanism.

Structure (Camb), 2002 Oct, 10(10), 1371 - 81
Studies on the reaction mechanism of riboflavin synthase: X-ray crystal structure of a complex with 6-carboxyethyl-7-oxo-8-ribityllumazine; Gerhardt S et al.; Riboflavin synthase catalyzes the disproportionation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione . We have determined the structure of riboflavin synthase from Schizosaccharomyces pombe in complex with the substrate analog, 6-carboxyethyl-7-oxo-8-ribityllumazine at 2.1 A resolution . In contrast to the homotrimeric solution state of native riboflavin synthase, we found the enzyme to be monomeric in the crystal structure . Structural comparison of the riboflavin synthases of S . pombe and Escherichia coli suggests oligomer contact sites and delineates the catalytic site for dimerization of the substrate and subsequent fragmentation of the pentacyclic intermediate . The pentacyclic substrate dimer was modeled into the proposed active site, and its stereochemical features were determined . The model suggests that the substrate molecule at the C-terminal domain donates a four-carbon unit to the substrate molecule bound at the N-terminal domain of an adjacent subunit in the oligomer.

EMBO J, 2002 Oct 15, 21(20), 5567 - 76
Site-specific ORC binding, pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins; Kong D et al.; Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins . Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Delta3 site, weakly to the Delta6 site and not at all to the remaining sequences . In situ, the footprint over Delta3 is extended during G(1) phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin . Moreover, this footprint extends into the adjacent Delta2 site, where leading strand DNA synthesis begins . Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre-replication complex and initiates DNA synthesis, plus an additional novel origin element (Delta9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle . Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa.

EMBO J, 2002 Oct 15, 21(20), 5516 - 26
Pre-spliceosome formation in S.pombe requires a stable complex of SF1-U2AF(59)-U2AF(23); Huang T et al.; We have initiated a biochemical analysis of splicing complexes in extracts from the fission yeast Schizosaccharomyces pombe . Extracts of S.pombe contain high levels of the spliceosome-like U2/5/6 tri-snRNP, which dissociates into mono-snRNPs in the presence of ATP, and supports binding of U2 snRNP to the 3' end of introns, yielding a weak ATP-independent E complex and the stable ATP-dependent complex A . The requirements for S.pombe complex A formation (pre-mRNA sequence elements, protein splicing factors, SF1/BBP and both subunits of U2AF) are analogous to those of mammalian complex A . The S.pombe SF1/BBP, U2AF(59) and U2AF(23) are tightly associated in a novel complex that is required for complex A formation . This pre-formed SF1- U2AF(59)-U2AF(23) complex may represent a streamlined mechanism for recognition of the branch site, pyrimidine tract and 3' splice site at the 3' end of introns.

Mol Microbiol, 2002 Oct, 46(1), 49 - 62
A nuclear protein in Schizosaccharomyces pombe with homology to the human tumour suppressor Fhit has decapping activity; Salehi Z et al.; A number of eukaryotic proteins are already known to orchestrate key steps of mRNA metabolism and translation via interactions with the 5' m7GpppN cap . We have characterized a new type of histidine triad (HIT) motif protein (Nhm1) that co-purifies with the cap-binding complex eIF4F of Schizosaccharomyces pombe . Nhm1 is an RNA-binding protein that binds to m7GTP-Sepharose, albeit with lower specificity and affinity for methylated GTP than is typical for the cap-binding protein known as eukaryotic initiation factor 4E . Sequence searches have revealed that proteins with strong sequence similarity over all regions of the new protein exist in a wide range of eukaryotes, yet none has been characterized up to now . However, other proteins that share specific motifs with Nhm1 include the human Fhit tumour suppressor protein and the diadenosine 5', 5"'-P1, P4-tetraphosphate asymmetrical hydrolase of S . pombe . Our experimental work also reveals that Nhm1 inhibits translation in a cell-free extract prepared from S . pombe, and that it is therefore a putative translational modulator . On the other hand, purified Nhm1 manifests mRNA decapping activity, yet is physically distinct from the Saccharomyces cerevisiae decapping enzyme Dcp1 . Moreover, fluorescence and immunofluorescence microscopy show that Nhm1 is predominantly, although not exclusively, nuclear . We conclude that Nhm1 has evolved as a special branch of the HIT motif superfamily that has the potential to influence both the metabolism and the translation of mRNA, and that its presence in S . pombe suggests the utilization of a novel decapping pathway.

Nat Cell Biol, 2002 Oct, 4(10), 816 - 20
Astral microtubules monitor metaphase spindle alignment in fission yeast; Oliferenko S et al.; Segregating genetic material along the longest axis of the cell ensures that there is a sufficient distance between daughter chromosomes at the point of cytokinesis . Monitoring the orientation of the mitotic spindle can be subjected to cell cycle controls . In the fission yeast Schizosaccharomyces pombe, the existence of such a cell-cycle checkpoint has been proposed to delay the metaphase to anaphase transition when spindle poles are not properly oriented with respect to the actomyosin ring . Here we show, by using a fission yeast mutant compromised in its assembly of astral microtubules, that in the absence of astral microtubules short metaphase spindles are unable to orient themselves with respect to the long axis of the cell and are delayed in spindle elongation . This astral defect engages a spindle orientation checkpoint because deletion of the transcription factor Atf1, which is involved in maintaining this checkpoint, allows misaligned asterless metaphase spindles to elongate . We propose that astral microtubules are involved directly in monitoring orientation of the metaphase spindle and in controlling the timing of elongation in fission yeast.

Biochim Biophys Acta, 2002 Sep 27, 1577(3), 395 - 400
Cloning, expression and functional characterization of Schizosaccharomyces pombe TFIIB; Tamayo E et al.; The transcription factor TFIIB has been identified and cloned from the yeast Schizosaccharomyces pombe . The cloned polypeptide is highly homologous to human TFIIB and to Saccharomyces cerevisiae TFIIB . S . pombe TFIIB is a 340-amino-acid-long protein and it possesses a repeated motif of 75 amino acids near the carboxy-terminal region . The purified recombinant protein is able to bind to the TBP-DNA promoter complex in gel retardation experiments . Recombinant S . pombe TFIIB is active in in vitro transcription assays, since it can complement the transcription activity of a S . pombe cell extract in which TFIIB was depleted by using antibodies.

Biochem Biophys Res Commun, 2002 Oct 4, 297(4), 854 - 62
Regulation and the role of Cu,Zn-containing superoxide dismutase in cell cycle progression of Schizosaccharomyces pombe; Lee J et al.; Regulation and the role of the sod1+ gene encoding CuZnSOD were investigated in fission yeast Schizosaccharomyces pombe . The amount of sod1+ mRNA decreased in the stationary phase, consistent with the decrease in enzyme activity . The transcript increased by treatment with oxidants such as H(2)O(2) and menadione (MD) . Induction by H(2)O(2) was rapid and transient, being dependent on Wis1-Spc1-Atf1 pathway of signal transduction, whereas induction by MD was slow and sustained longer, being independent of Wis1 pathway . Wis1 and Spc1 also turned out to down-regulate sod1+ gene at the stationary phase . Tetrad analysis following sod1+ gene disruption revealed that the sod1Delta cells were not viable, even on rich media . Repression of the sod1+ gene expression by thiamine through nmt1 promoter resulted in the arrest of cell cycle progression following S phase, possibly between G(2) and cytokinesis . The current and previous observations that the viability of Schizosaccharomyces pombe cells, unlike Saccharomyces cerevisiae, critically depends on the action of oxidative defense enzymes in the cytosol, such as CuZnSOD and glutathione reductase, suggest that S . pombe can serve as a good model system to study the effect of oxidative stress on cell proliferation.

J Biochem (Tokyo), 2002 Oct, 132(4), 635 - 41
Characterization of recombinant YakC of Schizosaccharomyces pombe showing YakC defines a new family of aldo-keto reductases; Morita T et al.; The yakC gene in Schizosaccharomyces pombe, which encodes yakC protein (YakC), a potential member of an aldo-keto reductase (AKR) family, was cloned and expressed in Escherichia coli cells . The recombinant YakC purified to homogeneity catalyzed the reduction of 2-nitrobenzaldehyde (k(cat), 44.1 s(-1), K(m), 0.185 +/- 0.018 mM), 2-phthalaldehyde (19.8, 0.333 +/- 0.032), and pyridine-2-aldehyde (7.64, 0.302 +/- 0.028) . Neither pyridoxal nor other compounds examined acted as substrates . NADPH, but not NADH, was a hydrogen donor . The enzyme is a monomer with a molecular weight of 38,900 +/- 6,600 (SDS-PAGE) . The amino acid sequence deduced from yakC showed the highest (34%) identity with that of pyridoxal reductase (AKR8A1) among the identified AKRs . Twenty-one function-unknown proteins showed 40% or higher identity to the deduced amino acid sequence: DR2261 protein of Deionococcus radiodurans showed the highest (50%) identity . The predicted secondary structure of YakC is similar to that of human aldose reductase, a representative AKR . The results establish YakC as the first member of a new AKR family, AKR13 . The yeast cells contained enzyme(s) other than YakC and pyridoxal reductase with the ability to reduce 2-nitrobenzaldehyde: total (100%) activity in the crude extract consisted of about 23% YakC, about 44% pyridoxal reductase, and about 33% other enzyme(s).

J Biochem (Tokyo), 2002 Oct, 132(4), 513 - 7
Yeast protein kinase C; Perez P et al.; The mammalian protein kinase C (PKC) superfamily plays regulatory roles in many different cellular processes . However, due to the many members that exist in cells, it is very complicated to present experimental evidence of the particular function of each member . In contrast, yeasts have only one or two PKC members and genetic tools have unveiled their role as main regulators of cell integrity . In this review, we will discuss the function of yeast protein kinase C homologues, their mechanism of activation and the signalling pathways that they regulate in two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe.

J Cell Sci, 2002 Nov 1, 115(Pt 21), 4081 - 96
Localization of the (1,3)beta-D-glucan synthase catalytic subunit homologue Bgs1p/Cps1p from fission yeast suggests that it is involved in septation, polarized growth, mating, spore wall formation and spore germination; Cortes JC et al.; Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)beta-D-glucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth . To study this possibility, double mutants of cps1-12 and cdc septation mutants were made . The double mutants displayed several hypersensitive phenotypes and altered actin distribution . Epistasis analysis showed mutations prior to septum synthesis were dominant over cps1-12, while cps1-12 was dominant over the end of septation mutant cdc16-116, suggesting Bgs1p is involved in septum cell-wall (1,3)beta-D-glucan synthesis at cytokinesis . We have studied the in vivo physiological localization of Bgs1p in a bgs1delta strain containing a functional GFP-bgs1(+) gene (integrated single copy and expressed under its own promoter) . During vegetative growth, Bgs1p always localizes to the growing zones: one or both ends during cell growth and contractile ring and septum during cytokinesis . Bgs1p localization in cdc septation mutants indicates that Bgs1p needs the medial ring and septation initiation network (SIN) proteins to localize properly with the rest of septation components . Bgs1p localization in the actin mutant cps8-188 shows it depends on actin localization . In addition, Bgs1p remains polarized in the mislocalized growing poles and septa of tea1-1 and tea2-1 mutants . During the meiotic process of the life cycle, Bgs1p localizes to the mating projection, to the cell-to-cell contact zone during cell fusion and to the neck area during zygote formation . Also, Bgs1p localization suggests that it collaborates in forespore and spore wall synthesis . During spore germination, Bgs1p localizes first around the spore during isotropic growth, then to the zone of polarized growth and finally, to the medial ring and septum . At the end of spore-cell division, the Bgs1p displacement to the old end occurs only in the new cell . All these data show that Bgs1p is localized to the areas of polarized cell wall growth and so we propose that it might be involved in synthesizing the lineal (1,3)beta-D-glucan of the primary septum, as well as a similar lineal (1,3)beta-D-glucan when other processes of cell wall growth or repair are needed.

Nat Struct Biol, 2002 Oct, 9(10), 719 - 24
The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization; Leung GC et al.; The small family of polo-like kinases (Plks) includes Cdc5 from Saccharomyces cerevisiae, Plo1 from Schizosaccharomyces pombe, Polo from Drosophila melanogaster and the four mammalian genes Plk1, Prk/Fnk, Snk and Sak . These kinases control cell cycle progression through the regulation of centrosome maturation and separation, mitotic entry, metaphase to anaphase transition, mitotic exit and cytokinesis . Plks are characterized by an N-terminal Ser/Thr protein kinase domain and the presence of one or two C-terminal regions of similarity, termed the polo box motifs . These motifs have been demonstrated for Cdc5 and Plk1 to be required for mitotic progression and for subcellular localization to mitotic structures . Here we report the 2.0 A crystal structure of a novel domain composed of the polo box motif of murine Sak . The structure consists of a dimeric fold with a deep interfacial cleft and pocket, suggestive of a ligand-binding site . We show that this domain forms homodimers both in vitro and in vivo, and localizes to centrosomes and the cleavage furrow during cytokinesis . The requirement of the polo domain for Plk family function and the unique physical properties of the domain identify it as an attractive target for inhibitor design.

J Biochem Mol Biol, 2002 Jul 31, 35(4), 409 - 13
Transcription of Schizosaccharomyces pombe thioltransferase-1 in response to stress conditions; Kim M et al.; Thioltransferase, also known as glutaredoxin, is an enzyme that catalyzes the reduction of a variety of disulfide compounds . In Schizosaccharomyces pombe, two thioltransferases were reported and the cDNA of one of the thioltransferases (thioltransferase-1) was cloned . Using a Northern blot assay, we investigated the thioltransferase transcription in response to various stress conditions . When the culture was shifted to a high temperature, the thioltransferase transcription was not significantly changed compared to the unshifted 30 degrees culture . Treatment of zinc chloride to exponentially-growing cells remarkably increased the thioltransferase transcription, whereas the treatment of mercury chloride greatly reduced the transcription . Treatment of hydrogen peroxide and cadmium chloride caused no significant effects on the transcription of the thioltransferase . These results suggest that the transcription of thioltransferase-1 in S . pombe is induced in response to metal stress that is caused by zinc chloride, but not in response to heat stress or oxidative stress that is caused by hydrogen peroxide.

J Biol Chem, 2002 Nov 29, 277(48), 46676 - 86 Epub 2002 Sep 18.
Ctr6, a vacuolar membrane copper transporter in Schizosaccharomyces pombe; Bellemare DR et al.; Aerobic organisms possess efficient systems for the transport of copper . This involves transporters that mediate the passage of copper across biological membranes to reach essential intracellular copper-requiring enzymes . In this report, we identify a new copper transporter in Schizosaccharomyces pombe, encoded by the ctr6(+) gene . The transcription of ctr6(+) is induced under copper-limiting conditions . This regulation is mediated by the cis-acting promoter element CuSE (copper-signaling element) through the copper-sensing transcription factor Cuf1 . An S . pombe strain bearing a disrupted ctr6Delta allele displays a strong reduction of copper,zinc superoxide dismutase activity . When the ctr6+ gene is overexpressed from the thiamine-inducible nmt1(+) promoter, the cells are unable to grow on medium containing exogenous copper . Surprisingly, this copper-sensitive growth phenotype is not due to an increase of copper uptake at the cell surface . Instead, copper delivery across the plasma membrane is reduced . Consistently, this results in repressing ctr4(+) gene expression . By using a functional ctr6(+) epitope-tagged allele expressed under the control of its own promoter, we localize the Ctr6 protein on the membrane of vacuoles . Furthermore, we demonstrate that Ctr6 is an integral membrane protein that can trimerize . Moreover, we show that Ctr6 harbors a putative copper-binding Met-X-His-Cys-X-Met-X-Met motif in the amino terminus, which is essential for its function . Our findings suggest that under conditions in which copper is scarce, Ctr6 is required as a means to mobilize stored copper from the vacuole to the cytosol.

Mol Cells, 2002 Aug 31, 14(1), 43 - 9
Regulation of Schizosaccharomyces pombe gene encoding copper/zinc superoxide dismutase; Lee YY et al.; Copper/zinc superoxide dismutase (Cu/Zn SOD) is an abundant enzyme that scavenges superoxide radicals . To independently examine the regulation of the Cu/Zn SOD gene of the fission yeast Schizosaccharomyces pombe, the 882 bp upstream region of the Cu/Zn SOD gene was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357R, which generated the fusion plasmid pSC601 . Cupric chloride (4.5 microM), aluminum chloride (10 mM), cadmium chloride (30 microM, 50 microM), mercuric chloride (1 microM), zinc chloride (11 mM), and hydrogen peroxide (0.3 mM) enhanced the synthesis of beta-galactosidase from the fusion plasmid . These results indicate that the expression of the S . pombe Cu/Zn SOD gene is, therefore, regulated by various metal ions, however superoxide-generating menadione did not affect the expression of the S . pombe Cu/Zn SOD gene . The expression of the S . pombe Cu/Zn SOD gene is also regulated by the transcription factor Pap1.

Mol Cell Biol, 2002 Oct, 22(20), 7134 - 46
Phosphorylation of eukaryotic initiation factor 2 by heme-regulated inhibitor kinase-related protein kinases in Schizosaccharomyces pombe is important for fesistance to environmental stresses; Zhan K et al.; Protein synthesis is regulated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in response to different environmental stresses . One member of the eIF2alpha kinase family, heme-regulated inhibitor kinase (HRI), is activated under heme-deficient conditions and blocks protein synthesis, principally globin, in mammalian erythroid cells . We identified two HRI-related kinases from Schizosaccharomyces pombe which have full-length homology with mammalian HRI . The two HRI-related kinases, named Hri1p and Hri2p, exhibit autokinase and kinase activity specific for Ser-51 of eIF2alpha, and both activities were inhibited in vitro by hemin, as previously described for mammalian HRI . Overexpression of Hri1p, Hri2p, or the human eIF2alpha kinase, double-stranded-RNA-dependent protein kinase (PKR), impeded growth of S . pombe due to elevated phosphorylation of eIF2alpha . Cells from strains with deletions of the hri1(+) and hri2(+) genes, individually or in combination, exhibited a reduced growth rate when exposed to heat shock or to arsenic compounds . Measurements of in vivo phosphorylation of eIF2alpha suggest that Hri1p and Hri2p differentially phosphorylate eIF2alpha in response to these stress conditions . These results demonstrate that HRI-related enzymes are not unique to vertebrates and suggest that these eIF2alpha kinases are important participants in diverse stress response pathways in some lower eukaryotes.

