Schizosaccharomyces

J Cell Sci, 2002 May 1, 115(Pt 9), 1847 - 57
The novel HECT-type ubiquitin-protein ligase Pub2p shares partially overlapping function with Pub1p in Schizosaccharomyces pombe; Tamai KK et al.; The fission yeast Schizosaccharomyces pombe has three putative ubiquitin-protein ligases of the Nedd4/Rsp5 family, named Pub1p, Pub2p and Pub3p . Pub1p has been reported to be involved in cell cycle regulation and proliferation under acidic pH conditions . Here we characterize Pub2p, which contains a conserved HECT domain and a WW domain but lacks a C2 domain . Transcription of the pub2(+) gene was constitutive and further enhanced by nitrogen starvation . A pub2-null mutation gave no remarkable phenotypes, but intensified temperature sensitivity in a pub1Delta background . Moderately overexpressed pub2(+) suppressed the temperature sensitivity of pub1Delta cells, which suggests that the function of Pub2p overlaps with that of Pub1p . Overexpression of pub2(+) by a strong nmt1 promoter in wild-type strains caused growth arrest and cell elongation, probably owing to defects in G2 progression or the G2/M transition . Unlike Pub1p, however, overexpression of Pub2p did not reduce the levels of Cdc25p . Pub2-GFP was found throughout the cell, especially at the cell surface in the polar regions . Pub2p contains a conserved cysteine residue (Cys639) in its putative catalytic HECT domain that can be thiol-ubiquitinated . Substitution of Cys639 by alanine (Pub2CA) caused a functional defect, because growth arrest and cell elongation were not induced by overexpression of Pub2CA . A chimeric Pub1 protein, in which the HECT domain was replaced by the Pub2 HECT domain, completely suppressed the temperature sensitivity of pub1Delta cells, suggesting that the HECT domain of Pub2p has the catalytic activity of a ubiquitin ligase . We conclude that Pub2p is a HECT-type ubiquitin-protein ligase that shares partially overlapping function with Pub1p.

J Biol Chem, 2002 Jun 21, 277(25), 22950 - 8 Epub 2002 Apr 15.
Fep1, an iron sensor regulating iron transporter gene expression in Schizosaccharomyces pombe; Pelletier B et al.; Schizosaccharomyces pombe cells acquire iron under high affinity conditions through the action of a cell surface ferric reductase encoded by the frp1(+) gene and a two-component iron-transporting complex encoded by the fip1(+) and fio1(+) genes . When cells are grown in the presence of iron, transcription of all three genes is blocked . A conserved regulatory element, 5'-(A/T)GATAA-3', located upstream of the frp1(+), fip1(+), and fio1(+) genes, is necessary for iron repression . We have cloned a novel gene, termed fep1(+), which encodes an iron-sensing transcription factor . Binding studies reveal that the putative DNA binding domain of Fep1 expressed as a fusion protein in Escherichia coli specifically interacts with the 5'-(A/T)GATAA-3' sequence in an iron-dependent manner . In a fep1 Delta mutant strain, the fio1(+) gene is highly expressed and is unregulated by iron . Furthermore, the fep1 Delta mutation increases activity of the cell surface iron reductase and renders cells hypersensitive to the iron-dependent free radical generator phleomycin . Mutations in the transcriptional co-repressors tup11(+) and tup12(+) are phenocopies to fep1(+) . Indeed, strains with both tup11 Delta and tup12 Delta deletions fail to sense iron . This suggests that in the presence of iron and Fep1, the Tup11 and Tup12 proteins may act as co-repressors for down-regulation of genes encoding components of the reductive iron transport machinery.

Biochim Biophys Acta, 2002 Mar 19, 1574(2), 210 - 4
A potential membrane protein involved in pre-tRNA splicing of Schizosaccharomyces pombe; Kim M et al.; We had previously isolated six pre-tRNA splicing mutants of Schizosaccharomyces pombe named ptp1 to ptp6 . To investigate the molecular mechanism of tRNA splicing, we cloned the ptp4(+) gene by complementation of the temperature-sensitive growth defect . The ptp4(+) gene consists of three exons and encodes a putative protein of 218 amino acids with a molecular mass of 24.4 kDa . Analysis of the amino acid sequence reveals that the protein is a potential membrane protein with four membrane-spanning regions . The ptp4(+) shows significant similarity to the Saccharomyces cerevisiae putative protein YOR311C . Expression of the ptp4(+) gene in the ptp4(-) mutant restores the ability to splice tRNA . Northern blot analysis showed that the ptp4(+) gene is expressed in both mating-type cells of S . pombe . These results suggest that the Ptp4(+) could be a component involved in tRNA splicing.

