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