Mol Cell Biol, 2002 Oct, 22(20), 7105 - 19
The 14-3-3 proteins Rad24 and Rad25 negatively regulate Byr2 by affecting its localization in Schizosaccharomyces pombe; Ozoe F et al.; In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis . In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase {MAPK} kinase kinase) . Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells . In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells . Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells . Rad24 and Rad25 had no effect on the activity of constitutively active Byr1(S214DT218D) . Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined . The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation . Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants . In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein . Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation . Deletion of rad24 or rad25 fastened the timing of Byr2 translocation . Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.

Yeast, 2002 Sep 30, 19(13), 1139 - 51
Genome-wide search of Schizosaccharomyces pombe genes causing overexpression-mediated cell cycle defects; Tallada VA et al.; Genetic studies in yeasts enable an in vivo analysis of gene functions required for the cell division cycle (cdc genes) in eukaryotes . In order to characterize new functions involved in cell cycle regulation, we searched for genes causing cell division defects by overexpression in the fission yeast Schizosaccharomyces pombe . By using this dominant genetic strategy, 26 independent clones were isolated from a Sz . pombe cDNA library . The cloned cDNAs were partially sequenced and identified by computer analysis . The 26 clones isolated corresponded to 21 different genes . Among them, six were genes previously characterized in Sz . pombe, 11 were homologues to genes identified and characterized in other organisms, and four represented genes with unknown functions . In addition to known cell cycle regulators encoding inhibitory protein kinases (wee1, pka1) and DNA checkpoint proteins (Pcna, rad24), we have identified genes that are involved in a number of cellular processes . This includes protein synthesis (ribosomal proteins L7, L10, L29, L41, S6, S11, S17 and the PolyA-Binding Protein PABP), protein degradation (UBI3), nucleolar rRNA expression (fib, imp1, dbp2), cell cytoskeleton (act1) and glycolysis (pfk1) . The interference caused in the cell cycle by overexpression of these genes may elucidate novel mechanisms coupling different cellular processes with the control of the cell division . The effect caused by some of them is described in more detail .

Nucleic Acids Res, 2002 Sep 15, 30(18), 4022 - 31
Topoisomerase III is required for accurate DNA replication and chromosome segregation in Schizosaccharomyces pombe; Oh M et al.; The deletion of the top3(+) gene leads to defective nuclear division and lethality in Schizosaccharo myces pombe . This lethality is suppressed by concomitant loss of rqh1(+), the RecQ helicase . Despite extensive investigation, topoisomerase III function and its relationship with RecQ helicase remain poorly understood . We generated top3 temperature-sensitive (top3-ts) mutants and found these to be defective in nuclear division and cytokinesis and to be sensitive to DNA-damaging agents . A temperature shift of top3-ts cells to 37 degrees C, or treatment with hydroxyurea at the permissive temperature, caused an increase in 'cut' (cell untimely torn) cells and elevated rates of minichromosome loss . The viability of top3-ts cells was decreased by a temperature shift during S-phase when compared with a similar treatment in other cell cycle stages . Furthermore, the top3-ts mutant was not sensitive to M-phase specific drugs . These results indicate that topoisomerase III may play an important role in DNA metabolism during DNA replication to ensure proper chromosome segregation . Our data are consistent with Top3 acting downstream of Rqh1 to process the toxic DNA structure produced by Rqh1.

EMBO Rep, 2002 Oct, 3(10), 975 - 81 Epub 2002 Sep 13.
Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha; Muchardt C et al.; In mammalian cells, as in Schizosaccharomyces pombe and Drosophila, HP1 proteins bind histone H3 tails methylated on lysine 9 (K9) . However, whereas K9-methylated H3 histones are distributed throughout the nucleus, HP1 proteins are enriched in pericentromeric heterochromatin . This observation suggests that the methyl-binding property of HP1 may not be sufficient for its heterochromatin targeting . We show that the association of HP1alpha with pericentromeric heterochromatin depends not only on its methyl-binding chromo domain but also on an RNA-binding activity present in the hinge region of the protein that connects the conserved chromo and chromoshadow domains . Our data suggest the existence of complex heterochromatin binding sites composed of methylated histone H3 tails and RNA, with each being recognized by a separate domain of HP1alpha.

Curr Genet, 2002 Sep, 41(6), 367 - 78 Epub 2002 Aug 01.
The genetic complexity of chitin synthesis in fungi; Roncero C; Chitin synthesis is a process maintained across the fungal kingdom that, thanks to the power of genetic manipulation of yeast cells, is now beginning to be understood . Chitin synthesis is based on the regulation of distinct chitin synthase isoenzymes whose number ranges from one in Schizosaccharomyces pombe to seven in some filamentous fungi, such as Aspergillus fumigatus . This high diversity makes it difficult to find a unique model of regulation . However, the results available suggest common themes in regulation . The arrival of the genomic era, together with the development of fungal genetic technology should allow experimental approaches to this process.

Biol Chem, 2002 Jun, 383(6), 983 - 7
Functional genomics of C190T single nucleotide polymorphism in human N-acetyltransferase 2; Zhu Y et al.; N-acetyltransferase 2 (NAT2) catalyzes N-acetylation and O-acetylation of many drugs and environmental carcinogens . Genetic polymorphisms in the NAT2 gene have been associated with differential susceptibility to cancers and drug toxicity from these compounds . Single nucleotide polymorphisms (SNPs) have been identified in the human NAT2 coding region . A new allele, NAT2*19, possessing the C190T (R64W) exchange, was recently identified . In order to understand the effect of this new SNP, recombinant NAT2*4 (reference) and NAT2*19 were expressed in yeast (Schizosaccharomyces pombe) . The C190T (R64W) SNP in NAT2*19 caused substantial reduction in the NAT2 protein level and stability, but did not cause significant reduction in transformation efficiency or mRNA level . The enzymatic activities for N-acetylation of two arylamine carcinogens (2-aminofluorene, 4-aminobiphenyl), and a sulfonamide drug (sulfamethazine) were over 100-fold lower for NAT2 19 compared to reference NAT2 4 . Kinetic studies showed a reduction in Vmax but no significant change in substrate Km . In addition, the SNP caused significant reduction in the O-acetylation of the N-hydroxy-2-amino-1-methyl-6-phenylimidazo {4,5-b} pyridine . These results show that NAT2*19 possessing the C190T (R64W) SNP encodes a slow acetylator phenotype for both N- and O-acetylation, due to a reduction in the amount and stability of the NAT2 19 allozyme.

Mol Biol Cell, 2002 Sep, 13(9), 2977 - 89
Role of fission yeast Tup1-like repressors and Prr1 transcription factor in response to salt stress; Greenall A et al.; In Schizosaccharomyces pombe, the Sty1 mitogen-activated protein kinase and the Atf1 transcription factor control transcriptional induction in response to elevated salt concentrations . Herein, we demonstrate that two repressors, Tup11 and Tup12, and the Prr1 transcription factor also function in the response to salt shock . We find that deletion of both tup genes together results in hypersensitivity to elevated cation concentrations (K(+) and Ca(2+)) and we identify cta3(+), which encodes an intracellular cation transporter, as a novel stress gene whose expression is positively controlled by the Sty1 pathway and negatively regulated by Tup repressors . The expression of cta3(+) is maintained at low levels by the Tup repressors, and relief from repression requires the Sty1, Atf1, and Prr1 . Prr1 is also required for KCl-mediated induction of several other Sty1-dependent genes such as gpx1(+) and ctt1(+) . Surprisingly, the KCl-mediated induction of cta3(+) expression occurs independently of Sty1 in a tup11Delta tup12Delta mutant and so the Tup repressors link induction to the Sty1 pathway . We also report that in contrast to a number of other Sty1- and Atf1-dependent genes, the expression of cta3(+) is induced only by high salt concentrations . However, in the absence of the Tup repressors this specificity is lost and a range of stresses induces cta3(+) expression.

Mol Genet Genomics, 2002 Aug, 267(6), 792 - 6 Epub 2002 Jul 03.
Poly(A) site choice during mRNA 3'-end formation in the Schizosaccharomyces pombe wos2 gene; Munoz MJ et al.; In the fission yeast Schizosaccharomyces pombe, the wos2 gene encodes p23, a highly conserved protein which functions as a co-chaperone for the heat shock protein Hsp90 . This p23 protein binds to Hsp90, but its activities and regulatory mechanisms are still unclear . Northern analysis has shown that the wos2 gene produces three transcripts of about 1.1, 0.9 and 0.8 kb, which are expressed differentially depending on the growth temperature . The largest and the smallest transcripts were most abundant at 25 degrees C, whereas the 0.9-kb transcript predominated at 37 degrees C . A time-course analysis indicated that this 0.9-kb species rapidly increased in abundance after a shift from 25 degrees C to 37 degrees C, reaching a maximum after 15 min . A shift back to 25 degrees C resulted in a decline in the amount of this transcript, albeit at a slower rate . Expression analysis of wos2:ura4 and nmt1:wos2 constructs showed that the 3' untranslated region of wos2 alone directs the formation of these multiple, discrete wos2 mRNAs . Sequence analysis of cDNAs derived from these mRNAs showed that the use of different polyadenylation sites results in the production of the three differently sized wos2 transcripts . In the case of the 0.9- and 0.8-kb mRNA species, these sites lie in a predicted hairpin loop in the mRNA, suggesting that polyadenylation signals in wos2 transcripts may be mediated by RNA secondary structure . The possibility that differential thermal stability of these hairpin structures could influence polyadenylation site choice during formation of the 3'-ends of the mRNAs is discussed.

Genes Genet Syst, 2002 Jun, 77(3), 147 - 57
The fission yeast RPA21 subunit of RNA polymerase I: an evolutionarily conserved subunit interacting with ribosomal DNA (rDNA) transcription factor Rrn3p for recruitment to rDNA promoter; Imazawa Y et al.; Recruitment of RNA polymerases to the cognate promoter is a key step for the transcription initiation of specific genes in eukaryotes . Recently, RNA polymerase I (pol I) of Saccharomyces cerevisiae was shown to be recruited to the rDNA promoter via interaction between Rrn3p, a conserved transcription factor for rDNA, and A43, a subunit specific to pol I . The question of whether a similar interaction for pol I recruitment is conserved in other eukaryotes remains to be answered . We show here that Schizosaccharomyces pombe rpa21(+) encodes a protein of apparent molecular mass 21 kD which shows 36% identity to the A43 subunit of pol I in S . cerevisiae, and that rpa21(+) is essential for cell growth . To gain further insight into the functions of RPA21, we isolated a total of 22 temperature-sensitive (ts) mutants of rpa21(+) and found that most of the substitutions causing the ts phenotype are clustered in the N-terminal half of RPA21 . The ts mutants showed a markedly reduced amount of primary transcripts of rDNA immediately after temperature shift-up . Over-expression of S . pombe rrn3(+) in the ts mutants suppressed the growth defect in an allele-specific manner . Therefore, we conclude that S . pombe RPA21 plays a functional role similar to that of A43 in S . cerevisiae and that the mechanism of recruitment of pol I to the rDNA promoter by the interaction of a specific pol I subunit with Rrn3p is evolutionarily conserved.

Biol Cell, 2002 Jun, 94(3), 127 - 37
Mac1, a fission yeast transmembrane protein localizing to the poles and septum, is required for correct cell separation at high temperatures; Grandin N et al.; Schizosaccharomyces pombe represents a genetic model system for studying cell polarity and division in eukaryotes . We report here the identification of Mac1, a novel fission yeast protein that localized predominantly to the cell tips and septum . Sequences corresponding to roughly the first 180 amino acids of Mac1, which exhibited weak homology to the transmembrane domains of the Aspergillus Pall protein {Mol . Microbiol . 30 (1998) 259}, were found to specify localization to the cell periphery . The other 574 amino acids of Mac1 localized to the cytoplasm when expressed alone, thus suggesting that the N-terminal part of Mac1 functions as a plasma membrane anchor for the rest of the protein . In pom1 null mutant cells, which never switch from unipolar to bipolar growth but, instead, grow exclusively at the randomly chosen end {Genes Dev . 12 (1998) 1356}, Mac1 was, nevertheless, found at both poles, thus suggesting that Mac1 does not specifically localize to the sites of growth . mac1 null mutant cells had no overt phenotype at 22-32 degrees C, but, nevertheless, displayed a marked decrease in viability at 34-36 degrees C, accompanied by severe separation defects . Overexpression of mac1 resulted in similar defects . Our data suggest that a correct dosage of Mac1 is needed for correct cell separation at elevated temperatures of growth.

Prog Nucleic Acid Res Mol Biol, 2002, 72, 41 - 94
Initiation of eukaryotic DNA replication: regulation and mechanisms; Nasheuer HP et al.; The accurate and timely duplication of the genome is a major task for eukaryotic cells . This process requires the cooperation of multiple factors to ensure the stability of the genetic information of each cell . Mutations, rearrangements, or loss of chromosomes can be detrimental to a single cell as well as to the whole organism, causing failures, disease, or death . Because of the size of eukaryotic genomes, chromosomal duplication is accomplished in a multiparallel process . In human somatic cells between 10,000 and 100,000 parallel synthesis sites are present . This raises fundamental problems for eukaryotic cells to coordinate the start of DNA replication at each origin and to prevent replication of already duplicated DNA regions . Since these general phenomena were recognized in the middle of the 20th century the regulation and mechanisms of the initiation of eukaryotic DNA replication have been intensively investigated . These studies were carried out to find the essential factors involved in the process and to determine their functions during DNA replication . These studies gave rise to a model of the organization and the coordination of DNA replication within the eukaryotic cell . The elegant experiments carried out by Rao and Johnson (1970) (1), who fused cells in different phases of the cell cycle, showed that G1 cells are competent for replication of their chromosomes, but lack a specific diffusible factor required to activate their replicaton machinery and showed that G2 cells are incompetent for DNA replication . These findings suggested that eukaryotic cells exist in two states . In G1 phase, cells are competent to initiate DNA replication, which is subsequently triggered in S phase . After completion of S phase, cells in G2 are no longer able to initiate DNA replication and they require a transition through mitosis to reenable initiation of DNA replication to take place in the next S phase . The Xenopus cell-free replication system has proved a good model system in which to study DNA replication in vitro as well as the mechanism preventing rereplication within a single cell cycle (2) . Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor, which binds to chromatin before the G1-S transition and which is displaced during replication (2, 3) . These results were supported by genetic and biochemical experiments in Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) (4, 5) . The investigation of cell division cycle mutants and the budding yeast origin of replication resulted in the concept of a prereplicative and a postreplicative complex of initiation proteins (6-9) . These three individual concepts have recently started to merge and it has become obvious that initiation in eukaryotes is generally governed by the same ubiquitous mechanisms.

Nature, 2002 Sep 5, 419(6902), 82 - 6
Actin dynamics in the contractile ring during cytokinesis in fission yeast; Pelham RJ et al.; Cytokinesis in many eukaryotes requires a contractile ring of actin and myosin that cleaves the cell in two . Little is known about how actin filaments and other components assemble into this ring structure and generate force . Here we show that the contractile ring in the fission yeast Schizosaccharomyces pombe is an active site of actin assembly . This actin polymerization activity requires Arp3, the formin Cdc12, profilin and WASP, but not myosin II or IQGAP proteins . Both newly polymerized actin filaments and pre-existing actin cables can contribute to the initial assembly of the ring . Once formed, the ring remains a dynamic structure in which actin and other ring components continuously assemble and disassemble from the ring every minute . The rate of actin polymerization can influence the rate of cleavage . Thus, actin polymerization driven by the Arp2/3 complex and formins is a central process in cytokinesis . Our studies show that cytokinesis is a more dynamic process than previously thought and provide a perspective on the mechanism of cell division.

J Cell Biol, 2002 Sep 2, 158(5), 901 - 14 Epub 2002 Sep 03.
The CeCDC-14 phosphatase is required for cytokinesis in the Caenorhabditis elegans embryo; Gruneberg U et al.; In all eukaryotic organisms, the physical separation of two nascent cells must be coordinated with chromosome segregation and mitotic exit . In Saccharomyces cerevisiae and Schizosaccharomyces pombe this coordination depends on a number of genes that cooperate in intricate regulatory pathways termed mitotic exit network and septum initiation network, respectively . Here we have explored the function of potentially homologous genes in a metazoan organism, Caenorhabditis elegans, using RNA-mediated interference . Of all the genes tested, only depletion of CeCDC-14, the C . elegans homologue of the budding yeast dual-specificity phosphatase Cdc14p (Clp1/Flp1p in fission yeast), caused embryonic lethality . We show that CeCDC-14 is required for cytokinesis but may be dispensable for progression of the early embryonic cell cycles . In response to depletion of CeCDC-14, embryos fail to establish a central spindle, and several proteins normally found at this structure are mislocalized . CeCDC-14 itself localizes to the central spindle in anaphase and to the midbody in telophase . It colocalizes with the mitotic kinesin ZEN-4, and the two proteins depend on each other for correct localization . These findings identify the CDC14 phosphatase as an important regulator of central spindle formation and cytokinesis in a metazoan organism.