Mol Biol Cell, 2002 Apr, 13(4), 1203 - 14
The spindle pole body protein Cdc11p links Sid4p to the fission yeast septation initiation network; Tomlin GC et al.; The Schizosaccharomyces pombe septation initiation network (SIN) signals the onset of cell division from the spindle pole body (SPB) and is regulated by the small GTPase Spg1p . The localization of SIN components including Spg1p to the SPB is required for cytokinesis and is dependent on Sid4p, a constitutive resident of SPBs . However, a direct interaction between Sid4p and other members of the SIN has not been detected . To understand how Sid4p is linked to other SIN components, we have begun to characterize an S . pombe homolog of the Saccharomyces cerevisiae SPB protein Nud1p . We have determined that this S . pombe Nud1p homolog corresponds to Cdc11p, a previously uncharacterized SIN element . We report that Cdc11p is present constitutively at SPBs and that its function appears to be required for the localization of all other SIN components to SPBs with the exception of Sid4p . The Cdc11p C terminus localizes the protein to SPBs in a Sid4p-dependent manner, and we demonstrate a direct Cdc11p-Sid4p interaction . The N-terminus of Cdc11p is required for Spg1p binding to SPBs . Our studies indicate that Cdc11p provides a physical link between Sid4p and the Spg1p signaling pathway.

Mol Biol Cell, 2002 Apr, 13(4), 1132 - 43
The Schizosaccharomyces pombe aurora-related kinase Ark1 interacts with the inner centromere protein Pic1 and mediates chromosome segregation and cytokinesis; Leverson JD et al.; The chromosomal passenger proteins aurora-B, survivin, and inner centromere protein (INCENP) have been implicated in coordinating chromosome segregation with cell division . This work describes the interplay between aurora, survivin, and INCENP orthologs in the fission yeast Schizosaccharomyces pombe and defines their roles in regulating chromosome segregation and cytokinesis . We describe the cloning and characterization of the aurora-related kinase gene ark1(+), demonstrating that it is an essential gene required for sister chromatid segregation . Cells lacking Ark1p exhibit the cut phenotype, DNA fragmentation, and other defects in chromosome segregation . Overexpression of a kinase-defective version of Ark1, Ark1-K147R, inhibits cytokinesis, with cells exhibiting an elongated, multiseptate phenotype . Ark1p interacts physically and/or genetically with the survivin and INCENP orthologs Bir1p and Pic1p . We identified Pic1p in a two-hybrid screen for Ark1-K147R interacting partners and went on to map domains in both proteins that mediate their binding . Pic1p residues 925-972 are necessary and sufficient for Ark1p binding, which occurs through the kinase domain . As with Ark1-K147R, overexpression of Ark1p-binding fragments of Pic1p leads to multiseptate phenotypes . We also provide evidence that the dominant-negative effect of Ark1-K147R requires Pic1p binding, indicating that the formation of Ark1p-Pic1p complexes is required for the execution of cytokinesis.

J Cell Sci, 2002 Apr 15, 115(Pt 8), 1651 - 62
Different mechanisms of cell polarisation in vegetative and shmooing growth in fission yeast; Niccoli T et al.; Schizosaccharomyces pombe cells have two polarised growth modes: an intrinsic vegetative growth mode, determined by an internal positioning mechanism and an extrinsic shmooing growth mode, activated by external pheromone . We have analysed the role of the cell end marker Tea1p, the CLIP170 like protein Tip1p, the kinesin like protein Tea2p and the Dyrk-like kinase Pom1p, during the switch between the two growth patterns, with the intention of studying the switch away from the vegetative growth mode . In vegetative growth these morphological factors are concentrated at cell ends, whereas during shmooing growth they are delocalised from the cell ends . In the absence of Tea1p, Tip1p and Tea2p, vegetative cells display microtubule and cell polarisation defects, but shmooing cells are indistinguishable from wild-type and shmoo more readily . These results suggest that Tea1p, Tip1p and Tea2p are not required for polarised growth during shmooing, but form part of the intrinsic vegetative growth mode that needs to be dismantled before cells can generate an extrinsic growth patterns . In contrast, Pom1p appears to have a role in the initial stages of the switch to the shmooing growth mode.

