Mol Biol Cell, 2004 Apr, 15(4), 1793 - 801 Epub 2004 Feb 06. HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes; Chang F et al.; Human immunodeficiency virus type 1 (HIV-1) Vpr is a 15-kDa accessory protein that contributes to several steps in the viral replication cycle and promotes virus-associated pathology . Previous studies demonstrated that Vpr inhibits G2/M cell cycle progression in both human cells and in the fission yeast Schizosaccharomyces pombe . Here, we report that, upon induction of vpr expression, fission yeast exhibited numerous defects in the assembly and function of the mitotic spindle . In particular, two spindle pole body proteins, sad1p and the polo kinase plo1p, were delocalized in vpr-expressing yeast cells, suggesting that spindle pole body integrity was perturbed . In addition, nuclear envelope structure, contractile actin ring formation, and cytokinesis were also disrupted . Similar Vpr-induced defects in mitosis and cytokinesis were observed in human cells, including aberrant mitotic spindles, multiple centrosomes, and multinucleate cells . These defects in cell division and centrosomes might account for some of the pathological effects associated with HIV-1 infection. J Biol Chem, 2004 Apr 16, 279(16), 16144 - 53 Epub 2004 Feb 06. Primer utilization by DNA polymerase alpha-primase is influenced by its interaction with Mcm10p; Fien K et al.; Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains . In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S . pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates . We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p . Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared . Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA . Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not . These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation . In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates . A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex . We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication. EMBO J, 2004 Feb 25, 23(4), 930 - 8 Epub 2004 Feb 12. Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast; Kaykov A et al.; Mating-type switching in the fission yeast Schizosaccharomyces pombe is initiated by a strand-specific imprint located at the mating-type (mat1) locus . We show that the imprint corresponds to a single-strand DNA break (SSB), which is site- but not sequence-specific . We identified three novel cis-acting elements, involved in the formation and stability of the SSB . One of these elements is essential for a replication fork pause next to mat1 and interacts in vivo with the Swi1 protein . Another element is essential for maintaining the SSB during cell cycle progression . These results suggest that the DNA break appears during the S-phase and is actively protected against repair . Consequently, during the following round of replication, a polar double-strand break is formed . We show that when the replication fork encounters the SSB, the leading-strand DNA polymerase is able to synthesize DNA to the edge of the SSB, creating a blunt-ended recombination intermediate. EMBO J, 2004 Feb 25, 23(4), 919 - 29 Epub 2004 Feb 12. Fission yeast Clp1p phosphatase affects G(2)/M transition and mitotic exit through Cdc25p inactivation; Wolfe BA et al.; The Cdc14 family of phosphatases specifically reverses proline-directed phosphorylation events . In Saccharomyces cerevisiae, Cdc14p promotes Cdk1p inactivation at mitotic exit by reversing Cdk1p-dependent phosphorylations . Cdk1p is a proline-directed kinase whose activity is required in all eukaryotes for the transit into mitosis . At mitotic commitment, Cdk1p participates in its own regulation by activating the mitotic inducing phosphatase, Cdc25p, and inhibiting the opposing kinase, Wee1p . We have investigated the ability of Schizosaccharomyces pombe Clp1p, a Cdc14p homolog, to disrupt this auto-amplification loop . We show here that Clp1p is required to dephosphorylate, destabilize, and inactivate Cdc25p at the end of mitosis . Clp1p promotes recognition of Cdc25p by the anaphase-promoting complex/cyclosome, an E3 ubiquitin ligase . Failure to inactivate and destabilize Cdc25p in late mitosis delays progression through anaphase, interferes with septation initiation network signaling, and additionally advances the commitment to mitotic entry in the next cycle . This may be a widely conserved mechanism whereby Cdc14 proteins contribute to Cdk1p inactivation. EMBO J, 2004 Feb 25, 23(4), 908 - 18 Epub 2004 Feb 05. Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1; Den Elzen NR et al.; The G2 DNA damage checkpoint delays mitotic entry via the upregulation of Wee1 kinase and the downregulation of Cdc25 phosphatase by Chk1 kinase, and resultant inhibitory phosphorylation of Cdc2 . While checkpoint activation is well understood, little is known about how the checkpoint is switched off to allow cell cycle re-entry . To identify proteins required for checkpoint release, we screened for genes in Schizosaccharomyces pombe that, when overexpressed, result in precocious mitotic entry in the presence of DNA damage . We show that overexpression of the type I protein phosphatase Dis2 sensitises S . pombe cells to DNA damage, causing aberrant mitoses . Dis2 abrogates Chk1 phosphorylation and activation in vivo, and dephosphorylates Chk1 and a phospho-S345 Chk1 peptide in vitro . dis2Delta cells have a prolonged chk1-dependent arrest and a compromised ability to downregulate Chk1 activity for checkpoint release . These effects are specific for the DNA damage checkpoint, because Dis2 has no effect on the chk1-independent response to stalled replication forks . We propose that inactivation of Chk1 by Dis2 allows mitotic entry following repair of DNA damage in the G2-phase. J Cell Sci, 2004 Feb 29, 117(Pt 6), 967 - 74 Epub 2004 Feb 03. A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast; Oltra E et al.; We have cloned a fission yeast (Schizosaccharomyces pombe) homologue of Ini, a novel RING-finger-like protein recently identified in rat that interacts with the connexin43 (cx43) promoter and might be important for the response of the cx43 gene to estrogen . S . pombe cells deleted for ini1(+) fail to form colonies and arrest with an elongated cell phenotype, indicating a cell cycle block . Cell cycle arrest is dependent on expression of Wee1, but not Rad3, suggesting that it occurs independently of the DNA damage checkpoint control . Analysis of mRNA intermediates in cells depleted for Ini1 demonstrates that Ini1 is required for pre-mRNA splicing . We observe an accumulation of pre-mRNA for six of seven genes analysed, suggesting that Ini1 is required for general splicing activity . Interestingly, loss of Ini1 results in cell death that is partially suppressed by elimination of the Wee1 kinase . Therefore, Wee1 might promote cell death in the absence of Ini1. Mol Genet Genomics, 2004 Mar, 271(2), 161 - 70 Epub 2004 Feb 03. The transcription factor Pap1/Caf3 plays a central role in the determination of caffeine resistance in Schizosaccharomyces pombe; Benko Z et al.; We previously identified four nuclear genes (caf1+-caf4+) in Schizosaccharomyces pombe, mutations in which confer resistance to caffeine and brefeldin A . caf1+, caf2+ and caf4+ were sequenced and found to be identical to the multidrug-resistance/stress-response genes hba1, crm1 and trr1, respectively . Here we show that caf3 is allelic to pap1, which encodes an AP-1-like transcription factor . The allele associated with caffeine resistance, caf3-89, contains a single-nucleotide exchange that results in a Leu-->Ser exchange in the NES (nuclear export signal) domain of the gene product . Due to this alteration, the modified protein can not be exported from the nucleus back into the cytoplasm, and thus accumulates in the nucleus . The activity of pap1/caf3 is shown to be necessary for manifestation of the caffeine resistance caused by mutations in the genes hba1/caf1 and crm1/caf2 . We also cloned two genes that confer caffeine resistance when carried on a multicopy plasmid . One of them turned out to be a truncated allele of pad1/bfr2/sks1, which codes for a subunit of the 26 S proteosome . The putative product of the other gene, designated caf5, has a structure highly similar to that of MFS permeases . It contains two groups of six transmembrane spanning domains each, with the conserved motifs WRW, PET and GAIGGPVLGP in the fifth and sixth domains . These results are all consistent with our earlier hypothesis, which suggested that the caf genes are functionally interlinked in a complex detoxification mechanism . caf5 and pad1 may also encode parts of this mechanism. Nucleic Acids Res, 2004 Feb 02, 32(2), 736 - 41 Print 2004. Fission yeast Arp6 is required for telomere silencing, but functions independently of Swi6; Ueno M et al.; The actin-related proteins (Arps), which are subdivided into at least eight subfamilies, are conserved from yeast to humans . A member of the Arp6 subfamily in Drosophila, Arp4/Arp6, co-localizes with heterochromatin protein 1 (HP1) in pericentric heterochromatin . Fission yeast Schizosaccharomyces pombe possesses both an HP1 homolog and an Arp6 homolog . However, the function of S.pombe Arp6 has not been characterized yet . We found that deletion of arp6(+) impaired telomere silencing, but did not affect centromere silencing . Chromatin immunoprecipitation assays revealed that Arp6 bound to the telomere region . However, unlike Drosophila Arp4/Arp6, S.pombe Arp6 was distributed throughout nuclei . The binding of Arp6 to telomere DNA was not affected by deletion of swi6(+) . Moreover, the binding of Swi6 to telomere ends was not affected by deletion of arp6(+) . These results suggest that Arp6 and Swi6 function independently at telomere ends . We propose that the Arp6-mediated repression mechanism works side by side with Swi6-based telomere silencing in S.pombe. J Mol Biol, 2004 Feb 13, 336(2), 551 - 65 Identification of functionally important residues of Arp2/3 complex by analysis of homology models from diverse species; Beltzner CC et al.; We constructed homology models from the crystal structure of bovine Arp2/3 complex and sequences from six phylogenetically diverse species (Arabidopsis thaliana, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe) representing over 800 million years of evolution and used conserved surface residues to search for functionally important structural elements . The folds of the seven subunits and their core residues are well conserved, as well as residues at subunit interfaces . Only 45% of solvent-exposed surface residues are conserved and only 15% are identical across the seven species . Arp residues expected to interact with nucleotide and with the first and second actin subunits in a daughter filament are conserved and similar to actin . Arp residues required to form an Arp dimer differ from actin and may contribute to the dissociated state of the Arps in the unactivated complex . Conserved patches of surface residues guided us to candidate sites for nucleation promoting factors to interact with Arp3, Arp2, and ARPC3 . Other conserved residues were used with experimental constraints to propose how residues on the subunits ARPC1, ARPC2, ARPC4 and ARPC5 might interact with the mother filament at branch junctions. Genome Biol . 