Biotechnol Bioeng, 2002 Oct 5, 80(1), 22 - 32
Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells; Isoai A et al.; The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe . A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA) . Biocatalysts were prepared using ASP327-10 and T . variabilis, and evaluated to demonstrate the feasibility of recombinant S . pombe for industrial application . The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment . Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T . variabilis decreased to 20-30% . Heat treatment was required before cell fixation by GDA to inactivate the catalase in S . pombe . This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S . pombe . To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis . Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S . pombe even in the absence of G418 . The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required . The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength . This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme .

Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12114 - 9 Epub 2002 Sep 03.
Dual localization of human DNA topoisomerase IIIalpha to mitochondria and nucleus; Wang Y et al.; The human TOP3alpha gene encoding DNA topoisomerase IIIalpha (hTop3alpha) has two potential start codons for the synthesis of proteins 1,001 and 976 aa residues in length . The sequence of the N-terminal region of the 1,001-residue form resembles signal peptide sequences for mitochondrial import, and fluorescence microscopy shows that the addition of as few as the first 34 aa of the 1,001-residue form of hTop3alpha to a green fluorescent protein can direct the chimeric protein to mitochondria . Biochemical analyses of subcellular fractions of HeLa cells further demonstrate that a distinctive fraction of hTop3alpha is present inside mitochondria, as evidenced by its resistance to proteinase K . This fraction constitutes several percent of the enzyme in the nuclear fraction, suggesting that the distribution of the mitochondrial and nuclear forms of hTop3alpha is roughly in proportion to the DNA contents of these cellular compartments . The presence of a type IA DNA topoisomerase in the mitochondria of other eukaryotes is supported by an examination of the amino acid sequences of mouse and Drosophila DNA topoisomerase IIIalpha and Schizosaccharomyces pombe DNA topoisomerase III . Given the presence of at least one type IA DNA topoisomerase in all forms of life examined to date, the finding of a type IA enzyme in mitochondria further supports the notion of a key role of such enzymes in DNA transactions.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 553 - 9
{Cloning of Candida albicans CaBEM1 and its role in filamentous growth of Saccharomyces cerevisiae}; Zhou Z et al.; The pathogenic fungus Candida albicans has a dimorphic transition in various environmental conditions . Many regulatory factors and several transduction pathways have been identified in controlling filamentous growth . G(1) cyclins Cln1 and Cln2 have been reported as involved in the control of morphogenesis in Saccharomyces cerevisiae . Diploid cln1/cln1 and cln2/cln2 strains completely lost the ability to form pseudohyphae . A C . albicans genomic DNA library was introduced into cln1/cln1 and cln2/cln2 mutants to screen genes which could complement its filamentous growth defect . In this screening a BEM1 homolog, CaBEM1, was identified . The CaBEM1 gene has an ORF of 1 899 bp, encoding a putative protein of 632 amino acids . The CaBem1 protein shares highest homology in amino acids with Bem1 (38%) of S . cerevisiae and Scd2 (32%) of Schizosaccharomyces pombe . Sequence analysis showed that the CaBem1 contains two N-terminal SH3 domains, a PX domain and a C-terminal PB1 domain . It is believed these domains are required for binding to proteins involved in polarized growth in S . cerevisiae and S . pombe . Ectopic expression of the CaBEM1 gene in diploid S . cerevisiae suppressed defects in filamentous growth of some mutants under nitrogen starvation conditions . This suppression bypassed MAPK pathway and cAMP/PKA pathway in filamentous growth . These results suggest that the CaBem1 protein may be a downstream component of these two signal transduction pathways of filament formation.

Genetics, 2002 Aug, 161(4), 1437 - 52
Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres; Nakamura TM et al.; Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance . Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance . On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization . Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway . However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes . Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection . Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres . Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres . Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.

Science, 2002 Sep 13, 297(5588), 1833 - 7 Epub 2002 Aug 22.
Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi; Volpe TA et al.; Eukaryotic heterochromatin is characterized by a high density of repeats and transposons, as well as by modified histones, and influences both gene expression and chromosome segregation . In the fission yeast Schizosaccharomyces pombe, we deleted the argonaute, dicer, and RNA-dependent RNA polymerase gene homologs, which encode part of the machinery responsible for RNA interference (RNAi) . Deletion results in the aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats . This is accompanied by transcriptional de-repression of transgenes integrated at the centromere, loss of histone H3 lysine-9 methylation, and impairment of centromere function . We propose that double-stranded RNA arising from centromeric repeats targets formation and maintenance of heterochromatin through RNAi.

J Mol Evol, 2002 Sep, 55(3), 302 - 13
Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease; Pellenz S et al.; The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element . The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF) . The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs . In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing . However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity . We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli . Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro . Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.

J Cell Sci, 2002 Sep 15, 115(Pt 18), 3575 - 86
Cytokinetic actomyosin ring formation and septation in fission yeast are dependent on the full recruitment of the polo-like kinase Plo1 to the spindle pole body and a functional spindle assembly checkpoint; Mulvihill DP et al.; In dividing cells, the assembly and contraction of the cytokinetic actomyosin ring (CAR) is precisely coordinated with spindle formation and chromosome segregation . Despite having a cell wall, the fission yeast Schizosaccharomyces pombe forms a CAR reminiscent of the structure responsible for the cleavage of cells with flexible boundaries . We used the myo2-gc fission yeast strain in which the chromosomal copy of the type II myosin gene, myo2(+), is fused to the gene encoding green fluorescent protein (GFP) to investigate the dynamics of Myo2 recruitment to the cytokinetic actomyosin ring in living cells . Analysis of CAR formation in relation to spindle pole body (SPB) and centromere separation enabled us to pinpoint the timing of Myo2 recruitment into a stable CAR structure to the onset of anaphase A . Depolymerisation of actin with latrunculin B did not affect the timing of Myo2 accumulation at the cell equator (although Myo2 no longer formed a ring), whereas depolymerisation of microtubules with either thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC) resulted in a delay of up to 90 minutes in CAR formation . Microtubule depolymerisation also delayed the localisation of other CAR components such as actin and Mid1/Dmf1 . The delay of cytokinesis in response to loss of microtubule integrity was abolished in cells lacking the spindle assembly checkpoint protein Mad2 or containing non-functional Cdc16, a component of the fission yeast septation initiation network (SIN) . The delay was also abolished in cells lacking Zfs1, a component of the previously described S . pombe cytokinesis checkpoint . Recruitment of the polo-related kinase, Plo1, a key regulator of CAR formation, to the SPBs was substantially reduced in TBZ in a Mad2-dependent manner . Loading of Cdc7, a component of the SIN and downstream of Plo1 in the cytokinesis pathway, onto the the SPBs was also delayed in TBZ to the same extent as CAR formation . We conclude that CAR formation is subject to regulation by the spindle assembly checkpoint via the loading of Plo1 onto the SPBs and the consequent activation of the SIN.

Biochem J, 2002 Dec 1, 368(Pt 2), 527 - 34
Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation; Tang Z et al.; The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans . SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila . However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts . In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation . By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen . This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay . We also found that the Srp1-Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1) . Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex . These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.

Yeast, 2002 Jun 15, 19(8), 703 - 11
Schizosaccharomyces pombe Pmf1p is structurally and functionally related to Mmf1p of Saccharomyces cerevisiae; Marchini A et al.; A novel family of small proteins, termed p14.5 or YERO57c/YJGFc, has been identified . Independent studies indicate that p14.5 family members are multifunctional proteins involved in several pathways, e.g . regulation of translation or activation of the protease mu-calpain . We have previously shown that Mmf1p, a p14.5 of the budding yeast Saccharomyces cerevisiae, is localized in the mitochondria and influences mitochondrial DNA stability . In addition, we have demonstrated that Mmf1p is functionally related to p14.5 of mammalian cells . To explore further the evolutionary conservation of the mitochondrial function(s) of the p14.5s we have extended our study to the fission yeast, Schizosaccharomyces pombe . In this organism two p14.5 homologous proteins are present: Pmf1p (pombe mitochondrial factor 1) and Hpm1p (homologous Pmf1p factor 1) . We have generated a specific Pmf1p antibody, which recognizes a single band of approximately 15 kDa in total cellular extracts . Cellular fractionation experiments indicate that Pmf1p localizes in the mitochondria as well as in the cytoplasm . We also show that Pmf1p shares several properties of S . cerevisiae Mmf1p . Indeed, Pmf1p restores the wild-type phenotype when expressed in delta mmf1 S . cerevisiae cells . Deletion of the leader sequence of Pmf1p abrogates its ability to localize in mitochondria and to functionally replace Mmf1p . Thus, these data together with our previous study show that the mitochondrial function(s) of the p14.5 family members are highly conserved in eukaryotic cells.

Curr Genet, 2002 Aug, 41(5), 342 - 8 Epub 2002 Jul 23.
Fission yeast Cdc23 interactions with DNA replication initiation proteins; Hart EA et al.; Schizosaccharomyces pombe Cdc23 is an essential DNA replication protein, conserved in eukaryotes and functionally homologous with Saccharomyces cerevisiae Dna43 (Mcm10) . We sought evidence for interactions between Cdc23 and the MCM2-7 complex, a component of both the pre-replicative complex and the replication fork . Cdc23 shows genetic interactions with four MCM subunits: cdc23-M36 and cdc23-1E2 alleles both show synthetic phenotypes with mcm2 (cdc19-P1) and mcm6 (mis5-268), and cdc23-M36 is synthetically lethal with mcm4 (cdc21-K46) and with mcm5 (nda4-108) . The wild-type cdc23 gene on multicopy plasmids can partially suppress temperature-dependent defects in mcm5 (nda4-108) . Two-hybrid analysis demonstrates interactions at the protein-protein level between Cdc23 and Mcm4, Mcm5 and Mcm6 . Cdc23 also interacts with four subunits of the Schizosaccharomyces pombe origin recognition complex (ORC) in yeast two-hybrid assay: Orc1, Orc2, Orc5 and Orc6 . We found no evidence for interaction between Cdc23 and the MCM recruitment factor Cdc18 (the homologue of Saccharomyces cerevisiae Cdc6) . Unlike Cdc18, Cdc23 mRNA shows no significant fluctuation in level through the cell cycle . These data suggest that fission yeast Cdc23 is an MCM-associated factor which has a role in the initiation of DNA replication.

Mol Biol Cell, 2002 Aug, 13(8), 2571 - 84
Crp79p, like Mex67p, is an auxiliary mRNA export factor in Schizosaccharomyces pombe; Thakurta AG et al.; The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex . We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe . Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus . Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm . Like Mex67p, Crp79-GFP binds poly(A)(+) RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent . All of these properties are essential for Crp79p to promote mRNA export . Crp79p import into the nucleus depends on the Ran system . A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p . Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p . We propose that Crp79p is a nonessential mRNA export carrier in S . pombe.

J Biol Chem, 2002 Oct 25, 277(43), 41183 - 91 Epub 2002 Aug 13.
Characterization of a Schizosaccharomyces pombe strain deleted for a sequence homologue of the human damaged DNA binding 1 (DDB1) gene; Zolezzi F et al.; Human damaged DNA-binding protein (DDB) is a heterodimer of p48/DDB2 and p127/DDB1 subunits . Mutations in DDB2 are responsible for Xeroderma Pigmentosum group E, but no mutants of mammalian DDB1 have been described . To study DDB1, the Schizosaccharomyces pombe DDB1 sequence homologue (ddb1(+)) was cloned, and a ddb1 deletion strain was constructed . The gene is not essential; however, mutant cells showed a 37% impairment in colony-forming ability, an elongated phenotype, and abnormal nuclei . The ddb1Delta strain was sensitive to UV irradiation, X-rays, methylmethane sulfonate, and thiabendazole, and these sensitivities were compared with those of the well characterized rad13Delta, rhp51Delta, and cds1Delta mutant strains . Ddb1p showed nuclear and nucleolar localization, and the aberrant nuclear structures observed in the ddb1Delta strain suggest a role for Ddb1p in chromosome segregation.

Mol Microbiol, 2002 Aug, 45(4), 1153 - 63
SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans; Kawasaki L et al.; In eukaryotic cells, environmental stress signals are transmitted by evolutionarily conserved MAPKs, such as Hog1 in the budding yeast Saccharomyces cerevisiae, Spc1 in the fission yeast Schizosaccharomyces pombe and p38/JNK in mammalian cells . Here, we report the identification of the Aspergillus nidulans sakA gene, which encodes a member of the stress MAPK family . The sakA gene is able to complement the S . pombe spc1- defects in both osmo-regulation and cell cycle progression . Moreover, SakA MAPK is activated in response to osmotic and oxidative stress in both S . pombe and A . nidulans . However, in contrast to hog1 and spc1 mutants, the sakA null mutant is not sensitive to high osmolarity stress, indicating a different regulation of the osmostress response in this fungus . On the other hand, the DeltasakA mutant shows development and cell-specific phenotypes . First, it displays premature steA-dependent sexual development . Second, DeltasakA mutant produces asexual spores that are highly sensitive to oxidative and heat shock stress and lose viability upon storage . Indeed, SakA is transiently activated early after induction of conidiation . Our results indicate that SakA MAPK is involved in stress signal transduction and repression of sexual development, and is required for spore stress resistance and survival.

Curr Genet, 2002 Jul, 41(4), 241 - 53 Epub 2002 Jun 25.
Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe; Didmon M et al.; The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery . Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals . To understand this aspect of cell signalling, we studied the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism . Here, we describe the development of yeast strains containing ura4 and lacZ reporter genes under the control of the pheromone-regulated sxa2 promoter and the use of these strains to isolate mutants defective in their ability to regulate signalling . Several different types of mutant were identified . Some mutants were defective in proteins already known to regulate the pheromone-signalling pathway (Rgs1, Map1, Map2) . Our approach also identified the MAP kinase phosphatase Pmp1 as a regulator of the pheromone-response pathway . Although previously shown to regulate other MAP kinase pathways in Sz . pombe, this is the first demonstration of a role for Pmp1 in pheromone signalling.

J Biol Chem, 2002 Oct 18, 277(42), 39585 - 93 Epub 2002 Aug 08.
Oligomerization-dependent association of the SAM domains from Schizosaccharomyces pombe Byr2 and Ste4; Ramachander R et al.; SAM (sterile alpha motif) domains are protein-protein interaction modules found in a large number of regulatory proteins . Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe . Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4 . Tu et al . (Tu, H., Barr, M., Dong, D . L., and Wigler, M . (1997) Mol . Cell . Biol . 17, 5876-5887) showed that the isolated SAM domain of Byr2 binds a fragment of Ste4 that contains both a leucine zipper (Ste4-LZ) domain as well as a SAM domain, suggesting that Byr2-SAM and Ste4-SAM may form a hetero-oligomer . Here, we show that the individual SAM domains of Ste4 and Byr2 are monomeric at low concentrations and bind to each other in a 1:1 stoichiometry with a relatively weak dissociation constant of 56 +/- 3 microm . Inclusion of the Ste4-LZ domain, which determines the oligomeric state of Ste4, has a dramatic effect on binding affinity, however . We find that the Ste4-LZ domain is trimeric and, when included with the Ste4-SAM domain, yields a 3:1 Ste4-LZ-SAM:Byr2-SAM complex with a tight dissociation constant of 19 +/- 4 nm . These results suggest that the Ste4-LZ-SAM protein may recognize multiple binding sites on Byr2-SAM, indicating a new mode of oligomeric organization for SAM domains . The fact that high affinity binding occurs only with the addition of an oligomerization domain suggests that it may be necessary to include ancillary oligomerization modules when searching for binding partners of SAM domains.

Nat Genet, 2002 Sep, 32(1), 143 - 7 Epub 2002 Aug 05.
The transcriptional program of meiosis and sporulation in fission yeast; Mata J et al.; Sexual reproduction requires meiosis to produce haploid gametes, which in turn can fuse to regenerate a diploid organism . We have studied the transcriptional program that drives this developmental process in Schizosaccharomyces pombe using DNA microarrays . Here we show that hundreds of genes are regulated in successive waves of transcription that correlate with major biological events of meiosis and sporulation . Each wave is associated with specific promoter motifs . Clusters of neighboring genes (mostly close to telomeres) are co-expressed early in the process, which reflects a more global control of these genes . We find that two Atf-like transcription factors are essential for the expression of late genes and formation of spores, and identify dozens of potential Atf target genes . Comparison with the meiotic program of the distantly related Saccharomyces cerevisiae reveals an unexpectedly small shared meiotic transcriptome, suggesting that the transcriptional regulation of meiosis evolved independently in both species.

Eur J Biochem, 2002 Aug, 269(15), 3847 - 55
Molecular interaction of neutral trehalase with other enzymes of trehalose metabolism in the fission yeast Schizosaccharomyces pombe; Soto T et al.; Trehalose metabolism is an essential component of the stress response in yeast cells . In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1+ (coding for trehalose-6-P synthase, Tps1p), ntp1+ (encoding neutral trehalase, Ntp1p) and tpp1+ (that codes for trehalose-6-P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes . Disruption of the gene tps1+ blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress . We propose that this association may reflect the Tps1p-dependent requirement for thermal activation of trehalase . Data reported here indicate that following a heat shock the enzyme activity of trehalase is associated with Ntp1p dimers or trimers but not with either Ntp1p monomers or with complexes involving Tps1p . These results raise the possibility that heat shock and osmotic stress activate trehalase differentially by acting in the first case through an specific mechanism involving Tps1p-Ntp1p complexes . This study provides the first evidence for the participation of the catabolic enzyme trehalase in the structural framework of a regulatory macromolecular complex containing trehalose-6-P synthase in the fission yeast.