J Cell Sci, 2002 Apr 15, 115(Pt 8), 1603 - 10
Control of localization of a spindle checkpoint protein, Mad2, in fission yeast; Ikui AE et al.; To ensure accurate chromosome segregation, the spindle checkpoint delays the onset of sister chromatid separation when the spindle is not attached to a kinetochore . Mad2, a component of the checkpoint, targets fission yeast Slp1/budding yeast Cdc20/human p55CDC and prevents it from promoting proteolysis, which is a prerequisite to sister chromatid separation . The protein is localized to unattached kinetochores in higher eukaryotes, and it is thought to be required for activation of the checkpoint as well . In this study, Mad2 and its target Slp1 were visualized in a tractable organism, fission yeast Schizosaccharomyces pombe . When cells were arrested at a prometaphase-like stage, the Mad2-Slp1 complex was stable and the two proteins were colocalized to unattached kinetochores . When the spindle attachment was completed, the complex was no longer detectable and only Mad2 was found associated to the spindle . These results would suggest that unattached kinetochores provide sites for assembly of the Mad2-Slp1 complex . During interphase, Mad2 was localized to the nuclear periphery as well as to the chromatin domain . This localization was abolished in a yeast strain lacking Mad1, a protein that physically interacts with Mad2 . Mad1 may anchor Mad2 to the nuclear membrane and regulate its entry into the nucleus.

J Bacteriol, 2002 May, 184(9), 2460 - 4
Identification of {2Fe-2S} clusters in microbial ferrochelatases; Dailey TA et al.; The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme . Prior to the present work, {2Fe-2S} clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases . Herein we present evidence that ferrochelatases from the bacteria Caulobacter crescentus and Mycobacterium tuberculosis possess {2Fe-2S} clusters . The enzyme from C . crescentus is a homodimeric, membrane-associated protein while the enzyme from M . tuberculosis is monomeric and soluble . The clusters of the C . crescentus and M . tuberculosis ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized {2Fe-2S} cluster-containing protein, including the ferrochelatase of the yeast Schizosaccharomyces pombe . Thus, the microbial ferrochelatases represent a new group of {2Fe-2S} cluster-containing proteins.

J Microbiol Methods, 2002 Jun, 50(1), 55 - 62
Electro-orientation of Schizosaccharomyces pombe in high conductivity media; Markx GH et al.; The orientation of microbial cells may be important in cell-cell interactions within microbial consortia . As part of our research programme aimed at the construction of Artificial Structured Microbial Consortia (ASMC), we have investigated the electro-orientation of Schizosaccharomyces pombe in AC electric fields, and studied the effects of the applied frequency, voltage, and distance between the electrodes, at different medium conductivities . It is shown that the electro-orientation of S . pombe in media with conductivities similar to that of growth media is feasible using microelectrodes . Oriented growth of S . pombe can be obtained when continuously exposed to AC electric fields in growth medium over extended periods.

Cell Struct Funct, 2001 Dec, 26(6), 555 - 65
Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe; D'souza VM et al.; The fission yeast Schizosaccharomyces pombe undergoes cell division through a medially placed actomyosin-based contractile ring . One of the key components of this ring is the F-actin based motor protein myosin II . The myosin II heavy chain Myo2p has two light-chain-binding domains, IQl and IQ2, which bind the essential light chain, Cdc4p, and the regulatory light chain, Rlc1p . Previously, we have reported the characterization of cells expressing Myo2p lacking the IQ2 domain that facilitates Myo2p interaction with Rlc1p . In this study, we have created and characterized S . pombe strains carrying precise deletions of IQ1 and the entire neck region encompassing the IQ1 and IQ2 domains . Surprisingly, we found that the entire neck region of Myo2p is dispensable for Myo2p function . Cells deleted for IQ1, IQ2 and the entire neck region of Myo2p do not display any obvious cytoskeletal abnormalities . Immunofluorescence studies indicated that Cdc4p localizes at the ring in early and late mitotic cells in a strain in which interactions of Cdc4p with both the myosin II heavy chains (Myo2p and Myp2p) are abolished . Unlike mutations in Rlc1p that are suppressed by a simultaneous deletion of its binding site on Myo2p, mutations in the essential light chain Cdc4p are not suppressed by deletion of its binding sites on Myo2p, suggesting that Cdc4p may have additional partners essential for cytokinesis . Consistent with this, we provide evidence that two other IQ-domain containing actomyosin ring proteins, Rng2p (an IQGAP-related protein) and Myo51p (a type V myosin heavy chain), physically interact with Cdc4p . We concluded that Cdc4p, a novel myosin light chain, interacts with multiple actomyosin ring components to effect cytokinesis.