2004;5(2):R7 . Epub 2004 Jan 15. A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes; Koonin EV et al.; BACKGROUND: Sequencing the genomes of multiple, taxonomically diverse eukaryotes enables in-depth comparative-genomic analysis which is expected to help in reconstructing ancestral eukaryotic genomes and major events in eukaryotic evolution and in making functional predictions for currently uncharacterized conserved genes . RESULTS: We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs (eukaryotic orthologous groups or KOGs) from seven eukaryotic genomes: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Encephalitozoon cuniculi . Conservation of KOGs through the phyletic range of eukaryotes strongly correlates with their functions and with the effect of gene knockout on the organism's viability . The approximately 40% of KOGs that are represented in six or seven species are enriched in proteins responsible for housekeeping functions, particularly translation and RNA processing . These conserved KOGs are often essential for survival and might approximate the minimal set of essential eukaryotic genes . The 131 single-member, pan-eukaryotic KOGs we identified were examined in detail . For around 20 that remained uncharacterized, functions were predicted by in-depth sequence analysis and examination of genomic context . Nearly all these proteins are subunits of known or predicted multiprotein complexes, in agreement with the balance hypothesis of evolution of gene copy number . Other KOGs show a variety of phyletic patterns, which points to major contributions of lineage-specific gene loss and the 'invention' of genes new to eukaryotic evolution . Examination of the sets of KOGs lost in individual lineages reveals co-elimination of functionally connected genes . Parsimonious scenarios of eukaryotic genome evolution and gene sets for ancestral eukaryotic forms were reconstructed . The gene set of the last common ancestor of the crown group consists of 3,413 KOGs and largely includes proteins involved in genome replication and expression, and central metabolism . Only 44% of the KOGs, mostly from the reconstructed gene set of the last common ancestor of the crown group, have detectable homologs in prokaryotes; the remainder apparently evolved via duplication with divergence and invention of new genes . CONCLUSIONS: The KOG analysis reveals a conserved core of largely essential eukaryotic genes as well as major diversification and innovation associated with evolution of eukaryotic genomes . The results provide quantitative support for major trends of eukaryotic evolution noticed previously at the qualitative level and a basis for detailed reconstruction of evolution of eukaryotic genomes and biology of ancestral forms. J Agric Food Chem, 2004 Feb 11, 52(3), 493 - 7 Gluconic acid consumption in wines by Schizosaccharomyces pombe and its effect on the concentrations of major volatile compounds and polyols; Peinado RA et al.; Schizosaccharomyces pombe 1379 (ATCC 26760) yeast strain in wine substantially increases acetaldehyde and 1,1-diethoxyethane concentrations and to decreases levo-2,3-butanediol, glycerol, acetoin, and gluconic acid concentrations . In this study, S . pombe has been used for the first time to reduce gluconic acid in wine under aerobic conditions . Only acetaldehyde and acetoin exhibited significantly higher levels in the wines containing gluconic acid . The high in vitro specific activity of alcohol dehydrogenase observed may be directly related to the high production of acetaldehyde by the studied fission yeast. Nucleic Acids Res, 2004 Jan 29, 32(2), 632 - 42 Print 2004. Functional analysis of subcellular localization and protein-protein interaction sequences in the essential DNA ligase I protein of fission yeast; Martin IV et al.; DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus . In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair . In this report, dual nuclear-mitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe . Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability . In the nucleus, Lig I interacts with the sliding clamp proliferating cell nuclear antigen (PCNA) via a conserved PCNA interacting sequence motif known as a PIP box . Deletion of the PIP motif from the N-terminus of the nuclear form of Cdc17 fails to abolish Cdc17 function, indicating that PCNA binding by Cdc17 is not an absolute requirement for completion of S-phase. Cell Struct Funct, 2003 Oct, 28(5), 399 - 417 Vesicle-mediated protein transport pathways to the vacuole in Schizosaccharomyces pombe; Takegawa K et al.; The vacuole of Saccharomyces cerevisiae plays essential roles not only for osmoregulation and ion homeostasis but also down-regulation (degradation) of cell surface proteins and protein and organellar turnover . Genetic selections and genome-wide screens in S . cerevisiae have resulted in the identification of a large number of genes required for delivery of proteins to the vacuole . Although the complete genome sequence of the fission yeast Schizosaccharomyces pombe has been reported, there have been few reports on the proteins required for vacuolar protein transport and vacuolar biogenesis in S . pombe . Recent progress in the S . pombe genome project of has revealed that most of the genes required for vacuolar biogenesis and protein transport are conserved between S . pombe and S . cerevisiae . This suggests that the basic machinery of vesicle-mediated protein delivery to the vacuole is conserved between the two yeasts . Identification and characterization of the fission yeast counterparts of the budding yeast Vps and Vps-related proteins have facilitated our understanding of protein transport pathways to the vacuole in S . pombe . This review focuses on the recent advances in vesicle-mediated protein transport to the vacuole in S . pombe. Plant Cell, 2004 Feb, 16(2), 422 - 34 Epub 2004 Jan 23. Two classes of the CDh1-type activators of the anaphase-promoting complex in plants: novel functional domains and distinct regulation; Tarayre S et al.; The Cdc20 and Cdh1/Fzr proteins are the substrate-specific activators of the anaphase-promoting complex (APC) . In Medicago truncatula, the MtCcs52A and MtCcs52B proteins represent two subgroups of the Cdh1-type activators, which display differences in their cell cycle regulation, structure, and function . The ccs52A transcripts are present in all phases of the cell cycle . By contrast, expression of ccs52B is restricted to late G2-phase and M-phase, and its induced overexpression in BY2 cells inhibited mitosis . MtCcs52A is active in Schizosaccharomyces pombe and binds to the S . pombe APC, whereas MtCcs52B does not because of differences in the N-terminal region . We identified a new functional domain, the Cdh1-specific motif conserved in the Cdh1 proteins that, in addition to the C-box and the terminal Ile and Arg residues, was essential for the activity and required for efficient binding to the APC . Moreover, we demonstrate that cyclin-dependent kinase phosphorylation sites adjacent to the C-box may regulate the interaction with the APC . In the different plant organs, the expression of Mtccs52A and Mtccs52B displayed differences and indicated the involvement of the APC in differentiation processes. Mol Genet Genomics, 2004 Mar, 271(2), 197 - 207 Epub 2004 Jan 21. Characterization of Schizosaccharomyces pombe mutants defective in vacuolar acidification and protein sorting; Iwaki T et al.; The vacuolar H+-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells . To investigate the functional roles of the V-ATPase in Schizosaccharomyces pombe, the gene vma1 encoding subunit A or vma3 encoding subunit c was disrupted . Both deletion mutants lost the capacity for vacuolar acidification in vivo, and showed sensitivity to neutral pH or high concentrations of divalent cations including Ca2+ . The delivery of FM4-64 to the vacuolar membrane and accumulation of Lucifer Yellow CH were strongly inhibited in the vma1 and vma3 mutants . Moreover, deletion of the S . pombe vma1+ or vma3+ gene resulted in pleiotropic phenotypes consistent with lack of vacuolar acidification, including the missorting of vacuolar carboxypeptidase Y, abnormal vacuole morphology, and mating defects . These findings suggest that V-ATPase is essential for endocytosis, ion and pH homeostasis, and for intracellular targeting of vacuolar proteins and vacuolar biogenesis in S . pombe. J Cell Sci, 2004 Feb 15, 117(Pt 5), 689 - 700 Epub 2004 Jan 20. Role of microtubules and tea1p in establishment and maintenance of fission yeast cell polarity; Sawin KE et al.; Microtubules and the protein tea1p have important roles in regulating cell polarity in the fission yeast Schizosaccharomyces pombe . Here, using combinations of drugs, environmental perturbations and genetic mutants, we demonstrate that once a cell polarity axis is established, microtubules have at best a minor role in maintaining the cortical actin cytoskeleton and the rate and direction of cell growth . In addition, we find that after perturbations that disrupt cell polarity and the cortical actin cytoskeleton, microtubules are not required for re-establishment of polarity per se . However, after such perturbations, the distribution of cytoplasmic microtubules plays an important role in dictating the position of sites of polarity re-establishment . Furthermore, this influence of microtubule distribution on site selection during polarity re-establishment requires the presence of tea1p, suggesting that tea1p is crucial for coupling microtubule distribution to the regulation of cell polarity . Our results suggest a model in which, at the cellular level, two distinct and separable mechanisms contribute to how tea1p regulates site selection during polarity re-establishment . First, tea1p remaining at cell tips after cortical depolarization can serve as a cortical landmark for microtubule-independent site selection; second, tea1p newly targeted to the cell cortex by association with microtubules can promote the formation of polarity axes de novo. Trends Cell Biol, 1993 Feb, 3(2), 60 - 5 Signal transduction during mating and meiosis in S . pombe; Nielsen O; When starved, the fission yeast Schizosaccharomyces pombe responds by producing mating factors or pheromones that signal to cells of the opposite sex to initiate mating . Like its distant relative Saccharomyces cerevisiae, cells of the two mating types of S . pombe each produce a distinct pheromone that binds to receptors on the opposite cell type to induce the morphological changes required for mating . While the pathways are basically very similar in the two yeasts, pheromone signalling in S . pombe differs in several important ways from that of the more familiar budding yeast . In this article, Olaf Nielsen describes the pheromones and their effects in S . pombe, and compares the signalling pathways of the two yeasts. RNA, 2004 Feb, 10(2), 240 - 53 The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF 65) is essential in vivo but dispensable for activity in vitro; Banerjee H et al.; The general splicing factor U2AF(65) recognizes the polypyrimidine tract (Py tract) that precedes 3' splice sites and has three RNA recognition motifs (RRMs) . The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein-protein contacts with splicing factors mBBP/SF1 and SAP155 . Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF(65) activity in vitro . However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155 . First, deletion of RRM3 from the human protein has no effect on Py-tract binding . Second, RRM123 and RRM12 select similar sequences from a random pool of RNA . Third, deletion of RRM3 has no effect on the splicing activity of U2AF(65) in vitro . However, deletion of the RRM3 domain of S . pombe U2AF(59) abolishes U2AF function in vivo . In addition, certain amino acid substitutions on the four-stranded beta-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro . We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns . We discuss the implications of these observations on previous models of U2AF function. Genes Cells, 2004 Jan, 9(1), 71 - 82 Pmr1, a P-type ATPase, and Pdt1, an Nramp homologue, cooperatively regulate cell morphogenesis in fission yeast: the importance of Mn2+ homeostasis; Maeda T et al.; Schizosaccharomyces pombe pmr1+ gene is homologous to Saccharomyces cerevisiae PMR1 gene, which encodes the P-type Ca2+/Mn2+-ATPase . Addition of Mn2+, as well as Ca2+, to the medium induced pmr1+ gene expression in a calcineurin-dependent manner . The pmr1 knockout (Deltapmr1) cells exhibited hypersensitivity to EGTA . A screen for high gene dosage-suppressors of the EGTA-hypersensitive phenotype of Deltapmr1 led to the identification of pdt1+ gene, which encodes an Nramp-related metal transporter . The Deltapmr1 cells showed round cell morphology . Although Deltapdt1 cells appeared normal in the regular medium, it showed round cell morphology similar to that of the Deltapmr1 cells when Mn2+ was removed from the medium . The removal of Mn2+ also exacerbated the round morphology of the Deltapmr1 cells . The Deltapmr1Deltapdt1 double mutants grew very slowly and showed extremely aberrant cell morphology with round, enlarged and depolarized shape . The addition of Mn2+, but not Ca2+, to the medium completely suppressed the morphological defects, while both Mn2+ and Ca2+ markedly improved the slow growth of the double mutants . These results suggest that Pmr1 and Pdt1 cooperatively regulate cell morphogenesis through the control of Mn2+ homeostasis, and that calcineurin functions as a Mn2+ sensor as well as a Mn2+ homeostasis regulator. J Gen Appl Microbiol, 2003 Oct, 49(5), 271 - 8 Sulphate metabolism of selenate-resistant Schizosaccharomyces pombe mutants; Banszky L et al.; Selenate-resistant mutants were obtained from several strains of Schizosaccharomyces pombe . The obtained mutants all belonged to the same genetic complementation group . They were low in sulphate uptake activity and in ATP sulphurylase activity . They grew on medium containing sulphite, thiosulphate, cysteine or glutathione but not methionine as the sole source of sulphur . From these results, the mutants were concluded to carry mutations in the ATP sulphurylase gene . Inability of the mutants to utilize methionine as a sulphur source is rationalized by the absence of the reverse transsulphurylation pathway in this organism; wild type strains must utilize methionine as a sulphur source after it is degraded to give rise to sulphate. J Biol Chem, 2004 Mar 26, 279(13), 12744 - 54 Epub 2004 Jan 13. Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance; Hamasaki-Katagiri N et al.; The neuronal calcium sensor (NCS) proteins (e.g . recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors . Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p) . Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9 . Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch . The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein . Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes . The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)) . Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical . Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p . Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin . In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent . We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p. Mol Biol Cell, 2004 Apr, 15(4), 1568 - 79 Epub 2004 Jan 12. TopBP1 and ATR colocalization at meiotic chromosomes: role of TopBP1/Cut5 in the meiotic recombination checkpoint; Perera D et al.; Mammalian TopBP1 is a BRCT domain-containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint . Here, we study its possible role during meiosis in mice . TopBP1 foci are abundant during early prophase I and localize mainly to histone gamma-H2AX-positive domains, where DNA double-strand breaks (required to initiate recombination) occur . Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint . In the synapsis-defective Fkbp6(-/-) mouse, TopBP1 heavily stains unsynapsed regions of chromosomes . We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue . Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint . Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint. J Biol Chem, 2004 Mar 26, 279(13), 12706 - 13 Epub 2004 Jan 12. Tsc1+ and tsc2+ regulate arginine uptake and metabolism in Schizosaccharomyces pombe; van Slegtenhorst M et al.; Mutations in either TSC1 or TSC2 cause tuberous sclerosis complex, an autosomal dominant disorder characterized by seizures, mental retardation, and benign tumors of the skin, brain, heart, and kidneys . Homologs for the TSC1 and TSC2 genes have been identified in mouse, rat, Fugu, Drosophila, and in the yeast Schizosaccharomyces pombe . Here we show that S . pombe lacking tsc1+ or tsc2+ have similar phenotypes including decreased arginine uptake, decreased expression of three amino acid permeases, and low intracellular levels of four members of the arginine biosynthesis pathway . Recently, the small GTPase Rheb was identified as a target of the GTPase-activating domain of tuberin in mammalian cells and in Drosophila . We show that the defect in arginine uptake in cells lacking tsc2+ is rescued by the expression of a dominant negative form of rhb1+, the Rheb homolog in S . pombe, but not by expressing wild-type rhb1+ . Expression of the tsc2+ gene with a patient-derived mutation within the GAP domain did not rescue the arginine uptake defect in tsc2+ mutant yeast . Taken together, these findings support a model in which arginine uptake is regulated through tsc1+, tsc2+, and rhb1+ in S . pombe and also suggest a role for the Tsc1 and Tsc2 proteins in amino acid biosynthesis and sensing. Biochem Biophys Res Commun, 2004 Jan 30, 314(1), 259 - 67 A two-component signal transduction system with a PAS domain-containing sensor is required for virulence of Mycobacterium tuberculosis in mice; Rickman L et al.; Mycobacterium tuberculosis, the causative organism of tuberculosis, encounters oxidative stress during phagocytosis by the macrophage and following macrophage activation during an acquired immune response, and also from internally generated sources of radical oxygen intermediates through intermediary metabolism . We have identified the SenX3 protein, a sensor in 1 of the 11 complete pairs of two-component signal transduction systems in M . tuberculosis, as a possible orthologue of the Mak2p protein from the fission yeast Schizosaccharomyces pombe that is known to sense peroxide stress . Moreover, the SenX3-RegX3 two-component system was the top scoring hit in a homology search with the Escherichia coli ArcB-ArcA global control system of aerobic genes . Using structural modelling techniques we have determined that SenX3 contains a PAS-like domain found in a variety of prokaryotic and eukaryotic sensors of oxygen and redox . Mutants with knock-outs of senX3 or of the accompanying transcriptional regulator regX3 were constructed and found to have reduced virulence in a mouse model of tuberculosis infection, the mutant bacteria persisting for up to 4 months post-infection; complemented mutants had regained virulence confirming that it was mutations of this two-component system that were responsible for the avirulent phenotype . This work identifies the PAS domain as a possible drug target for tuberculosis and mutations in the senX3-regX signal transduction system as potentially useful components of live vaccine strains. EMBO J, 2004 Jan 28, 23(2), 376 - 85 Epub 2004 Jan 08. Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA; Xu YZ et al.; Communication between U1 and U2 snRNPs is critical during pre-spliceosome assembly; yet, direct connections have not been observed . To investigate this assembly step, we focused on Prp5, an RNA-dependent ATPase of the DExD/H family . We identified homologs of Saccharomyces cerevisiae Prp5 in humans (hPrp5) and Schizosaccharomyces pombe (SpPrp5), and investigated their interactions and function . Depletion and reconstitution of SpPrp5 from extracts demonstrate that ATP binding and hydrolysis by Prp5 are required for pre-spliceosome complex A formation . hPrp5 and SpPrp5 are each physically associated with both U1 and U2 snRNPs; Prp5 contains distinct U1- and U2-interacting domains that are required for pre-spliceosome assembly; and, we observe a Prp5-associated U1/U2 complex in S . pombe . Together, these data are consistent with Prp5 being a bridge between U1 and U2 snRNPs at the time of pre-spliceosome formation. Planta, 2004 Apr, 218(6), 906 - 15 Epub 2004 Jan 08. Profilin inhibits pollen tube growth through actin-binding, but not poly-L-proline-binding; McKenna ST et al.; Previously, we have shown that excess profilin inhibits pollen tube growth at significantly lower concentrations than it blocks cytoplasmic streaming . To elucidate the mechanism by which profilin achieves this function, we have employed mutant profilins from Schizosaccharomyces pombe {J . Lu and T.D . Pollard (2001) Mol Biol Cell 12:1161-1175}, which have defects in actin-binding, ability to inhibit polymerization, and poly- l-proline (PLP)-binding . Using Lilium longiflorum L . pollen and S . pombe profilins as wild-type (wt) standards, mutant profilins have been injected into pollen tubes of Lilium, and examined for their effects on growth rate and cell morphology . Our results show that mutant Y5D (68% actin-binding; 1.1% PLP-binding) is indistinguishable from wt-standard profilins . However mutant K81F (2.7% actin-binding; 77% PLP-binding) and especially mutant K67E (<1% actin-binding; 100% PLP-binding) are significantly less effective than wt-standard profilins in their ability to inhibit pollen tube growth . PLP also inhibits pollen tube growth . However, PLP is not different from K67E/PLP combined, which has no actin-binding, suggesting that PLP does not function by binding to profilin . In addition, there are differences in the morphology and F-actin organization in cells injected with PLP versus wt-profilin . Whereas wt-profilin causes a fragmentation and marked reduction in the amount of F-actin {L . Vidali et al . (2001) Mol Biol Cell 12:2534-2545}, PLP generates an extensive disorganization without any apparent reduction in the amount of F-actin . We conclude that along with actin-binding activity of profilin, PLP-containing proteins also participate in the growth control process, and can do so independently of binding to profilin. Nucleic Acids Res, 2004 Jan 02, 32(1), 201 - 10 Print 2004. Domainal organization of the lower eukaryotic homologs of the yeast RNA polymerase II core subunit Rpb7 reflects functional conservation; Singh SR et al.; The subcomplex of Rpb4 and Rpb7 subunits of RNA pol II in Saccharomyces cerevisiae is known to be an important determinant of transcription under a variety of physiological stresses . In S.cerevisiae, RPB7 is essential for cell viability while rpb4 null strains are temperature sensitive at low and high temperatures . The rpb4 null strain also shows defect in sporulation and a predisposed state of pseudohyphal growth . We show here that, apart from S.cerevisiae Rpb7, the Rpb7 homologs from other lower eukaryotes like Schizosaccharomyces pombe, Candida albicans and Dictyostelium discoideum can complement for the absence of S.cerevisiae RPB7 . This is the first report where we have shown that both the C.albicans and D.discoideum homologs are functional orthologs of the yeast RPB7 . We also show that high expression levels of S.cerevisiae RPB7 and its homologs rescue the sporulation defect of rpb4 homozygous null diploids, but only some of them cause significant enhancement of the pseudohyphal phenotype . Structural modeling of Rpb7 and its homologs show a high degree of conservation in the overall structure . This study indicates a structural and functional conservation of different Rpb7 across species and also a conserved role of Rpb7 in the subcomplex with respect to nutritional stress. Nucleic Acids Res, 2004 Jan 02, 32(1), 115 - 26 Print 2004. The major role of human AP-endonuclease homolog Apn2 in repair of abasic sites in Schizosaccharomyces pombe; Ribar B et al.; The abasic (AP) sites, the major mutagenic and cytotoxic genomic lesions, induced directly by oxidative stress and indirectly after excision of damaged bases by DNA glycosylases, are repaired by AP-endonucleases (APEs) . Among two APEs in Saccharomyces cerevisiae, Apn1 provides the major APE activity, and Apn2, the ortholog of the mammalian APE, provides back-up activity . We have cloned apn1 and apn2 genes of Schizosaccharomyces pombe, and have shown that inactivation of Apn2 and not Apn1 sensitizes this fission yeast to alkylation and oxidative damage-inducing agents, which is further enhanced by Apn1 inactivation . We also show that Uve1, present in S.pombe but not in S.cerevisiae, provides the back-up APE activity together with Apn1 . We confirmed the presence of APE activity in recombinant Apn2 and in crude cell extracts . Thus S.pombe is distinct from S.cerevisiae, and is similar to mammalian cells in having Apn2 as the major APE. Genetics, 2003 Dec, 165(4), 2289 - 93 Fission yeast Mus81.Eme1 Holliday junction resolvase is required for meiotic crossing over but not for gene conversion; Smith GR et al.; Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over . The Mus81.Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain . We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over . Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81.Eme1 protein complex resolves Holliday junctions in meiotic cells. J Cell Sci, 2004 Jan 26, 117(Pt 3), 389 - 96 Forespore membrane assembly in yeast: coordinating SPBs and membrane trafficking; Shimoda C; In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, sporulation involves de novo synthesis of forespore membrane (FSM) within the cytoplasm of mother cells . The FSM ultimately becomes the plasma membrane of the developing ascospores . Several protein components of the FSM have been identified . Visualization of these proteins has demonstrated the dynamic nature of the genesis and development of the FSM . It begins to develop at the differentiated outer plaque of the spindle pole bodies (SPBs) and extends outwards, encapsulating each of the haploid nuclei produced by meiosis . Several coiled-coil proteins are specifically recruited to the SPBs and play indispensable roles in FSM assembly . Temporal and spatial coordination of meiotic nuclear divisions and membrane assembly is of special importance . Comparison of the processes of FSM assembly in these yeasts shows that the basic mechanism has been conserved, even though the individual proteins involved are often different . Understanding these dynamic aspects of yeast sporulation will help to elucidate a general mechanism for the cellularization of cytoplasm containing multiple nuclei. J Biol Chem, 2004 Mar 19, 279(12), 10892 - 900 Epub 2003 Dec 29. Schizosaccharomyces pombe carboxyl-terminal domain (CTD) phosphatase Fcp1: distributive mechanism, minimal CTD substrate, and active site mapping; Hausmann S et al.; Schizosaccharomyces pombe Fcp1 is an essential protein serine phosphatase that preferentially dephosphorylates Ser(2) of the RNA polymerase II C-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7) . Here we show that: (i) Fcp1 acts distributively during the hydrolysis of substrates containing tandem Ser(2)-PO(4) heptads; (ii) the minimal optimal CTD substrate for Fcp1 is a single heptad of phasing S(5)P(6)S(7)Y(1)S(2)P(3)T(4); and (iii) single alanine mutations of flanking residues Tyr(1) or Pro(3) result in 6-fold decrements in CTD phosphatase activity . Fcp1 belongs to the DXDX(T/V) family of phosphotransferases that act via an acyl-phosphoenzyme intermediate . An alanine scan of 11 conserved positions of S . pombe Fcp1 identifies Thr(174), Tyr(237), Thr(243), and Tyr(249) as important for phosphatase activity . Structure-activity relationships at these positions were determined by introducing conservative substitutions . Our results, together with previous mutational studies, highlight a constellation of 11 amino acids that are conserved in all Fcp1 orthologs and likely comprise the active site. J Biol Chem, 2004 Mar 12, 279(11), 9937 - 43 Epub 2003 Dec 29. Ddb1 is required for the proteolysis of the Schizosaccharomyces pombe replication inhibitor Spd1 during S phase and after DNA damage; Bondar T et al.; Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression . A mutant S . pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype . We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1 . However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear . It was shown that Ddb1 associates with the Cop9/signalosome . Moreover, the phenotypes of the Deltaddb1 strain are remarkably similar to the Deltacsn1 (or Deltacsn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome . Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase . Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1 . Here we show that a S . pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage . Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the Deltaddb1 strain . In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Deltaddb1 strain . Our results establish an essential role of Ddb1 in the proteolysis of Spd1 . In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome. Mol Biol Cell, 2004 Mar, 15(3), 1425 - 35 Epub 2003 Dec 29. ago1 and dcr1, two core components of the RNA interference pathway, functionally diverge from rdp1 in regulating cell cycle events in Schizosaccharomyces pombe; Carmichael JB et al.; In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1+, dcr1+, and rdp1+, have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation . In the present study, we present evidence that null mutants for ago1+ and dcr1+ but not rdp1+, exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects . Subsequent analyses showed that ago1+ and dcr1+ are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults . Because rdp1+ is dispensable for this process, the functions of ago1+ and dcr1+ in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation . This was further supported by the finding that ago1+ is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant . Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast . Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1+ function in S . pombe . This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes. J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Feb 5, 800(1-2), 121 - 6 Heterologous overexpression and purification of four common subunits of nuclear RNA polymerases I, II and III of Schizosaccharomyces pombe; Proshkin SA et al.; Four subunits of Schizosaccharomyces pombe RNA polymerases I-III shared by all three enzymes (Rpb5, Rpb8, Rpb10 and Rpc10 {Rpb12}) have been overexpressed in Escherichia coli expression vectors pQE or pET as hexahistidine fusions . The recombinant proteins have been purified to near homogeneity using metal-chelate affinity chromatography and gel filtration . Homogeneity and identity of the purified protein preparations was demonstrated by denaturing polyacrylamide gel electrophoresis and TOF-MALDI mass spectrometry . The proteins were obtained in large amounts, and their preparations are currently in use for monoclonal antibody production and physico-chemical studies of these individual components of eukaryotic transcription enzymes. Dev Biol, 2004 Jan 1, 265(1), 127 - 39 The minibrain kinase homolog, mbk-2, is required for spindle positioning and asymmetric cell division in early C . elegans embryos; Pang KM et al.; In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo . The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential . Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C . elegans . In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized . Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants . These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division. BMC Biochem . 2003 Dec 23;4(1):18. Riboflavin synthase of Schizosaccharomyces pombe . Protein dynamics revealed by 19F NMR protein perturbation experiments; Fischer M et al.; BACKGROUND: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway . Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin . Riboflavin synthase is therefore a potential target for the development of antiinfective drugs . RESULTS: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E . coli strain . The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation . The protein sediments at an apparent velocity of 4.1 S at 20 degrees C . The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit . The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg(-1) min(-1) with an apparent KM of 5.7 microM . 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states . 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins . CONCLUSION: Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits . The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts . Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione . The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism. Plant J, 2004 Jan, 37(2), 269 - 81 Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors; Weber M et al.; The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri . We therefore used Arabidopsis GeneChips to identify genes more active in roots of A . halleri as compared to A . thaliana under control conditions . The two genes showing highest expression in A . halleri roots relative to A . thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn2+ uptake system . The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT-PCR analyses . A . halleri roots also show higher NA synthase protein levels . Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (CapLC-ESI-QTOF-MS)-based NA analysis procedure and consistently found higher NA levels in roots of A . halleri . Expression of a NA synthase in Zn2+-hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance . Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A . halleri . Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species. Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15670 - 5 Epub 2003 Dec 15. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes; Kroken S et al.; Fungal type I polyketides (PKs) are synthesized by PK synthases (PKSs) and include well known secondary metabolites such as the anticholesterol drug lovastatin and the potent natural carcinogen aflatoxin . Other type I PKs are known to be virulence factors for some plant pathogens and pigments such as melanin . In this study, a phylogenomic approach was used to investigate the origin and diversity of fungal genes encoding putative PKSs that are predicted to synthesize type I PKs . The resulting genealogy, constructed by using the highly conserved PKS ketosynthase (KS) domain, indicated that: (i) . Species within subphylum Pezizomycotina (phylum Ascomycota) but not early diverging ascomycetes, like Saccharomyces cerevisiae (Saccharomycotina) or Schizosaccharomyces pombe (Taphrinomycotina), had large numbers (7-25) of PKS genes . (ii) . Bacteria and fungi had separate groups of PKS genes; the few exceptions are the likely result of horizontal gene transfer from bacteria to various sublineages of fungi . (iii) . The bulk of genes encoding fungal PKSs fell into eight groups . Four groups were predicted to synthesize variously reduced PKs, and four groups were predicted to make unreduced PKs . (iv) . Species within different classes of Pezizomycotina shared the same groups of PKS genes . (v) . Different fungal genomes shared few putative orthologous PKS genes, even between closely related genomes in the same class or genus . (vi) The discontinuous distributions of orthologous PKSs among fungal species can be explained by gene duplication, divergence, and gene loss; horizontal gene transfer among fungi does not need to be invoked. Mol Cell Biochem, 2003 Dec, 254(1-2), 117 - 24 Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation; Wiebe CA et al.; The Na+/H+ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells . In eukaryotes it functions to exchange one proton for a sodium ion . In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons . In this study we used the Na+/H+ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein . Twelve mutant forms of the Na+/H+ exchanger were examined for their ability to translocate protons as assessed by a Cytosensor microphysiometer . Mutation of the amino acid Histidine 367 resulted in defective proton translocation . The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na+/H+ exchanger . Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na+/H+ exchanger . These results confirm that polar amino acids are important in proton flux activity of Na+/H+ exchangers. Mol Cell Biol, 2004 Jan, 24(1), 398 - 406 Transcription termination factor reb1p causes two replication fork barriers at its cognate sites in fission yeast ribosomal DNA in vivo; Sanchez-Gorostiaga A et al.; Polar replication fork barriers (RFBs) near the 3' end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes . In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage . On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs . Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe . S . pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3' to 5' order . The transcription termination protein reb1 and its two binding sites, present at the 3' end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo . On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors . Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs . These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also . S . pombe is suggested to be a transitional species in which both mechanisms coexist. Curr Microbiol, 2003 Nov, 47(5), 404 - 7 Hierarchical chromatin structure of Schizosaccharomyces pombe revealed by atomic force microscopy; Kobori T et al.; Many structural studies on higher eukaryotic chromatin have been carried out, but chromatin structure in fungi remains unclear . Schizosaccharomyces pombe has been used for investigations of chromosome function; however, the structural details of S . pombe chromatin have not been clarified owing to its small nucleus . We used atomic force microscopy for nano-scale imaging of chromatin isolated from S . pombe . Topographic images indicated that nuclear chromatin contained at least three hierarchical structures: large-scale chromatin fibers, spherical domains in the fibers, and nodules in the domains . The average diameters of the domain and the nodule were 363 +/- 85.2 nm and 46.2 +/- 9.30 nm . Each structure comprising the hierarchy was similar to higher eukaryotic chromatin thus far observed, despite definite differences in chromatin organization at the nucleosomal level . The presence of histone H1 suggested that there might be an alternative to compensate for histone H1 lacking in S . pombe. Mol Biol Cell, 2004 Feb, 15(2), 851 - 60 Epub 2003 Dec 10. Global gene expression responses of fission yeast to ionizing radiation; Watson A et al.; A coordinated transcriptional response to DNA-damaging agents is required to maintain genome stability . We have examined the global gene expression responses of the fission yeast Schizosaccharomyces pombe to ionizing radiation (IR) by using DNA microarrays . We identified approximately 200 genes whose transcript levels were significantly altered at least twofold in response to 500 Gy of gamma IR in a temporally defined manner . The majority of induced genes were core environmental stress response genes, whereas the remaining genes define a transcriptional response to DNA damage in fission yeast . Surprisingly, few DNA repair and checkpoint genes were transcriptionally modulated in response to IR . We define a role for the stress-activated mitogen-activated protein kinase Sty1/Spc1 and the DNA damage checkpoint kinase Rad3 in regulating core environmental stress response genes and IR-specific response genes, both independently and in concert . These findings suggest a complex network of regulatory pathways coordinate gene expression responses to IR in eukaryotes. Genetics, 2003 Nov, 165(3), 1031 - 43 Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination; Grishchuk AL et al.; The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+ . We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores . Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S . pombe are required for normal levels of meiotic recombination . Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1 . It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability . Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles . Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion) . On the basis of our results, we present a model for the involvement of five RecA-like proteins of S . pombe in meiotic recombination and discuss their respective roles. J Biol Chem, 2004 Mar 5, 279(10), 9462 - 74 Epub 2003 Dec 10. The Schizosaccharomyces pombe corepressor Tup11 interacts with the iron-responsive transcription factor Fep1; Znaidi S et al.; The Schizosaccharomyces pombe fep1(+) gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations . This transcriptional response is altered only in strains harboring a combined deletion of both tup11(+) and tup12(+) genes . This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa . The tup11(+)- and tup12(+)-encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor . Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other . The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11 . Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association . Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1 . Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted beta-propeller structure of this motif . Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated . In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain . Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes. Bioinformatics, 2003 Dec 12, 19(18), 2502 - 4 Characterizing gene sets with FuncAssociate; Berriz GF et al.; SUMMARY: FuncAssociate is a web-based tool to help researchers use Gene Ontology attributes to characterize large sets of genes derived from experiment . Distinguishing features of FuncAssociate include the ability to handle ranked input lists, and a Monte Carlo simulation approach that is more appropriate to determine significance than other methods, such as Bonferroni or idak p-value correction . FuncAssociate currently supports 10 organisms (Vibrio cholerae, Shewanella oneidensis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Caenorhaebditis elegans, Drosophila melanogaster, Mus musculus, Rattus norvegicus and Homo sapiens) . AVAILABILITY: FuncAssociate is freely accessible at Source code (in Perl and C) is freely available to academic users 'as is'. Eukaryot Cell, 2003 Dec, 2(6), 1274 - 87 Sla1, a Schizosaccharomyces pombe homolog of the human La protein, induces ectopic meiosis when its C terminus is truncated; Tanabe K et al.; Sla1 is a Schizosaccharomyces pombe homolog of the human La protein . La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved . In this study, we show that the S . pombe La homolog (Sla1) is involved in regulating sexual development . Sla1 truncated in the C terminus (Sla1DeltaC) induced ectopic sporulation in the ras1Delta strain and several other sporulation-deficient mutants . The C terminus contains a nuclear localization signal . While full-length Sla1 localizes in the nucleus, Sla1DeltaC is found throughout the cell, suggesting the cytoplasmic localization of Sla1DeltaC is involved in its sporulation-inducing activity . Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation . The La motif (RRM1) is not involved in this activity . Strikingly, Sla1DeltaC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation . Sla1DeltaC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1DeltaC requires Mei2 to induce haploid meiosis . Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant . Two-hybrid analysis indicated that Pat1 interacts with Sla1DeltaC but not full-length Sla1 . Thus, Sla1DeltaC may block Pat1 activity . This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis . Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced . The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins. Eukaryot Cell, 2003 Dec, 2(6), 1151 - 61 Whole-genome analysis of two-component signal transduction genes in fungal pathogens; Catlett NL et al.; Two-component phosphorelay systems are minimally comprised of a histidine kinase (HK) component, which autophosphorylates in response to an environmental stimulus, and a response regulator (RR) component, which transmits the signal, resulting in an output such as activation of transcription, or of a mitogen-activated protein kinase cascade . The genomes of the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans encode one, three, and three HKs, respectively . In contrast, the genome sequences of the filamentous ascomycetes Neurospora crassa, Cochliobolus heterostrophus (Bipolaris maydis), Gibberella moniliformis (Fusarium verticillioides), and Botryotinia fuckeliana (Botrytis cinerea) encode an extensive family of two-component signaling proteins . The putative HKs fall into 11 classes . Most of these classes are represented in each filamentous ascomycete species examined . A few of these classes are significantly more prevalent in the fungal pathogens than in the saprobe N . crassa, suggesting that these groups contain paralogs required for virulence . Despite the larger numbers of HKs in filamentous ascomycetes than in yeasts, all of the ascomycetes contain virtually the same downstream histidine phosphotransfer proteins and RR proteins, suggesting extensive cross talk or redundancy among HKs. Yeast, 2003 Dec, 20(16), 1349 - 58 Role of Tea1p, Tea3p and Pom1p in the determination of cell ends in Schizosaccharomyces pombe; Niccoli T et al.; Schizosaccharomyces pombe cells are rod-shaped and grow along a single axis from their two ends . Microtubules extend from the cell centre terminating at the cell ends . The ERM(ezrin/radixin/moesin)-like proteins Tea1p and Tea3p, and the Dyrk-like kinase Pom1p are cell end markers involved in the regulation of growth and microtubular dynamics at the cell ends . We have analysed the relative contribution of these three proteins to the determination of cell ends as sites both for cell growth and for microtubular termination . Pom1Delta, in combination with Tea1Delta or Tea3Delta, has the greatest difficulty in relocalizing actin to the cell ends following actin depolymerization and generates the most defective growth pattern . Tea1Delta, in combination with Pom1Delta or Tea3Delta, displays the highest number of microtubules bending round the cell ends . Tea1DeltaPom1Delta, which has the most defective growth pattern and microtubules, also displays the highest number of branched cells . We show that Tea1p, Tea3p and Pom1p all contribute, to different extents, to the determination of cell ends, as sites for both cell growth and microtubular termination . We also show that the fission yeast cell relies on both the positioning of landmarks and a properly organized microtubule cytoskeleton to direct cell growth . Microbiology, 2003 Dec, 149(Pt 12), 3629 - 37 Genetic control of chlamydospore formation in Candida albicans; Nobile CJ et al.; The chlamydospore is a distinctive morphological feature of the fungal pathogen Candida albicans that can be induced to form in oxygen-limited environments and has been reported in clinical specimens . Chlamydospores are not produced by the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, so there is limited understanding of the pathways that govern their development . Here, the results of a forward genetic approach that begins to define the genetic control of chlamydospore formation are described . Six genes - ISW2, MDS3, RIM13, RIM101, SCH9 and SUV3 - are required for efficient chlamydospore formation, based on the phenotypes of homozygous insertion mutants and reconstituted strains . Mutations in ISW2, SCH9 and SUV3 completely abolish chlamydospore formation . Mutations in RIM13, RIM101 and MDS3 delay normal chlamydospore formation . The involvement of alkaline pH-response regulators Rim13p and Mds3p in chlamydospore formation is unexpected in view of the fact that chlamydospores in the inducing conditions used here are repressed in alkaline media. Mol Biol Cell, 2004 Mar, 15(3), 1160 - 71 Epub 2003 Dec 02. Meiosis-specific failure of cell cycle progression in fission yeast by mutation of a conserved beta-tubulin residue; Paluh JL et al.; The microtubule cytoskeleton is involved in regulation of cell morphology, differentiation, and cell cycle progression . Precisely controlled dynamic properties are required for these microtubule functions . To better understand how tubulin's dynamics are embedded in its primary sequence, we investigated in vivo the consequences of altering a single, highly conserved residue in beta-tubulin that lies at the interface between two structural domains . The residue differs between the cold-adapted Antarctic fish and temperate animals in a manner that suggests a role in microtubule stability . Fungi, like the Antarctic fish, have a phenylalanine in this position, whereas essentially all other animals have tyrosine . We mutated the corresponding residue in fission yeast to tyrosine . Temperature effects were subtle, but time-lapse microscopy of microtubule dynamics revealed reduced depolymerization rates and increased stability . Mitotic exit signaled by breakdown of the mitotic spindle was delayed . In meiosis, microtubules displayed prolonged contact to the cell cortex during horsetail movement, followed by completion of meiosis I but frequent asymmetric failure of meiosis II spindle formation . Our results indicate that depolymerization dynamics modulated through interdomain motion may be important for regulating a subset of plus-end microtubule complexes in Schizosaccharomyces pombe. Nucleic Acids Res, 2003 Dec 15, 31(24), 7141 - 9 A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation; Ono Y et al.; Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair . In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation . So far, only one mutant allele of the rad11(+) gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe . To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant . rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and gamma-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity . Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening . Moreover, Rad11-Myc bound to telomere in a ChIP assay . These results strongly suggest that RPA is directly involved in telomere maintenance. Nucleic Acids Res, 2003 Dec 15, 31(24), 7110 - 6 Functional significance of intermediate cleavages in the 3'ETS of the pre-rRNA from Schizosaccharomyces pombe; Ivakine E et al.; Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sites that are not always conserved closely in the course of evolution . Both in eukaryotes and bacteria genetic analyses and in vitro studies have strongly implicated RNase III-like enzymes in the processing of rRNA precursors . In Schizosacharomyces pombe, for example, the RNase III-like Pac1 nuclease has been shown to cleave the free 3'ETS at two known intermediate sites but, in the presence of RAC protein, the same RNA also is cleaved at the 3'-end of the 25 S rRNA sequence . In this study normal and mutant 3'ETS sequences were digested with the Pac1 enzyme to further evaluate its role in rRNA processing . Accurate cleavage at the known intermediate processing sites was dependent on the integrity of the helical structure at these sites as well as a more distal upper stem region in the conserved extended hairpin structure of the 3'ETS . The cleavage of mutant 3'ETS sequences also generally correlated with the known effects of these mutations on rRNA production, in vivo . One mutant, however, was efficiently processed in vivo but was not a substrate for the Pac1 nuclease, in vitro . In contrast, in the presence of RAC protein, the same RNA remained susceptible to Pac1 nuclease cleavage at the 3'-end of the 25 rRNA sequence, indicating that the removal of the 3'ETS does not require cleavage at the intermediate sites . These results suggest that basic maturation pathways may be less complex than previously reported raising similar questions about other intermediate processing sites, which have been identified by analyses of termini, and/or processing, in vitro. Curr Biol, 2003 Dec 2, 13(23), 2015 - 24 Regulated mRNA stability of the Cdk inhibitor Rum1 links nutrient status to cell cycle progression; Daga RR et al.; BACKGROUND: The survival of a cell depends on continuous sensing of the nutritional environment and appropriate coordination of the cell cycle . The fission yeast Schizosaccharomyces pombe is an excellent model system in which to study these processes . In the presence of nutrients, fission yeast cells grow and divide, spending most of their time in G2; when nutrients are limiting, they are promoted into mitosis and arrest the cell cycle in G1 . The molecular mechanisms underlying this response are currently unknown.RESULTS: Here, we show that expression of the fission yeast Cdk inhibitor Rum1, a key regulator of Cdc2/cyclin B in G1, is subject to regulated mRNA stability in response to nutrient deprivation . In complete minimal medium, rum1 mRNAs are very unstable . Following nitrogen starvation, rum1 mRNAs are rapidly stabilized, allowing the accumulation of Rum1 protein to delay the G1 phase of the subsequent cell cycle . Instability of rum1 mRNAs in complete minimal medium depends on the presence of AU-rich elements in the 3'UTR . We also show that lack of this mechanism has consequences in the mitotic cell cycle, in meiosis, and in the control of ploidy.CONCLUSION: We propose that mRNA stability is an important mechanism to fine tune the expression of the rum1 gene, in order to allow the production of appropriate levels of Rum1 protein in response to changes in the nutritional environment. Mol Genet Genomics, 2004 Feb, 271(1), 82 - 90 Epub 2003 Dec 03. Fission yeast Cdc37 is required for multiple cell cycle functions; Westwood PK et al.; The identification of a Schizosaccharomyces pombe homologue of the cdc37 gene is described . The gene product is most similar to the budding yeast homologue, but shows similarity to metazoan Cdc37 proteins, with a region of high similarity at the extreme N-terminus . Gene transplacement experiments in diploid cells followed by tetrad dissection show that the gene is essential . Depletion of the gene product after switching off expression of cdc37 from the regulatable nmt81 promoter results in cessation of growth and division . The cells arrest heterogeneously, with a significant proportion showing mitotic defects; paradoxically, a proportion of the cells show a short-cell phenotype consistent with an advanced cell cycle. Curr Genet, 2004 Mar, 45(3), 170 - 5 Epub 2003 Dec 02. Fungal proteomics: mapping the mitochondrial proteins of a Trichoderma harzianum strain applied for biological control; Grinyer J et al.; Mitochondria are essential for cellular functions across organisms . A proteomic approach was taken to separate and identify mitochondrial proteins from a strain of Trichoderma harzianum with well established biocontrol properties . We optimized a method for the preparation of a sample enriched with mitochondria by ensuring efficient cell lysis and including several washing steps to minimize cytoplasmic contamination . We separated hundreds of proteins, using two-dimensional gel electrophoresis and identified 31 protein spots from T . harzianum, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatrography with mass spectrometry . Over 50% of the identified proteins were known to localize in the mitochondria . Three protein spots were identified from T . harzianum and a further five protein spots from the genera Trichoderma and Hypocrea . The remaining protein spots were identified by cross-species identification from other filamentous fungi, including Neurospora crassa and Aspergillus spp, and yeasts, including Saccharomyces cerevisiae and Schizosaccharomyces pombe . In total, we identified 25 protein spots from T . harzianum, representing proteins that have not been characterized in existing Trichoderma protein databases . To our knowledge, this is the first two-dimensional mitochondrial protein map of a filamentous fungus. FEBS Lett, 2003 Dec 4, 555(2), 335 - 40 Cyclophilin sensitivity to sanglifehrin A can be correlated to the same specific tryptophan residue as cyclosporin A; Pemberton TJ et al.; Sanglifehrin A (SFA) is a recently discovered immunosuppressant drug that shares its intracellular target with the major immunosuppressant drug cyclosporin A (CsA) . Both bind to and inhibit the cyclophilins, a diverse family of proteins found throughout nature that share a conserved catalytic domain . Although they share this common protein target, the mechanism of action of the cyclophilin-SFA complex has been reported as distinct from that of the well-studied cyclophilin-CsA complex . The X-ray structure of a macrolide analogue of SFA's cyclic region complexed with cyclophilin A has recently been resolved, but this left the placement of the linear region of SFA unresolved . Using five cyclophilins from the fission yeast Schizosaccharomyces pombe, and a mutant of one of these proteins, SpCyp3-F128W, we have shown that the sensitivity of cyclophilins to SFA can be correlated to the same specific tryptophan residue that has previously been identified to correlate to CsA sensitivity, and that the tail of SFA may be responsible for mediating this sensitivity. J Mol Biol, 2003 Dec 12, 334(5), 1117 - 31 Sub-families of alpha/beta barrel enzymes: a new adenine deaminase family; Ribard C et al.; No gene coding for an adenine deaminase has been described in eukaryotes . However, physiological and genetical evidence indicates that adenine deaminases are present in the ascomycetes . We have cloned and characterised the genes coding for the adenine deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe . The A.nidulans gene was expressed in Escherichia coli and the purified enzyme shows adenine but not adenosine deaminase activity . The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine deaminases rather than to the bacterial adenine deaminases . The latter are related to allantoinases, ureases and dihydroorotases . The fungal adenine deaminases and the homologous adenosine deaminases differ in a number of residues, some of these being clearly involved in substrate specificity . Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity . These results imply that adenine deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the alpha/beta barrel scaffold. Mutat Res, 2003 Nov 27, 532(1-2), 227 - 43 Apoptosis-like yeast cell death in response to DNA damage and replication defects; Burhans WC et al.; In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast . Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells . Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death . ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs) . These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes . This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage . Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex . Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them . This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer . The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication. Mutat Res, 2003 Nov 27, 532(1-2), 137 - 55 Regulation of alternative replication bypass pathways at stalled replication forks and its effects on genome stability: a yeast model; Barbour L et al.; Replication-blocking lesions result in increased genomic instability by stalling replication forks . Eukaryotic cells appear to have evolved several surveillance and repair/bypass mechanisms to ensure that replication can be resumed at these stalled forks . In the yeast Saccharomyces cerevisiae, the helicases Srs2 and Sgs1 appear to play a role in controlling the processing and stabilization of stalled replication forks . These proteins appear to be tightly regulated throughout the cell cycle and play a direct role in DNA-damage checkpoints . This allows the cells to determine the best mechanism to reestablish replication at the stalled fork: by shuttling the lesion into the RAD6-dependent pathway that can lead to error-free or error-prone bypass; or by using homologous recombination . Under conditions where both the RAD6-dependent pathway and recombination are disabled, the cells can bypass the lesion using a novel damage avoidance mechanism that is controlled by Mgs1 . Replication fork bypass processes appear to be highly conserved within eukaryotes, with homologs for SGS1 and MGS1 found in both Schizosaccharomyces pombe and mammalian cells. Mutat Res, 2003 Nov 27, 532(1-2), 59 - 73 Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis; Kai M et al.; Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats . Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype . Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants . Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa) . Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator . This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks.Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling . Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication . These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress . These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development. Mutat Res, 2003 Nov 27, 532(1-2), 21 - 7 Cdc7 kinases (DDKs) and checkpoint responses: lessons from two yeasts; Duncker BP et al.; Principally characterized for its requirement in the initiation of DNA replication, compelling evidence from two yeast model organisms now points to a central role for the Dbf4/Cdc7 kinase complex in S-phase checkpoint responses . Among the key findings supporting this view are observations that orthologs Dfp1 (Schizosaccharomyces pombe) and Dbf4 (Saccharomyces cerevisiae) interact with equivalent checkpoint kinases Cds1 and Rad53, respectively, and that mutants for Dbf4 and Cdc7 in these species are sensitive to genotoxic agents . Recently, these findings have been extended through mutational analyses of conserved regions in both Dfp1 and Dbf4, leading to the identification of distinct motifs which mediate cellular responses to DNA damage and replication fork arrest . The present review is a comparative survey of data obtained from studies conducted with S . pombe and S . cerevisae, and a consideration of models for the role played by Dbf4/Cdc7 in checkpoint responses. Mol Genet Genomics, 2004 Feb, 271(1), 60 - 71 Epub 2003 Nov 29. DSC1-MCB regulation of meiotic transcription in Schizosaccharomyces pombe; Cunliffe L et al.; Meiosis is initiated from the G1 phase of the mitotic cell cycle, and consists of pre-meiotic S-phase followed by two successive nuclear divisions . Here we show that control of gene expression during pre-meiotic S-phase in the fission yeast Schizosaccharomyces pombe is mediated by a DNA synthesis control-like transcription factor complex (DSC1), which acts upon M lu1 cell cycle box (MCB) promoter motifs . Several genes, including rec8+, rec11+, cdc18+, and cdc22+, which contain MCB motifs in their promoter regions, are found to be co-ordinately regulated during pre-meiotic S-phase . Both synthetic and native MCB motifs are shown to confer meiotic-specific transcription on a heterologous reporter gene . A DSC1-like transcription factor complex that binds to MCB motifs was also identified in meiotic cells . The effect of mutating and over-expressing individual components of DSC1 (cdc10+, res1+, res2+, rep1+ and rep2+) on the transcription of cdc22+, rec8+ and rec11+ during meiosis was examined . We found that cdc10+, res2+, rep1+ and rep2+ are required for correct meiotic transcription, while res1+ is not required for this process . This work demonstrates a role for MCB motifs and a DSC1-like transcription factor complex in controlling transcription during meiosis in fission yeast, and suggests a mechanism for how this specific expression occurs. Mol Cell Proteomics, 2004 Mar, 3(3), 209 - 25 Epub 2003 Nov 25. Synergistic computational and experimental proteomics approaches for more accurate detection of active serine hydrolases in yeast; Baxter SM et al.; An analysis of the structurally and catalytically diverse serine hydrolase protein family in the Saccharomyces cerevisiae proteome was undertaken using two independent but complementary, large-scale approaches . The first approach is based on computational analysis of serine hydrolase active site structures; the second utilizes the chemical reactivity of the serine hydrolase active site in complex mixtures . These proteomics approaches share the ability to fractionate the complex proteome into functional subsets . Each method identified a significant number of sequences, but 15 proteins were identified by both methods . Eight of these were unannotated in the Saccharomyces Genome Database at the time of this study and are thus novel serine hydrolase identifications . Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family, designated as Fsh (family of serine hydrolase), identified here for the first time . OVCA2, a potential human tumor suppressor, and DYR-SCHPO, a dihydrofolate reductase from Schizosaccharomyces pombe, are members of this family . Comparing the combined results to results of other proteomic methods showed that only four of the 15 proteins were identified in a recent large-scale, "shotgun" proteomic analysis and eight were identified using a related, but similar, approach (neither identifies function) . Only 10 of the 15 were annotated using alternate motif-based computational tools . The results demonstrate the precision derived from combining complementary, function-based approaches to extract biological information from complex proteomes . The chemical proteomics technology indicates that a functional protein is being expressed in the cell, while the computational proteomics technology adds details about the specific type of function and residue that is likely being labeled . The combination of synergistic methods facilitates analysis, enriches true positive results, and increases confidence in novel identifications . This work also highlights the risks inherent in annotation transfer and the use of scoring functions for determination of correct annotations. Biochem Biophys Res Commun, 2003 Dec 12, 312(2), 414 - 20 Role of guanine nucleotide exchange factors for Rho family GTPases in the regulation of cell morphology and actin cytoskeleton in fission yeast; Iwaki N et al.; Rho GTPases regulate fundamental processes including cell morphology and migration in various organisms . Guanine nucleotide exchange factor (GEF) has a crucial role in activating small GTPase by exchange GDP for GTP . In fission yeast Schizosaccharomyces pombe, six members of the Rho small GTPase family were identified and reported to be involved in cell morphology and polarized cell growth . We identified seven genes encoding Rho GEF domain from genome sequence and analyzed . Overexpressions of identified genes in cell lead to change of morphology, suggesting that all of them are involved in the regulation of cell morphology . Although all of null mutants were viable, two of seven null cells had morphology defects and five of seven displayed altered actin cytoskeleton arrangements . Most of the double mutants were viable and biochemical analysis revealed that each of GEFs bound to several small G proteins . These data suggest that identified Rho GEFs are involved in the regulation of cell morphology and share signals via small GTPase Rho family. Plant Physiol, 2003 Dec, 133(4), 1873 - 81 Epub 2003 Nov 20. Overexpression of SOD2 increases salt tolerance of Arabidopsis; Gao X et al.; The yeast (Schizosaccharomyces pombe) SOD2 (Sodium2) gene was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter . Transformants were selected for their ability to grow on medium containing kanamycin . Southern- and northern-blot analyses confirmed that SOD2 was transferred into the Arabidopsis genome . There were no obvious morphological or developmental differences between the transgenic and wild-type (wt) plants . Several transgenic homozygous lines and wt plants (control) were evaluated for salt tolerance and gene expression . Overexpression of SOD2 in Arabidopsis improved seed germination and seedling salt tolerance . Analysis of Na+ and K+ contents of the symplast and apoplast in the parenchyma cells of the root cortex and mesophyll cells in the spongy tissue of the leaf showed that transgenic lines accumulated less Na+ and more K+ in the symplast than the wt plants did . The photosynthetic rate and the fresh weight of the transgenic lines were distinctly higher than that of wt plants after NaCl treatment . Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants. J Basic Microbiol, 2003, 43(6), 473 - 82 Ksg1, a homologue of the phosphoinositide-dependent protein kinase 1, controls cell wall integrity in Schizosaccharomyces pombe; Graub R et al.; It has previously been shown that the Schizosaccharomyces pombe mutant ksg1-358 has a mating and sporulation defect at 30 degrees C and that it is temperature sensitive for growth at 35 degrees C . However the molecular basis for these phenotypes remained largely unknown . In this study we show that ksg1-358 mutant cells lysed at the non-permissive temperature, which could be prevented by sorbitol . Overexpression of ksg1 using the nmt1-promoter showed slow growth and cells became swollen when incubated at 35 degrees C under low inositol conditions . Interestingly, in a two-hybrid assay we found that the ksg1-protein interacted with Pck1p, a protein implicated in regulating cell wall integrity in S . pombe . Genetic complementation assays showed that an overexpression of pck2, the homologue of pck1 involved in the regulation of cell wall synthesis, could partially rescue ksg1-358 phenotypes . We digested the ksg1-358 cell wall using beta-glucanase . We found that the ksg1-358 mutant was more resistant to cell lysis at 30 degrees C than the wildtype strain h972, which was similar to a pck1-deletion strain . A ksg1-overexpressing strain was hypersensitive towards beta-glucanase treatment similar to a pck2-deletion strain . The pck1-deletion partially rescued beta-glucanase hypersensitivity of the ksg1-overexpressing strain but the pck2-deletion increased it . The ksg1-358 mutation increased beta-glucanase resistance of a pck1-overexpressing strain but it had no effect on a pck2-overexpressing strain . Our results provide evidence that ksg1 is a novel regulator of cell wall integrity in the fission yeast Schizosaccharomyces pombe . They further suggest that Ksg1p acts in a pathway with Pck1p, possibly upstream and through direct interaction. Nat Cell Biol, 2003 Dec, 5(12), 1111 - 6 Epub 2003 Nov 16. Hsk1-Dfp1 is required for heterochromatin-mediated cohesion at centromeres; Bailis JM et al.; Heterochromatin performs a central role in chromosome segregation and stability by promoting cohesion at centromeres . Establishment of both heterochromatin-mediated silencing and cohesion requires passage through S phase, although the mechanism is unknown . Here we demonstrate that Schizosaccharomyces pombe Hsk1 (CDC7), a conserved Dbf4-dependent protein kinase (DDK) that regulates replication initiation, interacts with and phosphorylates the heterochromatin protein 1 (HP1) equivalent Swi6 (ref . 