Biochim Biophys Acta, 2002 Aug 19, 1577(1), 164 - 70
Characterization, expression and regulation of a third gene encoding glutathione S-transferase from the fission yeast; Shin YH et al.; A third gene encoding glutathione S-transferase (GSTIII) was cloned from the fission yeast Schizosaccharomyces pombe . The nucleotide sequence determined was found to contain 2110 base pairs including an open reading frame of 242 amino acids that would encode a protein of a molecular mass of 26,620 Da . The cloned GSTIII gene could be expressed in S . pombe, S . cerevisiae and Escherichia coli cells which gave 1.4-, 2.1-, and 3.0-fold higher GST activity in an assay using 1-chloro-2,4-dinitrobenzene as a substrate, respectively . The cloned GSTIII gene caused higher survivals of S . pombe cells on solid media with cadmium chloride or mercuric chloride . The GSTIII protein has 16% and 18% homologies with the GSTI and GSTII proteins, respectively . To independently monitor the regulation of the GSTIII gene, its 1168 bp upstream region and N-terminal 33 amino acid-coding region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 . The synthesis of beta-galactosidase from the fusion plasmid pGY357 was greatly enhanced by cadmium chloride (50 microM), cupric chloride (10 microM), aluminum chloride (5 mM, 10 mM), mercuric chloride (1 microM), and zinc chloride (10 mM) . However, the synthesis of beta-galactosidase from the fusion plasmid pGY357 was not affected by superoxide-generating menadione, and o-dinitrobenzene, whereas they could significantly induce the expression of the GSTI and GSTII genes of S . pombe . The overproduced Pap1 inhibited the induction of beta-galactosidase synthesis from the fusion plasmid pGY357 by cadmium chloride, which is opposite to the previously known role of Pap1 in the response to oxidative stress . Our results collectively indicate that the three GST genes of S . pombe are subjected to different regulatory mechanisms . The major role of the GSTIII protein in S . pombe may be the detoxification of various metals.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10330 - 4 Epub 2002 Jul 29.
Absolute requirement of spermidine for growth and cell cycle progression of fission yeast (Schizosaccharomyces pombe); Chattopadhyay MK et al.; Schizosaccharomyces pombe cells that cannot synthesize spermidine or spermine because of a deletion-insertion in the gene coding for S-adenosylmethionine decarboxylase (Deltaspe2) have an absolute requirement for spermidine for growth . Flow cytometry studies show that in the absence of spermidine an overall delay of the cell cycle progression occurs with some accumulation of cells in the G(1) phase; as little as 10(-6) M spermidine is sufficient to maintain normal cell cycle distribution and normal growth . Morphologically some of the spermidine-deprived cells become spherical at an early stage with little evidence of cell division . On further incubation in the spermidine-deprived medium, growth occurs in most of the cells, not by cell division but rather by cell elongation, with an abnormal distribution of the actin cytoskeleton, DNA (4', 6-diamidino-2-phenylindole staining), and calcofluor-staining moieties . More prolonged incubation in the spermidine-deficient medium leads to profound morphological changes including nuclear degeneration.

J Steroid Biochem Mol Biol, 2002 Jun, 81(2), 173 - 9
Development of a test system for inhibitors of human aldosterone synthase (CYP11B2): screening in fission yeast and evaluation of selectivity in V79 cells; Ehmer PB et al.; Aldosterone synthase (CYP11B2) is a mitochondrial cytochrome P450 enzyme catalyzing the last steps of aldosterone production in the adrenal cortex . A new pharmacological approach for the treatment of the aldosterone induced effects in congestive heart failure and all forms of hyperaldosteronism could be the use of CYP11B2 inhibitors . In search for such compounds, it was our goal to develop a cellular enzyme assay suitable for screening high numbers of compounds . An assay procedure for the evaluation of inhibitors using the human CYP11B2 expressed in fission yeast Schizosaccharomyces pombe was established and a series of 10 compounds was tested in this whole cellular system . Human 11beta-hydroxylase (CYP11B1), which catalyzes the production of glucocorticoids, shows more than 90% homology compared to human CYP11B2 . As this enzyme should not be affected, strong inhibitors of CYP11B2 have to be tested for selectivity . For that purpose, an assay procedure with V79MZ cells that express human CYP11B1 and CYP11B2, respectively, was integrated into the evaluation process . Using these screening procedures a potent and rather selective non-steroidal inhibitor of human CYP11B2 was detected with an IC(50) value of 59nM . We also identified a very potent inhibitor of both enzymes showing a stronger inhibitory activity against the cortisol producing CYP11B1.

Genetics, 2002 Jul, 161(3), 1053 - 63
Role of the Tsc1-Tsc2 complex in signaling and transport across the cell membrane in the fission yeast Schizosaccharomyces pombe; Matsumoto S et al.; Heterozygous inactivation of either human TSC1 or TSC2 causes tuberous sclerosis (TSC), in which development of benign tumors, hamartomas, occurs via a two-hit mechanism . In this study, fission yeast genes homologous to TSC1 and TSC2 were identified, and their protein products were shown to physically interact like the human gene products . Strains lacking tsc1(+) or tsc2(+) were defective in uptake of nutrients from the environment . An amino acid permease, which is normally positioned on the plasma membrane, aggregated in the cytoplasm or was confined in vacuole-like structures in Deltatsc1 and Deltatsc2 strains . Deletion of tsc1(+) or tsc2(+) also caused a defect in conjugation . When a limited number of the cells were mixed, they conjugated poorly . The conjugation efficiency was improved by increased cell density . Deltatsc1 cells were not responsive to a mating pheromone, P-factor, suggesting that Tsc1 has an important role in the signal cascade for conjugation . These results indicate that the fission yeast Tsc1-Tsc2 complex plays a role in the regulation of protein trafficking and suggest a similar function for the human proteins . We also show that fission yeast Int6 is involved in a similar process, but functions in an independent genetic pathway.

FEBS Lett, 2002 Jul 31, 524(1-3), 79 - 86
Cmk2, a novel serine/threonine kinase in fission yeast; Alemany V et al.; The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family . The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent . The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary . Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.

Mol Cells, 2002 Jun 30, 13(3), 389 - 98
Cloning and characterization of the kinesin-related protein, Krp1p, in Schizosaccharomyces pombe; Jeong JW et al.; Kinesin have been cloned in many organisms . They played important roles in the transport of cell organelles, polarized growth, and secretion . We report here the identification of a kinesin-related protein in Schizosaccharomyces pombe, which was named kinesin-related protein (Krplp) . The primer sequences were driven from the highly conserved area of the kinesin genes in other organisms . We cloned kinesin genes from S . pombe using the PCR technique . Sequence analysis revealed that krp1+ has a 1,665 bp open-reading frame (ORF) that encoded a protein that consisted of 554 amino acids with a molecular weight of 61,900 . It is homologous to the proteins that belong to the kinesin heavy chain (KHC) superfamily {GenBank accession No . AF156966 (genomic DNA) and AF247188 (mRNA)} . To characterize Krplp, the gene was disrupted and overexpressed in S . pombe . Cells that contained a krp1+ null allele were viable . Overexpression of Krp1p resulted in the inhibition of mitotic growth; cells became elongated, branched, and formed aberrant septa . To identify proteins that interact with Krplp, the yeast two-hybrid system was used . As a result, the novel protein, designated kinesin associated protein (Kap1p), was identified and showed structural homology to the proteins of the myosin family (GenBank accession No . AF351206) . The data from the overexpression and two-hybrid study of Krplp may provide information that Krplp can have roles in cytokinesis with myosin.

Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 970 - 4
Hrp3, a chromodomain helicase/ATPase DNA binding protein, is required for heterochromatin silencing in fission yeast; Jae Yoo E et al.; Hrp3, a paralog of Hrp1, is a novel member of the CHD1 (chromo-helicase/ATPase-DNA binding 1) protein family of Schizosaccharomyces pombe . Although it has been considered that CHD1 proteins are required for chromatin modifications in transcriptional regulations, little is known about their roles in vivo . In this study, we examined the effects of Hrp3 on heterochromatin silencing using several S . pombe reporter strains . The phenotypic analysis revealed that hrp3(+) is not an essential gene for cell viability . However, Hrp3 is required for transcriptional repression at silence loci of mat3 . A chromatin immunoprecipitation assay showed that Hrp3 directly associates with mat3 chromatin . Thus, our results strongly suggest that Hrp3 is involved in heterochromatin silencing and plays a direct role as a chromatin remodeling factor at mat3 in vivo.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 51 - 7
Functional characterization of 4'-phosphopantetheinyl transferase genes of bacterial and fungal origin by complementation of Saccharomyces cerevisiae lys5; Mootz HD et al.; Lysine biosynthesis in yeast requires the posttranslational conversion of the alpha-aminoadipate semialdehyde reductase Lys2 by the 4'-phosphopantetheinyl transferase (PPTase) Lys5 from the inactive apo-form into the catalytically active holo-form . In this reaction, the peptidyl carrier domain of Lys2 is modified at a conserved serine residue side chain with the 4'-phosphopantetheine moiety derived from coenzyme A . We have deleted the lys5 gene in Saccharomyces cerevisiae to investigate the substrate specificity of various heterologous PPTase genes of bacterial and fungal origin by testing their ability to complement lys5 in trans . Genes encoding PPTases Sfp and Gsp from Bacillus spp., which are involved in non-ribosomal peptide antibiotic synthesis, complemented the lys5 deletion, whereas ydcB of Bacillus subtilis, which encodes the acyl carrier protein synthase involved in fatty acid synthesis, could not . Two yet uncharacterized fungal genes, q10474 of Schizosaccharomyces pombe, meanwhile annotated as the putative lys7 gene, and npgA of Aspergillus nidulans, also complemented the lys5 deletion and have thus been functionally characterized as PPTases . The complementation system described also provides the basis for a simple method of functional characterization of PPTase candidate genes and their cloning from chromosomal DNA or cDNA libraries of diverse origin.

J Biol Chem, 2002 Sep 27, 277(39), 36853 - 62 Epub 2002 Jul 17.
The influence of the Cdc27 subunit on the properties of the Schizosaccharomyces pombe DNA polymerase delta; Bermudez VP et al.; Schizosaccharomyces pombe DNA polymerase (pol) delta contains four subunits, pol 3, Cdc1, Cdc27, and Cdm1 . In this report, we examined the role of Cdc27 on the structure and activity of pol delta . We show that the four-subunit complex is monomeric in structure, in contrast to the previous report that it was a dimer (Zuo, S., Bermudez, V., Zhang, G., Kelman, Z., and Hurwitz, J . (2000) J . Biol . Chem . 275, 5153-5162) . This discrepancy between the earlier and recent observations was traced to the marked asymmetric shape of Cdc27 . Cdc27 contains two critical domains that govern its role in activating pol delta . The N-terminal region (amino acids (aa) 1-160) binds to Cdc1 and its extreme C-terminal end (aa 362-369) interacts with proliferating cell nuclear antigen (PCNA) . Mutants of S . pombe pol delta, containing truncated Cdc27 derivatives deficient in binding to PCNA, supported DNA replication less processively than the wild-type complex . Fusion of a minimal PCNA-binding motif (aa 352-372) to C-terminally truncated Cdc27 derivatives restored processive DNA synthesis in vitro . In vivo, the introduction of these fused Cdc27 derivatives into cdc27Delta cells conferred viability . These data support the model in which Cdc27 plays an essential role in DNA replication by recruiting PCNA to the pol delta holoenzyme.

FEBS Lett, 2002 Jul 17, 523(1-3), 182 - 6
Modification of rRNA as a 'quality control mechanism' in ribosome biogenesis; Song X et al.; An efficiently expressed rDNA plasmid was used to quantitatively analyze the effect of base changes in modified positions associated with the peptidyl transferase center of the 25S rRNA from the yeast Schizosaccharomyces pombe . The results show that, unlike normal RNA and relative to a less conserved modified position outside the center, these mutant RNAs are highly unstable and rapidly degraded with little or no effect on cell growth . These results provide direct evidence that the positions of modification can be critical sites for nuclease attack . Taken together with previous genetic analyses of rRNA modification, they raise the possibility that rRNA modification may act, at least in part, as a quality control mechanism to help ensure that only functional RNA is incorporated into active ribosomes.

FEBS Lett, 2002 Jul 17, 523(1-3), 171 - 6
The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space; Olichon A et al.; Mutations in the OPA1 gene are associated with autosomal dominant optic atrophy . OPA1 encodes a dynamin-related protein orthologous to Msp1 of Schizosaccharomyces pombe and Mgm1p of Saccharomyces cerevisiae, both involved in mitochondrial morphology and genome maintenance . We present immuno-fluorescence and biochemical evidences showing that OPA1 resides in the mitochondria where it is imported through its highly basic amino-terminal extension . Proteolysis experiments indicate that OPA1 is present in the inter-membrane space and electron microscopy further localizes it close to the cristae . The strong association of OPA1 with membranes suggests its anchoring to the inner membrane.

Mol Microbiol, 2002 Jul, 45(2), 321 - 32
Cyclin-dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii; Khan F et al.; The Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission . We tested the role of TPK2, a homologue of the CDC2 cyclin-dependent kinases, in cell cycle regulation . TPK2 tagged with HA epitope (TPK2-HA-wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies . TPK2-HA-wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase . TPK2-HA-wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E . Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains . Overexpression of a dominant-negative mutant of TPK2 (TPK2-HA-dn) in T . gondii tachyzoites arrested replication . FACS analysis of tachyzoites expressing TPK2-HA-dn revealed an increase in the fraction of cells in S-phase when compared with TPK2-HA-wt transfected parasites . Expression of TPK2-HA-wt did not arrest tachyzoite replication . No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T . gondii from most other eukaryotic cells . These data suggest that TPK2 executes an essential function in T . gondii cell cycle and is likely to be the T . gondii CDC2 orthologue.

Biotechnol Bioeng, 2002 Jun 5, 78(5), 583 - 8
Microbial synthesis of semiconductor CdS nanoparticles, their characterization, and their use in the fabrication of an ideal diode; Kowshik M et al.; Cadmium sulfide nanoparticles were synthesized intracellularly by a Schizosaccharomyces pombe strain when challenged with 1 mM cadmium in solution . The nanoparticles, a known semiconducting material, exhibited an absorbance maximum at 305 nm . X-ray scattering data showed that the nanoparticles had a Wurtzite (Cd(16)S(20))-type hexagonal lattice structure and most of the nanoparicles were in the size range of 1-1.5 nm . The nanoparticles were used in the fabrication of a heterojunction with poly (p-phenylenevinylene) . The diode exhibited approximately 75 mA/cm(2) current at 10 V when forward biased and the breakdown occurred at approximately 15 V in the reverse biased mode . These characteristics are considered ideal for a diode .

Yeast, 2002 Jul, 19(10), 841 - 8
Two non-complementing genes encoding enzymatically active methylenetetrahydrofolate reductases control methionine requirement in fission yeast Schizosaccharomyces pombe; Naula N et al.; By transforming two methionine auxotrophic mutants from fission yeast Schizosaccharomyces pombe with a wild-type gene library, we defined two genes, met9 and met11, which both encode a methylenetetrahydrofolate reductase . The genes cannot complement each other . We detected single transcripts for both . In vitro measurements of enzymatic activities showed that the met11-encoded enzyme was responsible for only 15-20% of the total methylenetetrahydrofolate reductase activity . A strain in which gene met9 was disrupted required significantly more methionine for full growth and efficient mating and sporulation than the strain disrupted for gene met11 . The in vitro and in vivo data thus indicated that met9 was the major expressed gene . Our results are in accordance with the assumption that the two methylenetetrahydrofolate reductases generate the methyl groups necessary for methionine synthetase to convert homocysteine to methionine, and suggest that expression of the two genes is an important parameter in the control of methionine biosynthesis .

Yeast, 2002 Jun 30, 19(9), 803 - 10
Schizosaccharomyces pombe spPABP, a homologue of Saccharomyces cerevisiae Pab1p, is a non-essential, shuttling protein that facilitates mRNA export; Thakurta AG et al.; Poly(A)-binding proteins play important roles in mRNA metabolism in eukaryotic cells . We examined the role of the Schizosaccharomyces pombe homologue of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1p, in cellular growth and mRNA export . In contrast to PAB1, the sppabp gene is not essential for cellular viability . Like the human hPABP1 protein, spPABP is cytoplasmically localized and can shuttle between the nucleus and the cytoplasm . We found that a spPABP-GFP fusion protein expressed from a multicopy plasmid could suppress the growth and mRNA export defect of rae1-16 7 nup184-1 synthetic lethal mutations . However, about 20-25% of cells in the population exhibited a pronounced nuclear accumulation of poly(A)(+) RNA . The same cells also localized the spPABP-GFP fusion to the nucleus, suggesting that the shuttling ability of spPABP is related to its function in mRNA export . When a heterologous nuclear export activity from spMex67p was fused to spPABP-GFP fusion protein, it overcame the nuclear retention but did not increase nuclear mRNA export . We discuss the implications of these observations in relation to how spPABP could function in mRNA export . Published in 2002 by John Wiley & Sons, Ltd.

Planta, 2002 Jul, 215(3), 518 - 22 Epub 2002 Jun 06.
A WEE1 homologue from Arabidopsis thaliana; Sorrell DA et al.; Little is known about the genes that regulate cyclinB-Cdc2 complexes at the G2/M transition of the plant cell cycle although in yeast and animals cdc25 and wee1 are central regulators of cdc2 . Here we describe the isolation, by reverse transcription polymerase chain reaction (RT-PCR), of a WEE1 cDNA (AtWEE1) in Arabidopsis thaliana (L.) Heynh . Semi-quantitative RT-PCR showed that AtWEE1 expression was confined to actively dividing regions of the plant . The overexpression of AtWEE1 in fission yeast (Schizosaccharomyces pombe) caused cells to arrest, and to grow but not divide, resulting in very elongated cells . Our data provide evidence for a functional WEE1 in A . thaliana.