Cell Struct Funct, 2001 Dec, 26(6), 539 - 44
Studies in fission yeast on mechanisms of cell division site placement; Chang F; One fundamental problem in cytokinesis is how the plane of cell division is established . In this review, we describe our studies on searching for "signals" that position the cell division plane, using fission yeast Schizosaccharomyces pombe . First, we take a genetic approach to determine how the nucleus may position the contractile ring in fission yeast . mid1p appears to link the position of the ring with the nuclear position, as it is required for proper placement of the contractile ring and is localized in a band at the cell surface overlying the nucleus . Second, we study how microtubules may function in the establishment of cell polarity at the cell tips . tea1p may be deposited on the cell surface by microtubules and function to recruit proteins involved in making actin structures . These studies suggest how microtubules may direct the assembly of the contractile ring in animal cells.

J Biol Inorg Chem, 2002 Apr, 7(4-5), 526 - 32 Epub 2002 Feb 13.
Iron-sulfur cluster biosynthesis: characterization of Schizosaccharomyces pombe Isa1; Wu G et al.; Eukaryotic Isa1 is one of several mitochondrial proteins that have been implicated in Fe-S cluster assembly paths in vivo . We report the first biochemical characterization of an eukaryotic member of this family and discuss this in the context of results from in vivo studies and studies of bacterial homologues . Schizosaccharomyces pombe Isa1 is a multimeric protein carrying {2Fe-2S}(2+) clusters that have been characterized by Mossbauer and optical spectroscopic studies . Complex formation with a redox-active ferredoxin has been identified through crosslinking experiments and the coordination chemistry and stability of the native clusters has been investigated through site-directed mutagenesis and spectroscopic analysis . Electronic supplementary material to this paper, containing Mossbauer and UV-visible spectra for mutant Isa1 proteins, can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0330-2.

Biochemistry, 2002 Apr 16, 41(15), 5024 - 32
Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe; Wu G et al.; Genetic studies of bacteria and eukaryotes have led to identification of several gene products that are involved in the biosynthesis of protein-bound iron-sulfur clusters . One of these proteins, ISU, is homologous to the N-terminus of bacterial NifU . The mature forms of His-tagged wild-type and D37A Schizosaccharomyces pombe ISU1 were cloned and overexpressed as inclusion bodies in Escherichia coli . The recombinant D37A protein was purified under denaturing conditions and subsequently reconstituted in vitro . By use of a 5-fold excess of iron and sulfide the reconstituted product was found to be red-brown in color, forming a homodimer of 17 kDa per subunit with approximately two iron atoms per monomer determined by protein and iron quantitation . UV-vis absorption and Mossbauer spectroscopies (delta = 0.29 +/- 0.05 mm/s; DeltaE(Q) = 0.59 +/- 0.05 mm/s) were used to characterize D37A ISU1 and show the presence of {2Fe-2S}(2+) clusters in each subunit . Formation of the holo form of wild-type ISU1 was significantly less efficient using the same reconstitution conditions and is consistent with prior observations that the D37A substitution can stabilize protein-bound clusters . Relative to the human homologue, the yeast ISU is significantly less soluble at ambient temperatures . In both cases the native ISU1 is more sensitive to proton-mediated degradation relative to the D37A derivative . The lability of this family of proteins relative to {2Fe-2S} bearing ferredoxins most likely is of functional relevance for cluster transfer chemistry . Mossbauer parameters obtained for wild-type ISU1 (delta = 0.31 +/- 0.05 mm/s; DeltaE(Q) = 0.64 +/- 0.05 mm/s) were similar to those obtained for the D37A derivative . Cluster transfer from ISU1 to apo Fd is demonstrated: the first example of transfer from an ISU-type protein . A lower limit for k(2) of 80 M(-1) min(-1) was established for WT cluster transfer and a value of 18 M(-1) min(-1) for the D37A derivative . Finally, we have demonstrated through cross-linking studies that ferredoxin, an electron-transport protein, forms a complex with ISU1 in both apo and holo states . Cross-linking of holo ISU1 with holo Fd is consistent with a role for redox chemistry in cluster assembly and may mimic the intramolecular complex already defined in NifU.