6) . Hsk1 and its regulatory subunit Dfp1 function downstream of Swi6 localization to promote heterochromatin function and cohesion specifically at centromeres . This role for Hsk1-Dfp1 is separable from its replication initiation activity, providing a temporal link between S phase and centromere cohesion that is mediated by heterochromatin. J Cell Sci, 2003 Dec 15, 116(Pt 24), 4891 - 903 Individual microtubule dynamics contribute to the function of mitotic and cytoplasmic arrays in fission yeast; Sagolla MJ et al.; Schizosaccharomyces pombe is an excellent organism for studying microtubule dynamics owing to the presence of well-defined microtubule arrays that undergo dramatic rearrangements during various stages of the cell cycle . Using sensitive time-lapse video microscopy and kymographic analysis, we have determined the polymerization/depolymerization kinetics of individual microtubules within these arrays throughout the fission yeast cell cycle . Interphase bundles are composed of 4-7 microtubules that act autonomously, demonstrating that individual microtubules are responsible for mediating the functions ascribed to these arrays . The nucleation and growth of cytoplasmic microtubules is inhibited upon cellular transition into mitosis, leading to their gradual disappearance . At the onset of mitosis, microtubules form on the nuclear face of the spindle pole body and exhibit dramatically increased dynamics . The presence of these intra-nuclear astral microtubules (INA) is reminiscent of spindle assembly and the search and chromosome capture mechanism observed in metazoan cells . Consistent with other in vivo studies, we do not observe microtubule flux in the anaphase B spindle . Finally, the depolymerization of individual microtubules alternates between each half-spindle, resulting in spindle collapse during telophase . On the basis of these observations, we conclude that microtubules in these diverse cytoskeletal arrays have autonomous behaviors that are an essential component of any model describing cell-cycle-dependent changes in the behavior and function of microtubule arrays. RNA, 2003 Dec, 9(12), 1422 - 30 Translational recoding signals between gag and pol in diverse LTR retrotransposons; Gao X et al.; Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and Pol . To assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Candida albicans, and Arabidopsis thaliana . In contrast to the retroviruses, which typically encode pol in the -1 frame relative to gag, nearly half of the retroelements surveyed encode a single gag-pol open reading frame . This was particularly true for the Ty1/copia group retroelements . Most animal Ty3/gypsy retroelements, on the other hand, encode gag and pol in separate reading frames, and likely express Pol through +1 or -1 frameshifting . Conserved sequences conforming to slippery sites that specify viral ribosomal frameshifting were identified among retroelements with pol in the -1 frame . None of the plant retroelements encoded pol in the -1 frame relative to gag; however, two closely related plant Ty3/gypsy elements encode pol in the +1 frame . Interestingly, a group of plant Ty1/copia retroelements encode pol either in a +1 frame relative to gag or in two nonoverlapping reading frames . These retroelements have a conserved stem-loop at the end of gag, and likely express pol either by a novel means of internal ribosomal entry or by a bypass mechanism. Biochem Biophys Res Commun, 2003 Nov 21, 311(3), 691 - 5 But1 and But2 proteins bind to Uba3, a catalytic subunit of E1 for neddylation, in fission yeast; Yashiroda H et al.; NEDD8/Rub1 is the most homologous protein to ubiquitin among the ubiquitin-like proteins, and it is covalently linked to target proteins via the C-terminal glycine residue in a manner analogous to ubiquitylation . However, the mechanism(s) involved in the regulation of the NEDD8 ligation pathway remains elusive . Using the two-hybrid system, we isolated novel genes from the Schizosaccharomyces pombe cDNA library whose products bind to Uba3, which is a catalytic protein for E1-like activity of the NEDD8 pathway . We designated these genes but1(+) and but2(+) (for proteins that bind to Uba three) . But1 is a nuclear protein and its overexpression caused cell elongation, which is a common phenotype of the NEDD8 pathway defective mutant in S . pombe . Furthermore, overexpression of but1(+) in ned8-temperature sensitive mutant had a deleterious effect even under permissive temperatures . Our results suggest that But1 may have an inhibitory role in the NEDD8 pathway. Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 1078 - 83 LAMMER kinase homolog, Lkh1, is involved in oxidative-stress response of fission yeast; Park YD et al.; Previously, we reported that the LAMMER kinase homolog, Lkh1, is a negative regulator of filamentous growth and asexual flocculation in the fission yeast, Schizosaccharomyces pombe . Here, we report that the lkh1(+) null mutant is sensitive to oxidative stress because of a reduction in the expression of genes for antioxidant enzymes such as catalase (ctt1(+)) and Cu,Zn-superoxide dismutase (sod1(+)) . Furthermore, the lkh1(+) null mutant shows increased levels of intracellular peroxides under conditions of oxidative stress compared with wild-type cells . Interestingly, expression of the gene for the transcription factor Atf1 is reduced in the lkh1(+) null mutant under oxidative stress, whereas expression of the transcription factor Pap1 is not . We report the novel finding that Lkh1 is involved in the oxidative-stress response of the fission yeast, S . pombe, and regulates the expression of antioxidant enzymes via the transcription factor Atf1. Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 942 - 7 Ribosomal proteins S0 and S21 are involved in the stability of 18S rRNA in fission yeast, Schizosaccharomyces pombe; Sato M et al.; Stability of ribosomal RNA (rRNA) is not only essential for ribosome biogenesis but also crucial to the maintenance of proper translational level for cell viability . rRNA processing (maturation) is one of the key steps to derive functional rRNA, and to date, a large number of factors involved in this process have been identified . We investigated Rps0 binding proteins in fission yeast, Schizosaccharomyces pombe, and revealed that Rps0p is associated with Rps21 protein, similar to that of our previous observation in human cells . We demonstrated that both rps0(+)s and rps21(+) are essential genes for S . pombe analyzed by tetrad dissection assay . To study the functions of both genes, we established disruption strains transformed with inducible rescue plasmids . Using the strains our studies revealed that the loss of rps0(+)s or rps21(+) led to a deficiency of 40S ribosomal subunit formation . Additional functional studies indicate that this phenomenon is likely to be caused by insufficient 18S rRNA stability . The possible role of Rps0p and Rps21 that contribute to 18S rRNA maturation is further discussed. Eur J Biochem, 2003 Nov, 270(22), 4507 - 14 Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly; Kostova Z et al.; Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis . It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases . All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E . To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S . cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids . Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity . Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth . The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not . Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none . The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable . Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo . Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps. Yeast, 2003 Nov, 20(15), 1273 - 8 DNA sequence of the mat2,3 region of Schizosaccharomyces kambucha shares high homology with the corresponding sequence from Sz . pombe; Singh G et al.; To define conserved sequences for mat1 imprinting and silencing of the mat2,3 region of Schizosaccharomyces pombe, we determined the DNA sequence of the cognate region (mat2,3 region) of another fission yeast, Sz . kambucha, a yeast species isolated from Kambucha tea mix . The entire mat2,3 region shows more than 98% identity between the two species . Sequence similarity is even higher (99.3%) for mating-type cassettes; deduced amino acid sequences of three of the four Mat peptides (Pi, Pc and Mi) are identical between the two species, while the fourth (Mc) has a single amino acid polymorphism . Comparison of the sequence motif of the imprint site essential for mat1 switching shows that mat-P of Sz . kambucha has a sequence identical to the conserved motif present in Sz . pombe . However, this sequence motif of nine bases differs by one base for mat-M of Sz . kambucha . The sequence of the K region shows about 98% identity between the two species, with the cenH region showing 98.3% homology . Thus, the arrangement of the mat2,3 region in both yeasts is conserved and shows 1-2% nucleotide sequence variation throughout the region . The DNA sequence of the mat2,3 region from Sz . kambucha has been submitted to GenBank under Accession No . AY271822 . Mol Cell Proteomics, 2004 Feb, 3(2), 125 - 32 Epub 2003 Nov 15. A comparative analysis of an orthologous proteomic environment in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe; Roguev A et al.; The sequential application of protein tagging, affinity purification, and mass spectrometry enables highly accurate charting of proteomic environments by the characterization of stable protein assemblies and the identification of subunits that are shared between two or more protein complexes, termed here "proteomic hyperlinks." We have charted the proteomic environments surrounding the histone methyltransferase, Set1, in both yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . Although the composition of these nonessential Set1 complexes is remarkably conserved, they differ with respect to their hyperlinks to their proteomic environments . We speculate that conservation of the core components of protein assemblies and variability of hyperlinks represents a general principle in the molecular organization of eukaryotic proteomes. Nature, 2003 Nov 13, 426(6963), 198 - 203 DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA; Lei M et al.; Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions . The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals . Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation . Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown . Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S . pombe Pot1p complexed with ssDNA . The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding . The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA . Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups . The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus. Genome Res, 2003 Dec, 13(12), 2686 - 90 Epub 2003 Nov 12. Correlations between gene expression and gene conservation in fission yeast; Mata J et al.; Genes can be expressed at a wide range of levels, and they show different degrees of cross-species conservation . We compared gene expression levels to gene conservation by integrating microarray data from fission yeast (Schizosaccharomyces pombe) with lists of "core" genes (present in worm and budding and fission yeasts), "yeast-specific" genes (present in budding and fission yeasts, but not in worm), and "pombe-specific" genes (present in fission yeast only) . Whereas a disproportionate number of core genes are highly expressed in vegetatively growing cells, many pombe-specific genes are expressed at lower levels . This bias is less pronounced in cells undergoing sexual development, when many pombe-specific genes beco