J Biol Chem, 2002 Sep 13, 277(37), 33580 - 9 Epub 2002 Jul 10.
Elf1p, a member of the ABC class of ATPases, functions as a mRNA export factor in Schizosacchromyces pombe; Kozak L et al.; Rae1p and Mex67p/Tap are conserved mRNA export factors . We have used synthetic lethal genetic screens in Schizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67 in mRNA export . From these screens, we have isolated mutations in a putative S . pombe homologue of the Candida albicans elf1 gene . The elf1 of S . pombe is not an essential gene . When elf1 mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants . This inhibition can be suppressed by the multicopy expression of mex67 suggesting that Mex67p can substitute for the loss of Elf1p function . Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm . Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP . A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro . This mutant protein no longer functions in mRNA export . Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S . pombe.

Can J Microbiol, 2002 May, 48(5), 399 - 406
Physiological diversity and trehalose accumulation in Schizosaccharomyces pombe strains isolated from spontaneous fermentations during the production of the artisanal Brazilian cachaça; Gomes FC et al.; Twenty-seven Schizosaccharomyces pombe isolates from seven cachaca distilleries were tested for maximum temperature of growth and fermentation, osmotolerance, ethanol resistance, invertase production, and trehalose accumulation . Two isolates were selected for studies of trehalose accumulation under heat shock and ethanol stress . The S . pombe isolates were also characterized by RAPD-PCR . The isolates were able to grow and ferment at 41 degrees C, resisted concentrations of 10% ethanol, and grew on 50% glucose medium . Four isolates yielded invertase activity of more than 100 micromol of reducing sugar x mg(-1) x min(-1) . The S . pombe isolates were able to accumulate trehalose during stationary phase . Two isolates, strains UFMG-A533 and UFMG-A1000, submitted to a 15 min heat shock, were able to accumulate high trehalose levels . Strain UFMG-A533 had a marked reduction in viability during heat shock, but strain UFMG-A1000 preserved a viability rate of almost 20% after 15 min at 48 degrees C . No clear correlation was observed between trehalose accumulation and cell survival during ethanol stress . Strain UFMG-A1000 had higher trehalose accumulation levels than strain UFMG-A533 under conditions of combined heat treatment and ethanol stress . Molecular analysis showed that some strains are maintained during the whole cachaca production period; using the RAPD-PCR profiles, it was possible to group the isolates according to their isolation sites.

Acta Microbiol Immunol Hung, 2002, 49(2-3), 289 - 304
Regularities and irregularities in the cell cycle of the fission yeast, Schizosaccharomyces pombe (a review); Sveiczer A et al.; In an exponentially growing wild-type fission yeast culture a size control mechanism ensures that mitosis is executed only if the cells have reached a critical size . However, there is some scattering both in cell length at birth (BL) and in cycle time (CT) . By computational simulations we show here that this scattering cannot be explained solely by asymmetric cell division, therefore we assume that nuclear division is a stochastic, asymmetric process as well . We introduce an appropriate stochastic variable into a mathematical model and prove that this assumption is suitable to describe the CT vs . BL graph in a wild-type fission yeast population . In a double mutant of fission yeast (namely wee1-50 cdc25 delta) this CT vs . BL plot is even more curious: cycle time splits into three different values resulting in three clusters in this coordinate system . We show here that it is possible to describe these quantized cycles by choosing the appropriate values of the key parameters of mitotic entry and exit and even more the clustered behavior may be simulated by applying a further stochastic parameter.

Acta Microbiol Immunol Hung, 2002, 49(2-3), 279 - 83
Genetics of sulphate assimilation in Schizosaccharomyces pombe (a short review); Simonics T et al.; Sulphur plays an important role in yeasts, especially in the biosynthesis of methionine and cysteine . The inorganic sulphur source, sulphate, is taken up by the cells via the sulphate-permease(s) . After its transport, it is activated and subsequently reduced to sulphide or serves as a donor for sulphurylation reactions . Selenate anion (SeO4(2-)), which has the same metabolic pathway as sulphate, is toxic for the cells of Schizosaccharomyces pombe . We isolated selenate resistant mutants which cannot utilize sulphate, therefore they need organic sulphur source for growth . One of the selenate resistant mutants was successively transformed with S . pombe genomic libraries and the gene complementing the selenate resistance was identified as that of coding for the ATP-sulphurylase enzyme.

BMC Genomics . 2002 Jul 8;3(1):18 . Epub 2002 Jul 08.
Expression and genomic analysis of midasin, a novel and highly conserved AAA protein distantly related to dynein; Garbarino JE et al.; BACKGROUND: The largest open reading frame in the Saccharomyces genome encodes midasin (MDN1p, YLR106p), an AAA ATPase of 560 kDa that is essential for cell viability . Orthologs of midasin have been identified in the genome projects for Drosophila, Arabidopsis, and Schizosaccharomyces pombe . RESULTS: Midasin is present as a single-copy gene encoding a well-conserved protein of approximately 600 kDa in all eukaryotes for which data are available . In humans, the gene maps to 6q15 and encodes a predicted protein of 5596 residues (632 kDa) . Sequence alignments of midasin from humans, yeast, Giardia and Encephalitozoon indicate that its domain structure comprises an N-terminal domain (35 kDa), followed by an AAA domain containing six tandem AAA protomers (approximately 30 kDa each), a linker domain (260 kDa), an acidic domain (approximately 70 kDa) containing 35-40% aspartate and glutamate, and a carboxy-terminal M-domain (30 kDa) that possesses MIDAS sequence motifs and is homologous to the I-domain of integrins . Expression of hemagglutamin-tagged midasin in yeast demonstrates a polypeptide of the anticipated size that is localized principally in the nucleus . CONCLUSIONS: The highly conserved structure of midasin in eukaryotes, taken in conjunction with its nuclear localization in yeast, suggests that midasin may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus . The AAA domain of midasin is evolutionarily related to that of dynein, but it appears to lack a microtubule-binding site.

Biochemistry, 2002 Jul 16, 41(28), 8876 - 85
Iron-sulfur cluster biosynthesis . Kinetic analysis of {2Fe-2S} cluster transfer from holo ISU to apo Fd: role of redox chemistry and a conserved aspartate; Wu SP et al.; ISU-type proteins mediate cluster transfer to apo protein targets . Rate constants have been determined for cluster transfer from ISU to apo Fd for both Homo sapiens and Schizosaccharomyces pombe proteins, and cross reactions have also been examined . Substitution of a key aspartate residue of ISU is found to decrease the rate of cluster transfer by at least an order of magnitude (for wild-type Hs ISU cluster transfer to Hs apo Fd, k(2) approximately 540 M(-1) min(-1), relative 56 M(-1) min(-1) for D37A ISU) . This change in rate constant does not reflect any change in binding affinity of the ISU and Fd proteins . The pH dependencies of cluster transfer rates are similar for WT and D37A ISU, arguing against a role for Asp37 as a catalytic base, although evidence for general base catalysis mediating deprotonation of Cys from the apo target is supported by an observed pK(a) of 6.9 determined from the pH profiles for both WT and D37A ISU . Such a pK(a) value is at the lower limit for Cys and is common for solvent-accessible Cys thiols . The temperature dependence of the rate constant defining the cluster transfer reaction for wild type versus the aspartate derivative is distinct . Thermal activation parameters (DeltaH and DeltaS) are consistent with a solvent-accessible ISU-bound cluster, with desolvation as a principle barrier to cluster transfer . Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer to be rate-limiting . Fully oxidized cluster appears to be the natural state for transfer to target proteins . Reduced Fd does not readily reduce ISU-bound {2Fe-2S}(2+) and does not promote cluster transfer to an apo Fd target.

Mol Microbiol, 2002 Jul, 45(1), 243 - 54
Diethylmaleate activates the transcription factor Pap1 by covalent modification of critical cysteine residues; Castillo EA et al.; During the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms . The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro-oxidant hydrogen peroxide (H2O2) or the glutathione-depleting agent diethylmaleate (DEM) . As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501) . Surprisingly, our studies demonstrate that DEM generates a non-reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532) . This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy-terminal domain of Pap1 . Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether . Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.

Nature, 2002 Jul 4, 418(6893), 79 - 85
Sequence and analysis of chromosome 2 of Dictyostelium discoideum; Glockner G et al.; The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes . Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome . Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes . This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb) . If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D . discoideum genome . A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes . This analysis strengthens the view that the evolutionary position of D . discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

Genome Res, 2002 Jul, 12(7), 1048 - 59
The role of lineage-specific gene family expansion in the evolution of eukaryotes; Lespinet O et al.; A computational procedure was developed for systematic detection of lineage-specific expansions (LSEs) of protein families in sequenced genomes and applied to obtain a census of LSEs in five eukaryotic species, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the green plant Arabidopsis thaliana . A significant fraction of the proteins encoded in each of these genomes, up to 80% in A . thaliana, belong to LSEs . Many paralogous gene families in each of the analyzed species are almost entirely comprised of LSEs, indicating that their diversification occurred after the divergence of the major lineages of the eukaryotic crown group . The LSEs show readily discernible patterns of protein functions . The functional categories most prone to LSE are structural proteins, enzymes involved in an organism's response to pathogens and environmental stress, and various components of signaling pathways responsible for specificity, including ubiquitin ligase E3 subunits and transcription factors . The functions of several previously uncharacterized, vastly expanded protein families were predicted through in-depth protein sequence analysis, for example, small-molecule kinases and methylases that are expanded independently in the fly and in the nematode . The functions of several other major LSEs remain mysterious; these protein families are attractive targets for experimental discovery of novel, lineage-specific functions in eukaryotes . LSEs seem to be one of the principal means of adaptation and one of the most important sources of organizational and regulatory diversity in crown-group eukaryotes.

Gene, 2002 May 29, 291(1-2), 187 - 201
Characterization of a fission yeast subunit of an RNA polymerase I essential transcription initiation factor, SpRrn7h/TAF(I)68, that bridges yeast and mammals: association with SpRrn11h and the core ribosomal RNA gene promoter; Boukhgalter B et al.; Production of eukaryotic ribosomal RNAs (rRNAs) entails sequence-specific recognition of regulatory sequences in the rRNA gene promoter . A putative subunit of the Schizosaccharomyces pombe essential transcription initiation factor for rRNA synthesis has been identified that shares homology with both murine TAF(I)68 and Saccharomyces cerevisiae Rrn7p, subunits of their species' transcription initiation factor . Affinity purified putative SpRrn7h and associated factors, including a putative Rrn11p homolog, SpRrn11h, bear RNA polymerase I transcription initiation factor activity, and recombinant SpRrn7h associates with S . pombe core rDNA promoter sequences . In the first widespread search for putative homologs of SpRrn7h/murine TAF(I)68, and SpRrn11h/murine TAF(I)48, multiple ones were identified across eukaryotes . Analysis of residues conserved between the fission yeast and murine essential initiation factor subunits aided in these identifications . Sequences in the core rRNA gene promoter contributing to transcriptional activation were investigated, including a perfect TATAAA element located at -35.

EMBO J, 2002 Jul 1, 21(13), 3370 - 6
G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast; Zarzov P et al.; Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear . We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2 . This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase . We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset . This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2 . Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.

Cancer Detect Prev, 2002, 26(1), 10 - 4
Effect of nucleotide substitutions in N-acetyltransferase-1 on N-acetylation (deactivation) and O-acetylation (activation) of arylamine carcinogens: implications for cancer predisposition; Fretland AJ et al.; Genetic polymorphism in N-acetyltransferase-1 (NAT1) is associated with increased risk of various cancers, but epidemiological investigations are compromised by poor understanding of the relationship between NAT1 genotype and phenotype . Human reference NAT1*4 and 12 known human NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe) . Large reductions in the N-acetylation of 4-aminobiphenyl and the O-acetylation of N-hydroxy-2-aminofluorene were observed for recombinant NAT1 allozymes encoded by NAT1*14B, NAT1*15, NAT1*17, NAT1*19, and NAT1*22 . Each of these alleles exhibited substantially lower expression of NAT1 protein than the reference NAT1*4 and the other NAT1 alleles . These results show an important effect of the NAT1 genetic polymorphism on the N- and O-acetylation of arylamine carcinogens, suggesting modification of cancer susceptibility following exposures to arylamine carcinogens.

RNA, 2002 Jun, 8(6), 798 - 815
Characterization of interactions among the Cef1p-Prp19p-associated splicing complex; Ohi MD et al.; Schizosaccharomyces pombe (Sp) Cdc5p and its Saccharomyces cerevisiae (Sc) ortholog, Cef1p, are essential components of the spliceosome . In S . cerevisiae, a subcomplex of the spliceosome that includes Cef1p can be isolated on its own; this has been termed the nineteen complex (Ntc) because it contains Prp19p . Components of the Ntc include Cef1p, Snt309p, Syf2p/Ntc31p, Ntc30p/lsy1p, Ntc20p and at least six unidentified proteins . We recently identified approximately 30 proteins that copurified with Cdc5p and Cef1p . Previously unidentified S . pombe proteins in this purification were called Cwfs for complexed with five and novel S . cerevisiae proteins were called Cwcs for complexed with Cef1p . Using these proteomics data coupled with available information regarding Ntc composition, we have investigated protein identities and interactions among Ntc components . Our data indicate that Cwc2p, Prp46p, Clf1p, and Syf1p most likely represent Ntc40p, Ntc50p, Ntc77p, and Ntc90p, respectively . We show that Sc Cwc2p interacts with Prp19p and is involved in pre-mRNA splicing . Sp cwf2+, the homolog of Sc CWC2, is allelic with the previously identified Sp prp3+ . We present evidence that Sp Cwf7p, an essential protein with obvious homologs in many eukaryotes but not S . cerevisiae, is a functional counterpart of Sc Snt309p and binds Sp Cwf8p (a homolog of Sc Prp19p) . Further, our data indicate that a mutation in the U-box of Prp19p disrupts these numerous protein interactions causing Cef1p degradation and Ntc instability.

Nucleic Acids Res, 2002 Jul 1, 30(13), 2940 - 9
Premature termination of RNA polymerase II mediated transcription of a seed protein gene in Schizosaccharomyces pombe; Chakraborty S et al.; The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription . We show that an intronic sequence derived from the plant seed protein gene (AmA1) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe . The 3'-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus . Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements . RT-PCR analyses detected transcripts up to the second SDE . This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe . The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions . Deletion of these cis-elements abolished 3'-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE . These results indicate that the bipartite sequence elements as signals for 3'-end processing in fission yeast act in tandem with other cis-acting elements . A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element . However, these sequences are not identical . Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611-949 nt . Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene.

Nucleic Acids Res, 2002 Jul 1, 30(13), 2772 - 81
Identification and characterization of transcription factor IIIA from Schizosaccharomyces pombe; Schulman DB et al.; Transcription factor IIIA (TFIIIA) is specifically required for transcription of 5S rRNA genes and is the archetypal C2H2 zinc finger protein . All known vertebrate TFIIIAs have a similar organization: nine zinc fingers, followed by a C-terminal domain of unknown structure . The zinc fingers of Saccharomyces cerevisiae TFIIIA are interrupted between fingers eight and nine by an 81-amino acid spacer . Aside from the amino acids required for zinc finger folding, TFIIIAs from different species are remarkably divergent, whereas the natural binding site, the internal control region of the 5S rRNA gene, is well conserved . We now describe the identification and characterization of TFIIIA from Schizosaccharomyces pombe . This protein is organized differently from its known homologs, in that it contains eight closely spaced zinc fingers, a ninth zinc finger missing a C-terminal Zn2+-coordinating histidine, a 53- amino acid spacer, and an unprecedented tenth zinc finger . We have confirmed the identity of this divergent protein as TFIIIA by showing that it binds specifically and with high affinity to the S.pombe 5S rRNA gene . Comparison of DNase I protection patterns produced by TFIIIA from multiple species suggests a novel mode of DNA recognition by the S.pombe protein . Recombinant S.pombe TFIIIA was also shown to support specific transcription of the 5S rRNA gene in vitro.

Virus Genes, 2002 Jun, 24(3), 257 - 66
Identification and characterization of the UL7 gene product of herpes simplex virus type 2; Nozawa N et al.; We have raised a rabbit polyclonal antiserum against a recombinant 6x His-tagged herpes simplex virus type 2 (HSV-2) UL7 fusion protein expressed in Escherichia coli . The antiserum specifically reacted with a 33 kDa protein in HSV-1 and HSV-2-infected cell lysates, and was used to characterize the UL7 gene product of HSV-2 . The UL7 protein was produced in the late phase of infection, and its synthesis was highly inhibited, but not abolished by the addition of acyclovir (ACV) . The UL7 protein associated with extracellular virions and also with all types of capsids, including A, B, and C capsids, though the association seemed to be weak . Indirect immunofluorescence studies revealed that at 9 h postinfection, UL7 specific fluorescence was detected in part or all of the nucleus, and the specific fluorescence colocalized with the scaffold protein ICP35 . However, at later times postinfection, the UL7 protein was mainly detected as a mass in a juxtanuclear cytoplasmic region . In addition, transmission immunoelectron microscopy (TIEM) confirmed the association of the UL7 protein with intracellular capsids and virions in HSV-2-infected cells . The HSV-2 UL7 protein contained a domain highly conserved in all herpesviruses, part of which exhibited a homology with domains in the fission yeast Schizosaccharomyces pombe DNA topoisomerase III . We discuss the possibility that the UL7 protein may play a supplementary role in the viral DNA cleavage/packaging process.