J Biol Chem, 2002 Jun 14, 277(24), 21213 - 20 Epub 2002 Apr 04.
Characterization of the CTD phosphatase Fcp1 from fission yeast . Preferential dephosphorylation of serine 2 versus serine 5; Hausmann S et al.; The C-terminal domain (CTD) of RNA polymerase II undergoes extensive phosphorylation and dephosphorylation at positions Ser2 and Ser5 during the transcription cycle . A single CTD phosphatase, Fcp1, has been identified in yeast and metazoans . Here we conducted a biochemical characterization of Fcp1 from the fission yeast Schizosaccharomyces pombe . The 723-amino acid Fcp1 protein was expressed at high levels in bacteria . Recombinant Fcp1 catalyzed the metal-dependent hydrolysis of para-nitrophenyl phosphate with a pH optimum of 5.5 (kcat = 2 s(-1); K(m) = 19 mm) . Deletion analysis showed that 139- and 143-amino acid segments could be deleted from the N and C termini of Fcp1, respectively, without affecting phosphatase activity . A segment containing amino acids 487-580, deletion of which abolished activity, embraces a BRCT domain present in all known Fcp1 orthologs . Mutations of residues Asp170 and Asp172 abrogated Fcp1 phosphatase activity; the essential aspartates are located within a 170DXDXT172 motif that defines a superfamily of metal-dependent phosphotransferases . We exploited defined synthetic CTD phosphopeptide substrates to show for the first time that: (i) Fcp1 CTD phosphatase activity is not confined to native polymerase II and (ii) Fcp1 displays an inherent preference for a particular CTD phosphorylation array . Using equivalent concentrations (25 microm) of CTD peptides of identical amino acid sequence and phosphoserine content, which differed only in the positions of phosphoserine within the heptad, we found that Fcp1 was 10-fold more active in dephosphorylating Ser2-PO4 than Ser5-PO4.

Microbiology, 2002 Apr, 148(Pt 4), 1225 - 32
Difference in substrate specificity divides the yeast alkali-metal-cation/H(+) antiporters into two subfamilies; Kinclova O et al.; Yeast plasma membrane Na(+)/H(+) antiporters (TC 2.A.36) share a high degree of similarity at the protein level . Expression of four antiporters (Saccharomyces cerevisiae Nha1p, Candida albicans Cnh1p, Zygosaccharomyces rouxii ZrSod2-22p and Schizosaccharomyces pombe sod2p) in a SACCH: cerevisiae mutant strain lacking both Na(+)-ATPase and Na(+)/H(+) antiporter genes made it possible to study the transport properties and contribution to cell salt tolerance of all antiporters under the same conditions . The ZrSod2-22p of the osmotolerant yeast Z . rouxii has the highest transport capacity for lithium and sodium but, like the SCHIZ: pombe sod2p, it does not recognize K(+) and Rb(+) as substrates . The SACCH: cerevisiae Nha1p and C . albicans Cnh1p have a broad substrate specificity for at least four alkali metal cations (Na(+), Li(+), K(+), Rb(+)), but their contribution to overall cell tolerance to high external concentration of toxic Na(+) and Li(+) cations seems to be lower compared to the antiporters of SCHIZ: pombe and especially Z . rouxii.

Microbiology, 2002 Apr, 148(Pt 4), 933 - 41
Potassium- or sodium-efflux ATPase, a key enzyme in the evolution of fungi; Benito B et al.; Potassium is the most abundant cation in cells . Therefore, plant-associated fungi and intracellular parasites are permanently or circumstantially exposed to high K(+) and must avoid excessive K(+) accumulation activating K(+) efflux systems . Because high K(+) and high pH are compatible in natural environments, free-living organisms cannot keep a permanent transmembrane DeltapH and cannot rely only on K(+)/H(+) antiporters, as do mitochondria . This study shows that the Schizosaccharomyces pombe CTA3 is a K(+)-efflux ATPase, and that other fungi are furnished with Na(+)-efflux ATPases, which also pump Na(+) . All these fungal ATPases, including those pumping only Na(+), form a phylogenetic group, IID or ENA, among P-type ATPases . By searching in databases and partial cloning of ENA genes in species of Zygomycetes and Basidiomycetes, the authors conclude that probably all fungi have these genes . This study indicates that fungal K(+)- or Na(+)-ATPases evolved from an ancestral K(+)-ATPase, through processes of gene duplication . In yeast hemiascomycetes these duplications have occurred recently and produced bifunctional ATPases, whereas in Neurospora, and probably in other euascomycetes, they occurred earlier in evolution and produced specialized ATPases . In Schizosaccharomyces, adaptation to Na(+) did not involve the duplication of the K(+)-ATPase and thus it retains an enzyme which is probably close to the original one . The parasites Leishmania and Trypanosoma have ATPases phylogenetically related to fungal K(+)-ATPases, which are probably functional homologues of the fungal enzymes.