J Biol Chem, 2002 Sep 6, 277(36), 33482 - 9 Epub 2002 Jun 25.
Comparison of Cak1p-like cyclin-dependent kinase-activating kinases; Tsakraklides V et al.; Cyclin-dependent kinases (cdks) coordinate progression through the eukaryotic cell cycle and require phosphorylation by a cdk-activating kinase (CAK) for full activity . In most eukaryotes Cdk7 is the catalytic subunit of a heterotrimeric CAK (Cdk7-cyclin H-Mat1) that is also involved in transcription as part of the transcription factor IIH complex . The Saccharomyces cerevisiae CAK, Cak1p, is a monomeric protein kinase with an atypical sequence and unusual biochemical properties compared with trimeric CAKs and other protein kinases . We sought to determine whether these properties were shared by a small group of monomeric CAKs that can function in place of CAK1 in S . cerevisiae . We found that Schizosaccharomyces pombe Csk1, Candida albicans Cak1, and Arabidopsis thaliana Cak1At, like Cak1p, all displayed a preference for cyclin-free cdk substrates, were insensitive to the protein kinase inhibitor 5'-fluorosulfonylbenzoyladenosine (FSBA), and were insensitive to mutation of a highly conserved lysine residue found in the nucleotide binding pocket of all protein kinases . The S . pombe and C . albicans kinases also resembled Cak1p in their kinetics of nucleotide and protein substrate utilization . Conservation of these unusual properties in fungi and plants points to shared evolutionary requirements not met by Cdk7 and raises the possibility of developing antifungal agents targeting CAKs.

J Mol Biol, 2002 May 17, 318(5), 1317 - 29
The structural basis of riboflavin binding to Schizosaccharomyces pombe 6,7-dimethyl-8-ribityllumazine synthase; Gerhardt S et al.; Riboflavin is an essential cofactor in all organisms . Its direct biosynthetic precursor, 6,7-dimethyl-8-ribityllumazine, is synthesised by the enzyme 6,7-dimethyl-8-ribityllumazine synthase . Recently, we have found that the enzyme from Schizosaccharomyces pombe binds riboflavin, the final product of the pathway with a relatively high affinity with a KD of 1.2 microM . Here, we report on the crystal structure of lumazine synthase from S . pombe with bound riboflavin and compare the binding mode with those of the substrate analogue inhibitor 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and of the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine . In all complexes the pyrimidinedione moieties of each respective ligand bind in a very similar orientation . Binding of riboflavin additionally involves a stacking interaction of the dimethylbenzene moiety with the side-chain of His94, a highly conserved residue in all lumazine synthases . The enzyme from Bacillus subtilis showed a KD of at least 1 mM whereas the very homologous enzyme from Saccharomyces cerevisiae had a comparable KD of 3.9 microM . Structural comparison of the S . cerevisiae, the S . pombe, and the mutant enzymes suggests that fine tuning of affinity is achieved by influencing this stacking interaction.

Genes Cells, 2002 Jul, 7(7), 619 - 27
Calcineurin phosphatase in signal transduction: lessons from fission yeast; Sugiura R et al.; Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+-dependent signalling to a wide variety of cellular responses . Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin . Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues . The structure of the protein is highly conserved from yeast to man . The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S . pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes . Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross-talk between the Pmk1 MAP kinase signalling, or the PI signalling . Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively . These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades.

J Biol Chem, 2002 Sep 6, 277(36), 33411 - 21 Epub 2002 Jun 21.
The Srk1 protein kinase is a target for the Sty1 stress-activated MAPK in fission yeast; Smith DA et al.; The fission yeast stress-activated Sty1/Spc1 MAPK pathway responds to a similar range of stresses as do the mammalian p38 and SAPK/JNK MAPK pathways . In addition, sty1(-) cells are sterile and exhibit a G(2) cell cycle delay, indicating additional roles of Sty1 in meiosis and cell cycle progression . To identify novel proteins involved in stress responses, a microarray analysis of the Schizosaccharomyces pombe genome was performed to find genes that are up-regulated following exposure to stress in a Sty1-dependent manner . One such gene identified, srk1(+) (Sty1-regulated kinase 1), encodes a putative serine/threonine kinase homologous to mammalian calmodulin kinases . At the C terminus of Srk1 is a putative MAPK binding motif similar to that in the p38 substrates, MAPK-activated protein kinases 2 and 3 . Indeed, we find that Srk1 is present in a complex with the Sty1 MAPK and is directly phosphorylated by Sty1 . Furthermore, upon stress, Srk1 translocates from the cytoplasm to the nucleus in a process that is dependent on the Sty1 MAPK . Finally, we show that Srk1 has a role in regulating meiosis in fission yeast; following nitrogen limitation, srk1(-) cells enter meiosis significantly faster than wild-type cells and overexpression of srk1(+) inhibits the nitrogen starvation-induced arrest in G(1).

J Mol Biol, 2002 Jun 21, 319(5), 1005 - 13
In vitro transcription and start site selection in Schizosaccharomyces pombe; Choi WS et al.; We have used the fission yeast Schizosaccharomyces pombe to establish both a biochemical and genetic system to study the roles of general transcription factors in transcription initiation . Extracts were prepared that faithfully transcribed S . pombe promoters and the results confirm that, in contrast to the budding yeast Saccharomyces cerevisiae, in vitro transcription in S . pombe initiates near to the TATA element . S . pombe transcription relies on upstream activation sequence elements and these can be replaced successfully with sites for binding Gal4-VP16 activators . Although it is mammalian-like in these respects, S . pombe initiation uses an unusual scanning mechanism . This directs initiation, preferentially using purines, within a narrow window approximately 25-40 base-pairs downstream from the edge of the TATA element . Genetic experiments showed that this scanning mechanism was associated with the properties of the TFIIB polypeptide . When human TFIIB was expressed in S . pombe, it was accepted by the endogenous transcription machinery and caused initiation to be restricted to the closer edge of this window, corresponding to the distance in humans . Preliminary experiments suggested that S . cerevisiae TFIIB was not accepted . The results enlarge the potential for using fission yeast to study the properties of general transcription factors such as TFIIB in choosing the sites at which transcription initiates . (c) 2002 Elsevier Science Ltd.

Mol Cell Biol, 2002 Jul, 22(14), 5141 - 56
Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes; Dellaire G et al.; A growing body of evidence supports the coordination of pre-mRNA processing and transcriptional regulation . We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes . PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles . RNA interference of Caenorhabditis elegans PRP4K indicates that it is essential in metazoans . In support of a role for PRP4K in pre-mRNA splicing, we identified PRP6, SWAP, and pinin as interacting proteins and demonstrated that PRP4K is a U5 snRNP-associated kinase . In addition, BRG1 and N-CoR, components of nuclear hormone coactivator and corepressor complexes, also interact with PRP4K . PRP4K coimmunoprecipitates with N-CoR, BRG1, pinin, and PRP6, and we present data suggesting that PRP6 and BRG1 are substrates of this kinase . Lastly, PRP4K, BRG1, and PRP6 can be purified as components of the N-CoR-2 complex, and affinity-purified PRP4K/N-CoR complexes exhibit deacetylase activity . We suggest that PRP4K is an essential kinase that, in association with the both U5 snRNP and N-CoR deacetylase complexes, demonstrates a possible coordination of pre-mRNA splicing with chromatin remodeling events involved in transcriptional regulation.

Curr Genet, 2002 May, 41(2), 82 - 8 Epub 2002 Apr 19.
Characterization of Cu, Zn-superoxide dismutase-deficient mutant of fission yeast Schizosaccharomyces pombe; Mutoh N et al.; A Cu, Zn-superoxide dismutase gene ( sod1+) deletion mutant of fission yeast Schizosaccharomyces pombe was constructed and its properties were investigated . Superoxide dismutase activity was not detected in the mutant on activity staining of polyacrylamide gels . The mutant showed cysteine or methionine and lysine auxotrophy, slow growth and sensitivity to menadione . While expression of the apt1+ gene, induction of which depends on the Pap1 transcription factor, was induced at the same concentration of menadione in both the wild-type cell and the sod1 mutant, expression of the gpx1+ gene, induction of which depends on the Atf1 transcription factor, was induced at a lower concentration of menadione in the mutant compared with the wild-type control . Expression of the sod1+ gene was induced by oxidative stress and no induction was observed in pap1, prr1 and spc1 mutants.

Curr Opin Cell Biol, 2002 Jun, 14(3), 286 - 98
The many faces of histone lysine methylation; Lachner M et al.; Diverse post-translational modifications of histone amino termini represent an important epigenetic mechanism for the organisation of chromatin structure and the regulation of gene activity . Within the past two years, great progress has been made in understanding the functional implications of histone methylation; in particular through the characterisation of histone methyltransferases that direct the site-specific methylation of, for example, lysine 9 and lysine 4 positions in the histone H3 amino terminus . All known histone methyltransferases of this type contain the evolutionarily conserved SET domain and appear to be able to stimulate either gene repression or gene activation . Methylation of H3 Lys9 and Lys4 has been visualised in native chromatin, indicating opposite roles in structuring repressive or accessible chromatin domains . For example, at the mating-type loci in Schizosaccharomyces pombe, at pericentric heterochromatin and at the inactive X chromosome in mammals, striking differences between these distinct marks have been observed . H3 Lys9 methylation is also important to direct additional epigenetic signals such as DNA methylation--for example, in Neurospora crassa and in Arabidopsis thaliana . Together, the available data strongly establish histone lysine methylation as a central modification for the epigenetic organisation of eukaryotic genomes.

EMBO J, 2002 Jun 17, 21(12), 3096 - 107
Schizosaccharomyces pombe NIMA-related kinase, Fin1, regulates spindle formation and an affinity of Polo for the SPB; Grallert A et al.; The Aspergillus nidulans protein kinase NIMA regulates mitotic commitment, while the human and Xenopus equivalents influence centrosome function . Two recessive, temperature-sensitive mutations in the Schizosaccharomyces pombe NIMA homologue, Fin1, blocked spindle formation at 37 degrees C . One of the two spindle pole bodies (SPBs) failed to nucleate microtubules . This phenotype was reduced by accelerating mitotic commitment through genetic inhibition of Wee1 or activation of either Cdc25 or Cdc2 . Polo kinase (Plo1) normally associates with the SPB of mitotic, but not interphase cells . cut12.s11 is a dominant mutation in an SPB component that both suppresses cdc25 mutants and promotes Plo1 association with the interphase SPB . Both cut12.s11 phenotypes were abolished by removing Fin1 function . Elevating Fin1 levels promoted Plo1 recruitment to the interphase SPB of wild-type cells and reduced the severity of the cdc25.22 phenotype . These data are consistent with Fin1 regulating Plo1 function during mitotic commitment . The fin1 mitotic commitment and spindle phenotypes resemble distinct nimA phenotypes in different systems and suggest that the function of this family of kinases may be conserved across species.

J Biol Chem, 2002 Sep 20, 277(38), 35523 - 31 Epub 2002 Jun 12.
Distinct roles for glutathione S-transferases in the oxidative stress response in Schizosaccharomyces pombe; Veal EA et al.; We have identified three genes, gst1(+), gst2(+), and gst3(+), encoding theta-class glutathione S-transferases (GSTs) in Schizosaccharomyces pombe . The gst1(+) and gst2(+) genes encode closely related proteins (79% identical) . Our analysis suggests that Gst1, Gst2, and Gst3 all have GST activity with the substrate 1-chloro-2,4-dinitrobenzene and that Gst3 has glutathione peroxidase activity . Although Gst1 and Gst2 have no detectable peroxidase activity, all three gst genes are required for normal cellular resistance to peroxides . In contrast, each mutant is more resistant to diamide than wild-type cells . The gst1Delta, gst2Delta, and gst3Delta mutants are also more sensitive to fluconazole, suggesting that GSTs may be involved in anti-fungal drug detoxification . Both gst2(+) and gst3(+) mRNA levels increase in stationary phase, and all three gst genes are induced by hydrogen peroxide . Indeed, gst1(+), gst2(+), and gst3(+) are regulated by the stress-activated protein kinase Sty1 . The Gst1 and Gst2 proteins are distributed throughout the cell and can form homodimers and Gst1-Gst2 heterodimers . In contrast, Gst3 is excluded from the nucleus and forms homodimers but not complexes with either Gst1 or Gst2 . Collectively, our data suggest that GSTs have separate and overlapping roles in oxidative stress and drug responses in fission yeast.

Biocell, 2002 Apr, 26(1), 1 - 13
Primitive forms of meiosis: the possible evolution of meiosis; Solari AJ; Meiosis is a basic process of most eukaryotes, as it forms with conjugation the basis of sexual reproduction . As sex seems to be present in the vast majority of eukaryotes, the origin of meiosis is presently unknown . Protists having optional or alternative sexual and asexual cycles seem to be the best targets for research on the evolution of meiosis . While the budding yeast Saccharomyces cerevisiae shows an elaborate and well-known meiotic process, the fission yeast Schizosaccharomyces pombe, has a much simpler meiosis, which may show some of the most primitive features of meiotic mechanisms . The present availability of whole genome sequences of many bacteria and some protists is revealing that eukaryotic sexual reproduction has recruited some prokaryotic processes for its own development . Some of these processes are analyzed and the basic role of chromosome linearity and telomere constitution in the development of meiosis is underlined.

Mol Biol Cell, 2002 Jun, 13(6), 2180 - 91
Schizosaccharomyces pombe pfh1+ encodes an essential 5' to 3' DNA helicase that is a member of the PIF1 subfamily of DNA helicases; Zhou JQ et al.; The Saccharomyces cerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans . S . cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA . Here we describe the isolation and characterization of pfh1+, a Schizosaccharomyces pombe gene that encodes a Pif1-like protein . Pfh1p was the only S . pombe protein with high identity to Saccharomyces Pif1p . Unlike the two S . cerevisiae Pif1 subfamily proteins, the S . pombe Pfh1p was essential . Like Saccharomyces Pif1p, a truncated form of the S . pombe protein had 5' to 3' DNA helicase activity . Point mutations in an invariant lysine residue in the ATP binding pocket of Pfh1p had the same phenotype as deleting pfh1+, demonstrating that the ATPase/helicase activity of Pfh1p was essential . Although mutant spores depleted for Pfh1p proceeded through S phase, they arrested with a terminal cellular phenotype consistent with a postinitiation defect in DNA replication . Telomeric DNA was modestly shortened in the absence of Pfh1p . However, genetic analysis demonstrated that maintenance of telomeric DNA was not the sole essential function of S . pombe Pfh1p.

Mol Biol Cell, 2002 Jun, 13(6), 2157 - 69
A polymer model for large-scale chromatin organization in lower eukaryotes; Ostashevsky J; A quantitative model of large-scale chromatin organization was applied to nuclei of fission yeast Schizosaccharomyces pombe (meiotic prophase and G2 phase), budding yeast Saccharomyces cerevisiae (young and senescent cells), Drosophila (embryonic cycles 10 and 14, and polytene tissues) and Caenorhabditis elegans (G1 phase) . The model is based on the coil-like behavior of chromosomal fibers and the tight packing of discrete chromatin domains in a nucleus . Intrachromosomal domains are formed by chromatin anchoring to nuclear structures (e.g., the nuclear envelope) . The observed sizes for confinement of chromatin diffusional motion are similar to the estimated sizes of corresponding domains . The model correctly predicts chromosome configurations (linear, Rabl, loop) and chromosome associations (homologous pairing, centromere and telomere clusters) on the basis of the geometrical constraints imposed by nuclear size and shape . Agreement between the model predictions and literature observations supports the notion that the average linear density of the 30-nm chromatin fiber is approximately 4 nucleosomes per 10 nm contour length.

Biochemistry, 2002 Jun 18, 41(24), 7841 - 8
Characterization of a novel mammalian phosphatase having sequence similarity to Schizosaccharomyces pombe PHO2 and Saccharomyces cerevisiae PHO13; Ndubuisil MI et al.; p34, a specific p-nitrophenyl phosphatase (pNPPase) was identified and purified from the murine cell line EL4 in a screen for the intracellular molecular targets of the antiinflammatory natural product parthenolide . A BLAST search analysis revealed that it has a high degree of sequence similarity to two yeast alkaline phosphatases . We have cloned, sequenced, and expressed p34 as a GST-tagged fusion protein in Escherichia coli and an EE-epitope-tagged fusion protein in mammalian cells . Using p-nitrophenyl phosphate (pNPP) as a substrate, p34 is optimally active at pH 7.6 with a K(m) of 1.36 mM and K(cat) of 0.052 min(-1) . Addition of 1 mM Mg(2+) to the reaction mixture increases its activity by 14-fold . Other divalent metal ions such as Co(2+) and Mn(2+) also stimulated the activity of the enzyme, while Zn(2+), Fe(2+), and Cu(2+) had no effect . Furthermore, both NaCl and KCl enhanced the activity of the enzyme, having maximal effect at 50 and 75 mM, respectively . The enzyme is inhibited by sodium orthovanadate but not by sodium fluoride or okadaic acid . Mutational analysis data suggest that p34 belongs to the group of phosphatases characterized by the sequence motif DXDX(T/V).

Biochemistry, 2002 Jun 18, 41(24), 7688 - 94
Purification and partial characterization of a DNA 3'-phosphatase from Schizosaccharomyces pombe; Jilani A et al.; Cells that depend on oxygen for survival constantly produce reactive oxygen species that attack DNA to produce a variety of lesions, including single-strand breaks with 3'-blocking groups such as 3'-phosphate and 3'-phosphoglycolate . These 3'-blocking ends prevent the activity of DNA polymerase and are generally removed by DNA repair proteins with 3'-diesterase activity . We report here the purification and partial characterization of a 45 kDa protein from Schizosaccharomyces pombe total extract based on the ability of this protein to process bleomycin- or H(2)O(2)-damaged DNA in vitro to allow DNA repair synthesis by DNA polymerase I . Further analysis revealed that the 45 kDa protein removes 3'-phosphate ends created by the Escherichia coli fpg AP lyase following the incision of AP site but is unable to process the 3'-alpha,beta unsaturated aldehyde generated by E . coli endonuclease III . The protein cannot cleave DNA bearing AP sites, suggesting that it is not an AP endonuclease or AP lyase . We conclude that the 45 kDa protein purified from S . pombe is a DNA 3'-phosphatase.

Biochem Biophys Res Commun, 2002 May 17, 293(4), 1209 - 16
Plant pre-mRNA splicing in fission yeast, Schizosaccharomyces pombe; Sarmah B et al.; Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the splicing machinery . We have analyzed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency of a plant gene AmA1 in Schizosaccharomyces pombe . Earlier, we reported the cloning of AmA1, a seed albumin gene from Amaranthus hypochondriacus {A . Raina, A . Datta, Proc . Natl . Acad . Sci . USA 89 (1992) 11774} . In the absence of an in vitro splicing system for plants, the expression of AmA1 genomic clone in S . pombe has been used to analyze splicing of intron constructs . We aim to focus on S . pombe as a possible alternative and examined its effectiveness as a host for plant gene splicing . The results show here that pre-mRNA transcripts of AmA1 gene underwent splicing in S . pombe . (c) 2002 Elsevier Science (USA).

Mol Cell Biol, 2002 Jul, 22(13), 4598 - 606
Two ras pathways in fission yeast are differentially regulated by two ras guanine nucleotide exchange factors; Papadaki P et al.; How a given Ras prreotein coordinates multiple signaling inputs and outputs is a fundamental issue of signaling specificity . Schizosaccharomyces pombe contains one Ras, Ras1, that has two distinct outputs . Ras1 activates Scd1, a presumptive guanine nucleotide exchange factor (GEF) for Cdc42, to control morphogenesis and chromosome segregation, and Byr2, a component of a mitogen-activated protein kinase cascade, to control mating . So far there is only one established Ras1 GEF, Ste6 . Paradoxically, ste6 null (ste6 Delta) mutants are sterile but normal in cell morphology . This suggests that Ste6 specifically activates the Ras1-Byr2 pathway and that there is another GEF capable of activating the Scd1 pathway . We thereby characterized a potential GEF, Efc25 . Genetic data place Efc25 upstream of the Ras1-Scd1, but not the Ras1-Byr2, pathway . Like ras1 Delta and scd1 Delta, efc25 Delta is synthetically lethal with a deletion in tea1, a critical element for cell polarity control . Using truncated proteins, we showed that the C-terminal GEF domain of Efc25 is essential for function and regulated by the N terminus . We conclude that Efc25 acts as a Ras1 GEF specific for the Scd1 pathway . While ste6 expression is induced during mating, efc25 expression is constitutive . Moreover, Efc25 overexpression renders cells hyperelongated and sterile; the latter can be rescued by activated Ras1 . This suggests that Efc25 can recruit Ras1 to selectively activate Scd1 at the expense of Byr2 . Reciprocally, Ste6 overexpression can block Scd1 activation . We propose that external signals can partly segregate two Ras1 pathways by modulating GEF expression and that GEFs can influence how Ras is coupled to specific effectors.

Mol Cell Biol, 2002 Jul, 22(13), 4477 - 90
A conserved domain of Schizosaccharomyces pombe dfp1(+) is uniquely required for chromosome stability following alkylation damage during S phase; Fung AD et al.; The fission yeast Dbf4 homologue Dfp1 has a well-characterized role in regulating the initiation of DNA replication . Sequence analysis of Dfp1 homologues reveals three highly conserved regions, referred to as motifs N, M, and C . To determine the roles of these conserved regions in Dfp1 function, we have generated dfp1 alleles with mutations in these regions . Mutations in motif N render cells sensitive to a broad range of DNA-damaging agents and replication inhibitors, yet these mutant proteins are efficient activators of Hsk1 kinase in vitro . In contrast, mutations in motif C confer sensitivity to the alkylating agent methyl methanesulfonate (MMS) but, surprisingly, not to UV, ionizing radiation, or hydroxyurea . Motif C mutants are poor activators of Hsk1 in vitro but can fulfill the essential function(s) of Dfp1 in vivo . Strains carrying dfp1 motif C mutants have an intact mitotic and intra-S-phase checkpoint, and epistasis analysis indicates that dfp1 motif C mutants function outside of the known MMS damage repair pathways, suggesting that the observed MMS sensitivity is due to defects in recovery from DNA damage . The motif C mutants are most sensitive to MMS during S phase and are partially suppressed by deletion of the S-phase checkpoint kinase cds1 . Following treatment with MMS, dfp1 motif C mutants exhibit nuclear fragmentation, chromosome instability, precocious recombination, and persistent checkpoint activation . We propose that Dfp1 plays at least two genetically separable roles in the DNA damage response in addition to its well-characterized role in the initiation of DNA replication and that motif C plays a critical role in the response to alkylation damage, perhaps by restarting or stabilizing stalled replication forks.

Fungal Genet Biol, 2002 Jun, 36(1), 59 - 70
The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus; Kessler MM et al.; Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals . To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries . We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection . Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs . Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A . fumigatus genes do not have orthologs in these fungal species . This method allowed the expeditious identification of genes in a fungal pathogen . The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.

J Biol Chem, 2002 Aug 16, 277(33), 30394 - 400 Epub 2002 Jun 05.
Surplus zinc is handled by Zym1 metallothionein and Zhf endoplasmic reticulum transporter in Schizosaccharomyces pombe; Borrelly GP et al.; Homeostatic mechanisms prevent the accumulation of free zinc in the cytoplasm, raising questions regarding where surplus zinc is stored and how it is delivered to and from these stores . A genetic screen for zinc hypersensitivity in Schizosaccharomyces pombe identified a missense mutation truncating Zhf, an endoplasmic reticulum transporter . These cells were approximately 5-fold more zinc-sensitive than other independent mutants . The targeted disruption of zhf prevented growth on low zinc medium and caused hypersensitivity to elevated zinc/cobalt but resistance to cadmium . The exposure to elevated zinc but not copper also promotes the accumulation of transcripts encoding a metallothionein designated Zym1 . The Sty1 pathway is required for maximal zym1 expression but is not obligatory for zinc perception . The targeted disruption of zym1 impaired cadmium tolerance but only slightly impaired zinc tolerance, whereas zym1 overexpression substantially rescued zinc hypersensitivity of zhf(-) cells . Four equivalents of zinc were displaced from Zym1 by up to 12 equivalents of p-(hydroxymercuri)phenylsulphonate . Zym1 thiols react rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) compared with bacterial zinc metallothionein (6.8 and 0.2 x 10(-4) s(-1), respectively) . Zym1 is unlike known fungal metallothioneins that are induced by and sequester copper but not zinc . Less zinc but normal cadmium was accumulated by zym1Delta, consistent with zinc sequestration by Zym1 in vivo.

J Biol Chem, 2002 Aug 16, 277(33), 30264 - 70 Epub 2002 Jun 05.
Physical interaction between recombinational proteins Rhp51 and Rad22 in Schizosaccharomyces pombe; Kim WJ et al.; In eukaryotes, Rad51 and Rad52 are two key components of homologous recombination and recombinational repair . These two proteins interact with each other . Here we investigated the role of interaction between Rhp51 and Rad22, the fission yeast homologs of Rad51 and Rad52, respectively, on the function of each protein . We identified a direct association between the two proteins and their self-interactions both in vivo and in vitro . We also determined the binding domains of each protein that mediate these interactions . To characterize the role of Rhp51-Rad22 interaction, we used random mutagenesis to identify the mutants Rhp51 and Rad22, which cannot interact each other . Interestingly, we found that mutant Rhp51 protein, which cannot interact with either Rad22 or Rti1 (G282D), lost its DNA repair ability . In contrast, mutant Rad22 proteins, which cannot specifically bind to Rhp51 (S379L and P381L), maintained their DNA repair ability . These results suggest that the interaction between Rhp51 and Rad22 is crucial for the recombinational repair function of Rhp51 . However, the significance of this interaction on the function of Rad22 remains to be characterized further.

Biochem J, 2002 Jun 15, 364(Pt 3), 869 - 74
Role of ATP-binding motifs on DNA-binding activity and biological function of Rhp51, a Rad51 homologue in fission yeast; Kim WJ et al.; Rhp51, a RecA and Rad51 homologue of Schizosaccharomyces pombe, plays a pivotal role in homologous recombination and recombinational repair . It has a set of the well-conserved type A and type B ATP-binding motifs, which are highly conserved in all RecA homologues . In a previous study {Kim, Lee, Park, Park and Park (2001) Nucleic Acids Res . 29, 1724-1732}, we reported that a single mutation of the conserved lysine in A motif {Lys(155)-->Ala (K155A)} destroyed the DNA repair ability of Rhp51 and that overexpression of this mutant protein conferred dominant negativity . In the present paper, we investigated DNA-binding properties of recombinant Rhp51 and its mutant proteins . Purified Rhp51 protein showed ATP-dependent double- and single-strand DNA-binding activities . To characterize the role of ATP-binding motifs, we generated Rhp51 K155A and Rhp51 Asp(244)-->Gln (D244Q), which have a single amino acid substitution in A and B motifs respectively . Interestingly, K155A and D244Q mutations impaired ATP-dependent DNA binding in a different manner . K155A lost the DNA binding itself, whereas D244Q maintained the binding ability but lost the ATP dependency . However, despite the difference in DNA-binding ability, both mutations failed to rescue the methylmethane sulphonate and UV sensitivity of the rhp51Delta mutant . Together, these results suggested that not only the DNA binding but also the ATP dependence in DNA binding is required for proper in vivo functioning of Rhp51.

Biosci Biotechnol Biochem, 2002 Apr, 66(4), 928 - 33
Alpha-glucosidase mutant catalyzes "alpha-glycosynthase"-type reaction; Okuyama M et al.; Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe alpha-glucosidase eliminated the hydrolytic activity . The mutant enzyme (D481G) was found to catalyze the formation of an alpha-glucosidic linkage from beta-glucosyl fluoride and 4-nitrophenyl (PNP) alpha-glucoside to produce two kinds of PNP alpha-diglucosides, alpha-isomaltoside and alpha-maltoside . The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP alpha-isomaltoside and alpha-maltoside, respectively . PNP monoglycosides, such as alpha-xyloside, alpha-mannoside, or beta-glucoside, acted as the substrate, but PNP alpha-galactoside and maltose could not . No detectable product was observed in the combination of alpha-glucosyl fluoride and PNP alpha-glucoside . This study is the first report on an "alpha-glycosynthase"-type reaction to form an alpha-glycosidic linkage.

Nucleic Acids Res, 2002 Jun 1, 30(11), 2546 - 54
Dominant genetic screen for cofactors that enhance antisense RNA-mediated gene silencing in fission yeast; Raponi M et al.; Specific gene silencing has been demonstrated in a number of organisms by the introduction of antisense RNA . Mutagenesis of host-encoded factors has begun to unravel the mechanism of several forms of RNA-mediated gene silencing and has suggested that it may have been conserved through evolution . This has led to the identification of certain host genes, which, when mutated, abrogate this phenomenon . Conversely, the identification of other factors that, when co-expressed or overexpressed, can enhance gene inhibition is equally important for both elucidating the mechanism of this process and enhancing gene silencing in recalcitrant systems . We have taken such a dominant genetic approach to identify several host-encoded factors that dramatically enhance target gene silencing when co-expressed with antisense RNA in fission yeast . The transcription factor thi1 and, surprisingly, the ATP-dependent RNA helicase ded1 were initially shown to enhance gene silencing in this system . Additionally, screening of a Schizosaccharomyces pombe cDNA library identified four novel antisense-enhancing sequences (aes factors) all of which are homologous to genes encoding proteins with natural affinities for nucleic acids . These findings demonstrate the utility of this strategy in identifying host-encoded factors that can modulate gene silencing when co-expressed with antisense RNA and possibly other forms of gene-silencing activators.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7472 - 7
A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway; Marchetti MA et al.; The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood . Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways . The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase epsilon) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition) . These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.

EMBO J, 2002 Jun 3, 21(11), 2807 - 18
The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast; Shimada M et al.; During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division . Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs . A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression . This delay is dependent on rec12+, namely on DSB formation . Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded . We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2) . This implies that these genes are involved in a checkpoint that provides time to repair DSBs . Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner . dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+ . We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.

Genes Dev, 2002 May 15, 16(10), 1195 - 208
Cdc2-cyclin B kinase activity links Crb2 and Rqh1-topoisomerase III; Caspari T et al.; The availability of a sister chromatid, and thus the cell cycle phase in which DNA double-strand breaks (DSBs) occur, influences the choice between homologous recombination (HR) or nonhomologous end joining (NHEJ) . The sequential activation and destruction of CDK-cyclin activities controls progression through the cell cycle . Here we provide evidence that the major Schizosaccharomyces pombe CDK, Cdc2-cyclin B, influences recombinational repair of radiation-induced DSBs during the G(2) phase at two distinct stages . At an early stage in HR, a defect in Cdc2 kinase activity, which is caused by a single amino acid change in cyclin B, affects the formation of Rhp51 (Rad51(sp)) foci in response to ionizing radiation in a process that is redundant with the function of Rad50 . At a late stage in HR, low Cdc2-cyclin B activity prevents the proper regulation of topoisomerase III (Top3) function, disrupting a recombination step that occurs after the assembly of Rhp51 foci . This effect of Cdc2-cyclin B kinase on Top3 function is mediated by the BRCT-domain-containing checkpoint protein Crb2, thus linking checkpoint proteins directly with recombinational repair in G(2) . Our data suggest a model in which CDK activity links processing of recombination intermediates to cell cycle progression via checkpoint proteins.

Genetics, 2002 May, 161(1), 461 - 8
A 160-bp palindrome is a Rad50.Rad32-dependent mitotic recombination hotspot in Schizosaccharomyces pombe; Farah JA et al.; Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity . We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic recombination hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe . The hotspot activity of the palindrome, but not the basal level of recombination, was abolished by a rad50 deletion, by a rad50S "separation of function" mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog . We propose that upon extrusion of the palindrome the Rad50.Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.

Mol Cells, 2002 Apr 30, 13(2), 347 - 50
The 5'-flanking AT-rich sequence of thioredoxin gene involved in high-frequency transformation of the fission yeast; Lee YJ et al.; During the cloning of a genomic DNA encoding mitochondrial thioredoxin (TRX) from the fission yeast Schizosaccharomyces pombe, its 5' flanking sequence was involved in the high-frequency of transformation . The recombinant plasmid pYEX that was constructed in the 2 mu plasmid-derived vector pYES2 gave rise to a significant high-frequency of transformation in S . pombe, compared to the vector alone . Plasmid pYEX contains 1,090 bp 5'-flanking sequences of the TRX gene that are ahead of the open-reading frame . Similar 5'-flanking sequences, which were inserted in the lacZ fusion vector YEp357R that contained the 2 mu origin of replication, also gave a high-frequency of transformation . Dissection of the 5'-flanking sequence of the TRX gene by the HindIII restriction site showed that the 782 bp flanking sequence (5' upstream of the HindIII site) was responsible for the high-frequency of transformation by the 2 mu plasmid-derived vector DNAs . The putative sequence that is involved in the high-frequency of transformation contains a very high ratio of A-T pairs . No known functions were assigned on the sequence, which was estimated from the GenBank database.

Mol Cells, 2002 Apr 30, 13(2), 315 - 21
Pap1-mediated regulation of thioredoxin gene from Schizosaccharomyces pombe; Cho YW et al.; The genomic DNA encoding thioredoxin (TRX) was previously isolated from the fission yeast Schizosaccharomyces pombe . In this investigation, regulation of the S . pombe TRX gene was studied in lacZ translational fusions . The synthesis of beta-galactosidase from the fusion plasmid pYKT24 was significantly enhanced by treatments with cadmium chloride, zinc chloride, and high temperatures . Synthesis of beta-galactosidase from the fusion plasmid was significantly decreased by higher concentrations (5 microM, 10 microM) of mercuric chloride, whereas it was enhanced by its lower concentration (1 microM) . Diamide affected the synthesis of beta-galactosidase in the same manner with mercuric chloride . However, high osmolarity had no effect on the beta-galactosidase synthesis from the fusion plasmid pYKT24 . Various fusion plasmids were constructed to carry serially deleted upstream regions of the TRX gene . Pap1 mediates the regulation of the S . pombe TRX gene . The upstream region, between 987 and 1,270 bp from the translational initiation point, is responsible for the regulation.

J Biomol NMR, 2002 Apr, 22(4), 333 - 47
Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints; Kikuchi J et al.; We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa) . Although the two DNA-binding domains in the Abpl protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abpl protein was poorly defined, due to the lack of interdomain distance restraints . Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies . Neither the NH residual dipolar couplings nor the backbone 15N T1/T2 data were sufficient to determine the overall structure of the Abpl protein, due to spectral overlap . We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures . The range of the observed T1/T2 ratios was wider (20-50 for the secondary structure regions of Abp 1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1.DNA complex is ellipsoid . This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.