Fungal Genet Biol, 2002 Apr, 35(3), 277 - 86
Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola; Waalwijk C et al.; Both mating-type loci from the wheat septoria leaf blotch pathogen Mycosphaerella graminicola have been cloned and sequenced . The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae . The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids . The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph . The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative alpha1-domain protein of 297 amino acids . Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S . pombe . For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269 . An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates . The primers were also used to screen a collection of field isolates in a multiplex PCR . An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.

Fungal Genet Biol, 2002 Apr, 35(3), 183 - 95
The DNA damage response in filamentous fungi; Goldman GH et al.; The mechanisms used by fungal cells to repair DNA damage have been subjects of intensive investigation for almost 50 years . As a result, the model yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae have led the way in yielding critical insights into the nature of the DNA damage response . At the same time, largely through the efforts of Etta Kafer, Hirokazu Inoue, and colleagues, a substantial collection of Aspergillus nidulans and Neurospora crassa DNA repair mutants has been identified and characterized in detail . As the analysis of these mutants continues and increasing amounts of annotated genome sequence become available, it is becoming readily apparent that the DNA damage response of filamentous fungi possesses several features that distinguish it from the model yeasts . These features are emphasized in this review, which describes the genes, regulatory networks, and processes that compose the fungal DNA damage response . Further characterization of this response will likely yield general insights that are applicable to animals and plants . Moreover, it may also become evident that the DNA damage response can be manipulated to control fungal growth.

J Biol Chem, 2002 Jun 14, 277(24), 21585 - 91 Epub 2002 Apr 01.
Identification of a novel non-structural maintenance of chromosomes (SMC) component of the SMC5-SMC6 complex involved in DNA repair; Fujioka Y et al.; Structural maintenance of chromosomes (SMC) proteins play central roles in chromosome organization and dynamics . They have been classified into six subtypes, termed SMC1 to SMC6, and function as heterodimer components of large protein complexes that also include several non-SMC proteins . The SMC2-SMC4 and SMC1-SMC3 complexes are also known as condensin and cohesin, respectively, but the recently identified SMC5 and SMC6 complex is less well characterized . Here, we report that NSE1 from Saccharomyces cerevisiae encodes a novel non-SMC component of the SMC5(Yol034wp)-SMC6(Rhc18p) complex corresponding to the 2-3-MDa molecular mass . Nse1p is essential for cell proliferation and localizes primarily in the nucleus . nse1 mutants are highly sensitive to DNA-damaging treatments and exhibit abnormal cellular morphologies, suggesting aberrant mitosis as a terminal morphological phenotype . These results are consistent with the reported features of the Schizosaccharomyces pombe SMC6 gene, rad18, which is thought to be involved in recombinational DNA repair . We conclude that Nse1p and the SMC5-SMC6 heterodimer together form a high molecular mass complex that is conserved in eukaryotes and required for both DNA repair and proliferation.

J Biol Chem, 2002 Jun 14, 277(24), 21291 - 9 Epub 2002 Mar 28.
A RAC protein-binding site in the internal transcribed spacer 2 of Pre-rRNA transcripts from Schizosaccharomyces pombe; Abeyrathne PD et al.; The interdependence of steps in the processing of the eukaryotic preribosomal rRNA transcripts indicate that rRNA processing, at least in part, acts as a quality control mechanism to help ensure that only functional rRNA is incorporated into mature ribosomes . In search of structural components that underlie this interdependence, we have isolated a large protein complex or RAC that contains an independent binding site for all four of the transcribed spacers in the nascent pre-rRNA . In this study the RAC-binding site in the internal transcribed spacer 2 sequence of Schizosaccharomyces pombe rRNA transcripts was identified, and the influence of this site on rRNA maturation was assessed . Modification exclusion analyses indicate that the protein complex interacts with a helical domain previously shown to contain features common to both the internal transcribed spacer 1 and the 3'-external transcribed spacer . Mutagenic analyses in vitro confirm an interaction with this sequence, and parallel analyses in vivo indicated a critical role in both the maturation of the rRNA components of the large subunit as well as the 18 S rRNA component of the small subunit . Hybridization analyses also indicated greatly elevated levels of unprocessed nascent RNA . These effects are contrasted with mutations in other regions of the secondary structure that resulted in some reduction of plasmid-derived mature rRNA but no elevated levels of the precursor molecules . The significance with respect to rRNA maturation and the interdependences in rRNA processing are discussed.