Biochim Biophys Acta, 2002 May 3, 1575(1-3), 143 - 7
Characterization and regulation of a second gene encoding thioredoxin from the fission yeast; Lee YJ et al.; A genomic DNA encoding a second thioredoxin (TRX2) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe . The cloned sequence contains 1823 bp and encodes a protein of 121 amino acids . It has extra N-terminal 17 amino acid residues compared to previously identified thioredoxin (TRX1), which are positively charged and hydrophobic amino acids . The additional N-terminal region contains a plausible prepeptidase cleavage site, indicating that the TRX2 protein exists in mitochondria . The cloned TRX2 gene produced functional TRX estimated with insulin reduction assay . The upstream region of the TRX2 gene was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357R . The 782 bp sequence in the region further upstream of the TRX2 gene was found to be inhibitory in its expression . Synthesis of beta-galactosidase from the fusion plasmid pYFX135-HRL was enhanced by the addition of aluminum chloride and ferrous chloride, indicating that the TRX2 protein is involved in stress response.

Biochim Biophys Acta, 2002 May 3, 1575(1-3), 31 - 9
Expression of the human homologue of the small nucleolar RNA-binding protein NHP2 gene during monocytic differentiation of U937 cells; Kang HS et al.; The human homologue of yeast NHP2, which is known to be a core protein component of yeast H/ACA small nucleolar ribonucleoprotein particles (snoRNPs), was identified by ODD-PCR as a 313-bp cDNA fragment exhibiting a distinct decrease in its expression level during TPA-induced differentiation of promonocytic U937 into monocytes and macrophages . Sequentially, a full-length cDNA of human NHP2 was isolated and the nucleotide sequence was determined . The NHP2 gene has a predicted 153 amino acid open reading frame, encoding 17.2 kDa protein that shares 38.4% and 44.4% identity with Saccharomyces cerevisiae NHP2 and Schizosaccharomyces pombe nhp2+, respectively . The TPA-induced differentiation of U937 cells, which also resulted in growth arrest, abruptly down-regulated the expression of NHP2 . Removal of TPA restored cell growth through the retrodifferentiation process and subsequent expression of NHP2 . NHP2 mRNA was markedly expressed in most tumor cells including Jurkat, K562, HL-60, U937, and HeLa S3 . In healthy human tissues, NHP2 mRNA was expressed at high levels in spleen, thymus, prostate, testis, ovary, small intestine, colon (mucosal lining), heart, brain, placenta, skeletal muscle, kidney and pancreas, and at low levels in liver, and very weakly in peripheral blood leukocyte and lung . NHP2 mRNA, undetectable in human peripheral T cells, was induced at a maximum level between late G(1) and S phase after polyclonal activation . The expression of NHP2 mRNA during the cell cycle progression of Jurkat T cells also reached a maximum between late G(1) and S phase . These results indicate that the human NHP2 gene may be regulated at the transcription level depending on tissue specificity and cellular proliferative status, and that the down-regulation of NHP2 expression during induced differentiation of U937 cells may result from the growth arrest accompanying the differentiation.

Braz J Med Biol Res, 2002 May, 35(5), 499 - 507
Centromere domain organization and histone modifications; Bjerling P et al.; Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles . Centromere structure appears to be organized in different, separable domains in order to accomplish these functions . Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences . Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms . The recent analysis of the centromere structure in the yeast S . pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere . The structural organization of the centromere is generally multilayered with a heterochromatin domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore . It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histones and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core . Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.

Int J Biochem Cell Biol, 2002 Sep, 34(9), 1031 - 4
RTS1-an eukaryotic terminator of replication; Vengrova S et al.; Eukaryotic replication termination generally occurs randomly in the region between two active origins . However, termination, or pausing of the replication forks has been observed at specific loci . Recently, a site-specific terminator of replication named RTS1 was shown to play an important role in mating-type switching in Schizosaccharomyces pombe . Mating-type switching in S . pombe relies on an imprinting event that chemically modifies one strand of the DNA at the mating-type locus mat1 . This imprint, that is formed only when mat1 is replicated in a specific direction, marks the DNA for a rearrangement leading to mating-type switching . The RTS1 element ensures that mat1 is replicated in the correct direction for imprinting and initiation of the subsequent mating-type switching event . This is the first replication terminator shown to play a role in cellular differentiation.

Curr Biol, 2002 Apr 30, 12(9), 751 - 6
Tea3p is a cell end marker activating polarized growth in Schizosaccharomyces pombe; Arellano M et al.; Eukaryotic cells are often polarized in their cytoplasmic structures, and this can be important for their function . The fission yeast Schizosaccharomyces pombe is a highly polarized cell that extends bipolarly along a single axis to generate a rod-shaped cell . It divides by medial fission to generate two equal-sized daughter cells that resume growth only at the old end . Once these cells have reached a particular length, they undergo NETO, new end take-off, whereby growth is activated at the other end to generate bipolarly extending cells . The activation and positioning of these growth zones are essential for maintaining growth in a straight line . Genetic analyses have identified many proteins involved in this process, like the cell end markers Tea1p and Pom1p and the kinases Orb2p/Shk1p/Pak1, Ssp1p, and Wee1p . Here, we describe tea3, a gene encoding a tea1-like protein with some similarities to ERM proteins . Tea3p is required for efficient NETO and for the proper placement of the septum . Like Pom1p, Tea3p localizes to cell ends, and its localization depends on microtubules and Tea1p . We propose that Tea3p is a novel cell end marker required specifically to activate polarized cell growth at the second end during NETO.

Curr Biol, 2002 Apr 30, 12(9), 724 - 9
Importance of a myosin II-containing progenitor for actomyosin ring assembly in fission yeast; Wong KC et al.; An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms {1-3} . Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells {4-5} . It is currently unknown if this myosin II-containing spot is important for cytokinesis . In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses . Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not . Maintenance of the myosin II-containing spot is independent of F-actin function . Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly {6} . Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring . Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division.

J Electron Microsc (Tokyo), 2000, 49(4), 569 - 78
Abnormal formation of the glucan network from regenerating protoplasts in Schizosaccharomyces pombe cps8 actin point mutant; Konomi M et al.; To study the close relationship between the actin cytoskeleton and cell wall formation, the process of cell wall formation in reverting protoplasts of the fission yeast, Schizosaccharomyces pombe, cps8 actin point mutant was investigated by ultra-high-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy (TEM) . The protoplast of the cps8 mutant began to form a glucan network in a unipolar manner and to secrete alpha-galactomannan . The site of cell wall formation grew in a cylindrical shape in the wild-type protoplast . The alpha-galactomannan did not fill in the intrafibrillar spaces completely, however, and the fibrils were exposed on the cell surface . UHR-LVSEM images indicated that the glucan fibrils were thin and rope-shaped, forming a looser network than the wild-type . TEM images indicated the finest fibrils were approximately 1.5 nm in diameter, the same diameter as the wild-type . These results suggest that the cps8 mutant was insufficient in developing cross-linkage with the glucan fibrils up to the wide ribbon shape as found in the wild-type {Osumi M et al . (1989) J . Electron Microsc . 38: 457-468; Osumi M (1998) Micron 29: 207-233} . These findings appear to indicate that the actin cytoskeleton controls formation of the glucan network and secretion of beta-1,6-glucan, and confirm the close relationship of the actin cytoskeleton and glucan formation.

Biochim Biophys Acta, 2002 Apr 12, 1574(3), 399 - 402
A second stress-inducible glutathione S-transferase gene from Schizosaccharomyces pombe; Cho YW et al.; A second glutathione S-transferase gene (GST II) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe . The nucleotide sequence determined contains 1908 bp including an open reading frame of 230 amino acids that would encode a protein of a molecular mass of 26843.4 Da . The amino acid sequence of the putative GST II is very homologous with that of the previously isolated GST gene (GST I) located in the same chromosome III of S . pombe . The cloned GST II gene produces the functional GST in S . pombe, and it gives much higher GST in the stationary phase than in the exponential phase . Regulation of the GST II gene was studied using the GST II-lacZ fusion . The synthesis of beta-galactosidase from the fusion plasmid is greatly enhanced by the treatments with oxidative stresses such as menadione and mercuric chloride . It is also induced by o-dinitrobenzene, one of the GST substrates . NO-generating S-nitroso-N-acetylpenicillamine has a weak induction effect on the expression of GST II gene . These results indicate that the S . pombe GST II gene is involved in the oxidative stress response and detoxification . However, physiological meaning on the existence of the two similar GST genes in S . pombe remains unknown yet.

Nat Cell Biol, 2002 May, 4(5), 384 - 8
Maintenance of replication forks and the S-phase checkpoint by Cdc18p and Orp1p; Murakami H et al.; S-phase and DNA damage checkpoint controls block the onset of mitosis when DNA is damaged or DNA replication is incomplete . It has been proposed that damaged or incompletely replicated DNA generates structures that are sensed by the checkpoint control pathway, although little is known about the structures and mechanisms involved . Here, we show that the DNA replication initiation proteins Orp1p and Cdc18p are required to induce and maintain the S-phase checkpoint in Schizosaccharomyces pombe . The presence of DNA replication structures correlates with activation of the Cds1p checkpoint protein kinase and the S-phase checkpoint pathway . By contrast, induction of the DNA damage pathway is not dependent on Orp1p or Cdc18p . We propose that the presence of unresolved replication forks, together with Orp1p and Cdc18p, are necessary to activate the Cds1p-dependent S-phase checkpoint.

Mol Cell, 2002 Apr, 9(4), 847 - 55
Meiotic DNA breaks at the S . pombe recombination hot spot M26; Steiner WW et al.; The ade6-M26 allele of Schizosaccharomyces pombe creates a well-defined meiotic recombination hot spot that requires a specific sequence, 5'-ATGACGT-3', and the Atf1*Pcr1 transcription factor for activity . We find that M26 stimulates the formation of meiosis-specific double-strand DNA breaks at multiple sites surrounding M26 . Like hot spot activity, breakage requires the M26 heptamer, Pcr1, and the general recombination factor Rec12 . When the M26 heptamer is moved to new positions within ade6, new break sites are observed spanning approximately 0.5-2 kb around the moved heptamer . Break frequency is strongly correlated with recombination frequency for these alleles . The occurrence of breaks at M26 suggests mechanistic similarities to hot spots in the distantly related yeast Saccharomyces cerevisiae.

Biochem J, 2002 Jul 1, 365(Pt 1), 1 - 6
A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning; Takahashi-Ando N et al.; Zearalenone (ZEN) is converted into a far less oestrogenic product by incubation with Clonostachys rosea IFO 7063 . An alkaline hydrolase responsible for the detoxification was purified to homogeneity from the fungus by a combination of salt precipitation and column chromatography methods . The purified enzyme was homodimeric with a subunit molecular mass of 30 kDa and contained an intra-subunit disulphide bridge . On the basis of the internal peptide sequences of the purified protein, we cloned the entire coding region of the gene (designated as zhd101) by PCR techniques . The ZEN degradation activity was detected in heterologous hosts (Schizosaccharomyces pombe and Escherichia coli) carrying the cloned gene . Zhd101 could be a promising genetic resource for in planta detoxification of the mycotoxin in important crops.

Genetics, 2002 Apr, 160(4), 1305 - 18
Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins; Gomez EB et al.; The six conserved MCM proteins are essential for normal DNA replication . They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases . It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity . To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast . Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex . Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences . Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin . We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.

Mol Microbiol, 2002 Apr, 44(2), 325 - 34
The p21-activated kinase, Shk1, is required for proper regulation of microtubule dynamics in the fission yeast, Schizosaccharomyces pombe; Qyang Y et al.; The p21-activated kinase, Shk1, is required for the proper establishment of cell polarity in the fission yeast, Schizosaccharomyces pombe . We showed recently that loss of the essential Shk1 inhibitor, Skb15, causes significant spindle defects in fission yeast, thus implicating Shk1 as a potential regulator of microtubule dynamics . Here, we show that cells deficient in Shk1 function have malformed interphase microtubules and mitotic microtubule spindles, are hypersensitive to the microtubule-destabilizing drug thiabendazole (TBZ) and cold sensitive for growth . TBZ treatment causes a downregulation of Shk1 kinase activity, which increases rapidly after release of cells from the drug, thus providing a correlation between Shk1 kinase function and active microtubule polymerization . Consistent with a role for Shk1 as a regulator of microtubule dynamics, green fluorescent protein (GFP)-Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles, as well as to cell ends and septum-forming regions of fission yeast cells . We show that loss of Tea1, a cell end- and microtubule-localized protein previously implicated as a regulator of microtubule dynamics in fission yeast, exacerbates the growth and microtubule defects resulting from partial loss of Shk1 and that Shk1 localizes to illicit growth tips produced by tea1 mutant cells . Our results demonstrate that Shk1 is required for the proper regulation of microtubule dynamics in fission yeast and implicate Tea1 as a potential Shk1 regulator.

Nucleic Acids Res, 2002 May 1, 30(9), 1952 - 8
Identification of histone H4-like TAF in Schizosaccharomyces pombe as a protein that interacts with WD repeat-containing TAF; Mitsuzawa H et al.; The general transcription factor TFIID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) . We previously identified two distinct WD repeat-containing TAFs, spTAF72 and spTAF73, in the fission yeast Schizosaccharomyces pombe . Here we report the identification of another S.pombe TAF, spTAF50, which is the S.pombe homolog of histone H4-like TAFs such as human TAF80, Drosophila TAF60 and Saccharomyces cerevisiae TAF60 . spTAF50 was identified in a two-hybrid screen as a protein that interacts with the C-terminal WD repeat-containing region of spTAF72 . Gene disruption revealed that spTAF50 is essential for cell viability . In vitro, spTAF50 bound to spTAF72 but less efficiently to spTAF73 . In S.pombe cells, spTAF50 was detected as a protein with an apparent molecular mass of approximately 50 kDa . Immunoprecipitation experiments demonstrated that spTAF50 is present in both the TFIID and SAGA-like complexes as in the case of spTAF72 . These results indicate that the C-terminal region of spTAF72, which largely consists of WD repeats, interacts with spTAF50 in the TFIID and SAGA-like complexes, suggesting a role for the WD repeat domain in the interaction between TAFs.

Mol Cell Biol, 2002 May, 22(10), 3537 - 48
The Schizosaccharomyces pombe rad60 gene is essential for repairing double-strand DNA breaks spontaneously occurring during replication and induced by DNA-damaging agents; Morishita T et al.; To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2 . This study characterizes one of these mutants, rad60-1 . The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60 . rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins . rad60-1 is hypersensitive to UV and gamma rays, epistatic to rhp51, and defective in the repair of DSBs caused by gamma-irradiation . The rad60-1 mutant is also temperature sensitive for growth . At the restrictive temperature (37 degrees C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain . The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells . rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A . R . Lehmann, M . Walicka, D . J . Griffiths, J . M . Murray, F . Z . Watts, S . McCready, and A . M . Carr, Mol . Cell . Biol . 15:7067-7080, 1995) . These results suggest that S . pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids . Rad60 may perform this function in concert with the SMC protein Rad18.

Mol Cell Biol, 2002 May, 22(10), 3292 - 300
c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage; Yoshida K et al.; The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage . The mechanisms by which c-Abl signals the induction of apoptosis are not understood . Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein . The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9 . c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents . The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-x(L) protein . The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis . These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.

Antioxid Redox Signal, 2002 Feb, 4(1), 123 - 40
Transcription factors regulating the response to oxidative stress in yeast; Moye-Rowley WS; A main avenue of defense against fungal infection uses oxidative killing of these and other microorganisms . Consequently, the ability of fungi to withstand an oxidative challenge has important implications for their ultimate pathogenicity in a host organism . Fungi also serve as an excellent model system for handling of reactive oxygen species in eukaryotic cells . For these reasons, a great deal of work has been invested in analyzing pathways involved in and the mechanisms regulating oxidative stress tolerance in fungi . The goal of this review is to discuss the current state of knowledge underlying the ability of fungal cells to mount a response to oxidative stress via activation of transcription factors . Studies in Saccharomyces cerevisiae have identified multiple transcriptional regulatory proteins that mediate tolerance to oxidative stress . Experiments focused on the fission yeast Schizosaccharomyces pombe have led to the discovery of protein kinase cascades highly related to mammalian stress-activated protein kinases . Recent studies on the pathogenic yeast Candida albicans have allowed analysis of the role of a critical oxidant-regulated transcription factor in this important human pathogen . Further understanding of oxidative stress resistance pathways in fungi is an important step toward understanding the molecular pathogenesis of these microorganisms.

Curr Biol, 2002 Apr 16, 12(8), R294 - 6
Genome sequencing: and then there were six; MacNeill SA; The genome of the fission yeast Schizosaccharomyces pombe has been sequenced, bringing the number of sequenced eukaryotic genomes to six . Analysis of the sequence predicts only 4824 protein coding genes, the smallest number yet recorded for a free-living eukaryote.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5527 - 32
cdk-7 Is required for mRNA transcription and cell cycle progression in Caenorhabditis elegans embryos; Wallenfang MR et al.; CDK7 is a cyclin-dependent kinase proposed to function in two essential cellular processes: transcription and cell cycle regulation . CDK7 is the kinase subunit of the general transcription factor TFIIH that phosphorylates the C-terminal domain (CTD) of RNA polymerase II, and has been shown to be broadly required for transcription in Saccharomyces cerevisiae . CDK7 can also phosphorylate CDKs that promote cell cycle progression, and has been shown to function as a CDK-activating kinase (CAK) in Schizosaccharomyces pombe and Drosophila melanogaster . That CDK7 performs both functions in metazoans has been difficult to prove because transcription is essential for cell cycle progression in most cells . We have isolated a temperature-sensitive mutation in Caenorhabditis elegans cdk-7 and have used it to analyze the role of cdk-7 in embryonic blastomeres, where cell cycle progression is independent of transcription . Partial loss of cdk-7 activity leads to a general decrease in CTD phosphorylation and embryonic transcription, and severe loss of cdk-7 activity blocks all cell divisions . Our results support a dual role for metazoan CDK7 as a broadly required CTD kinase, and as a CAK essential for cell cycle progression.

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