Yeast, 2002 Apr, 19(6), 521 - 7
The mei3 region of the Schizosaccharomyces pombe genome; Aves SJ et al.; Expression of the mei3 gene is sufficient to induce meiosis in the fission yeast Schizosaccharomyces pombe . The mei3 gene is located 0.64 Mb from the telomere of the left arm of Sz . pombe chromosome II . We have sequenced and analysed 107 kb of DNA from the mei3 genomic region . The sequence includes 14 known genes (bag1-B, csh3, dps1, gpt1, mei3, mfm3, pac1, prp31, rpl38-1, rpn3, rti1, spa1, spm1 and ubc4) and 26 other open reading frames (ORFs) longer than 100 codons: a density of one protein-coding gene per 2.7 kb . Twenty-one of the 40 ORFs (53%) have introns . In addition there is one lone Tf1 transposon long terminal repeat (LTR), tRNA(Trp) and tRNA(Ser) genes and a 5S rRNA gene . 14 of the novel ORFs show sequence similarities which suggest functions of their products, including a coatomer alpha-subunit, a catechol O-methyltransferase, protein kinase, asparagine synthetase, zinc metalloprotease, acetyltransferase, phosphatidylinositol 4-kinase, inositol polyphosphate phosphatase, GTPase-activating protein, permease, pre-mRNA splicing factor, 20S proteasome component and a thioredoxin-like protein . One predicted protein has similarity to the human Cockayne syndrome protein CSA and one with human GTPase XPA binding protein XAB1 . Three ORFs are likely to code for proteins because they have sequence similarity with hypothetical proteins, three encode predicted coiled-coil proteins and four are sequence orphans .

Yeast, 2002 Apr, 19(6), 485 - 98
Marker construction and cloning of a cut1-like sequence with ARS activity in the fission yeast Schizosaccharomyces japonicus; Bozsik A et al.; The dimorphic fission yeast Schizosaccharomyces japonicus has proved to be an excellent experimental model for the investigation of the eukaryotic cell . Here we show that it has a haplontic life cycle, in which the diploid phase is confined to the zygote . To make it amenable to genetic and molecular analysis, we generated genetic markers and cloned a genomic sequence which acts as ars when integrated into a plasmid . Diploids suitable for testing complementation and recombination between markers can be formed by protoplast fusion . The complementation tests and the recombination frequencies determined in octads of spores identified 28 non-allelic groups (genes) of mutations of the auxotrophic and mycelium-negative mutants . Two groups of linked markers were also identified . The cloned fragment, which expresses ars activity, encodes a putative amino acid sequence highly similar to a conserved domain of proteins Cut1 (Schizosaccharomyces pombe), BimB (Aspergillus nidulans) and Esp1 (Saccharomyces cerevisiae) .

J Cell Physiol, 2002 Apr, 191(1), 28 - 41
DNA mismatch repair and mutation avoidance pathways; Marti TM et al.; Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination . The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms . MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination . Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination . No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E . coli . Repair can be initiated by the heterodimers MSH2-MSH6 (MutSalpha) and MSH2-MSH3 (MutSbeta) . Interestingly, MSH3 (and thus MutSbeta) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR . MLH1-PMS1 (MutLalpha) is the major MutL homologous heterodimer . Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR . Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases delta and epsilon . MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1 . A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides . Such large loops cannot be processed by MMR .

Cancer, 2002 Mar 15, 94(6), 1808 - 14
Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin; Kinzel B et al.; BACKGROUND: In Schizosaccharomyces pombe, Hus1 is a component of the radiation sensitive (Rad) machinery that has been identified as playing a role in DNA repair and cell cycle G2/M checkpoint control pathways . Hus1 has been shown to exist in a discrete complex with at least two Rad family members, Rad1 and Rad9 . Furthermore, Hus1 is essential for checkpoint activation, since Hus1 mutants fail to arrest the cell cycle in response to DNA damage or unreplicated DNA . To establish the role and relevance of human Hus1 in cell cycle regulation, the authors applied antisense technology to selectively downregulate the expression of Hus1 mRNA . METHODS: Transfection of 2'-O-methoxyethyl-modified Hus1 antisense oligoribonucleotides into human H1299 nonsmall lung carcinoma cells was performed using Lipofectin as the carrier . The authors prepared RNA from transfected cells, and levels of Hus1 expression were analyzed by real time polymerase chain reaction . The growth and viability of cells treated with Hus1 antisense oligonucleotides in the presence or absence of cisplatin were analyzed and compared to controls . RESULTS: Transfection of selected Hus1 antisense oligonucleotides into p53 deficient H1299 cells resulted in significant downregulation of Hus1 mRNA, up to 80%; RNA analyses reveal a maximal Hus1 antisense activity at a concentration of 200 nM with an IC50 determined to be 90 nM . The design and transfection of oligonucleotides containing three mismatches to their corresponding antisense counterparts had no or only minor effects on Hus1 mRNA levels, showing the specificity of Hus1 mRNA downregulation . The cisplatin IC50 in untransfected H1299 cells was found to be 20 microM and could be reduced significantly to only 7 microM after transfection of a Hus1 antisense oligonucleotide . CONCLUSIONS: Experiments addressing the proliferation and viability of transfected H1299 cells suggest that downregulation of Hus1 by specific antisense oligonucleotides sensitizes human cells to treatment with the DNA damaging agent cisplatin .

Mol Genet Genomics, 2002 Mar, 267(1), 88 - 95 Epub 2002 Feb 08.
The deubiquitinating enzyme Ubp21p of fission yeast stabilizes a mutant form of protein kinase Prp4p; Richert K et al.; The protein kinase Prp4p of Schizosaccharomyces pombe is involved in control of the formation of active spliceosomes, phosphorylating the spliceosomal component Prp1p . The kinase domain of Prp4p is closely related to cyclin-dependent kinases (CDKs) and mitogen-activated kinases (MAPKs) . A mutational analysis of the highly conserved amino acid sequence ALKHP in subdomain XI of this kinase showed that structural features of this sequence are important for the function of the kinase . We identified ubp21 as a high-copy-number suppressor of a mutation in the ALKHP motif . Characterization of this gene revealed that it encodes a deubiquitinating enzyme belonging to the family of ubiquitin-specific processing proteases (Ubps) . The results presented in this report are consistent with the notion that the deubiquitinating activity of Ubp21p may be involved in regulating the steady-state levels of proteins including Prp4p.

Genes Cells, 2002 Mar, 7(3), 273 - 84
Level of the RNA polymerase II in the fission yeast stays constant but phosphorylation of its carboxyl terminal domain varies depending on the phase and rate of cell growth; Sakurai H et al.; BACKGROUND: The RNA polymerase II of the fission yeast Schizosaccharomyces pombe consists of 12 Rpb subunits, of which four (Rpb1, Rpb2, Rpb3 and Rpb11) form the assembly and catalytic core and five (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are shared among RNA polymerases I, II and III . The intracellular levels of three RNA polymerase forms should be interrelated, but the control of RNA polymerase formation remains mostly unknown . RESULTS: To reveal the physiological role and the synthesis control of each Rpb subunit, the intracellular levels of the Rpb proteins were examined in S . pombe growing at various phases under various conditions . Results indicate that the intracellular concentrations of the Rpb proteins stay constant at levels characteristic of the rate and phase of cell growth, and the relative level between the 12 subunits also remains constant, together implying that the intracellular concentration of RNA polymerase II stays constant, as in the case of prokaryotes . As an attempt to gain insights into the activity control of RNA polymerase II, we also analysed the phosphorylation level of the carboxyl-terminal domain (CTD) of the largest subunit Rpb1 . Phosphorylated forms of Tyr1 and Thr4 within 29 repeats of the YSPTSPS heptapeptide were detected in both slow-migrating IIo and fast-migrating IIa forms of Rpb1 on SDS-PAGE (polyacrylamide gel electrophoresis) . However, phosphorylated Ser2 and Ser5 were identified only in the IIo form, indicating that Ser phosphorylation contributes to the conformational change in CTD . The phosphorylation levels of Ser, Thr and Tyr all vary depending on the cell culture conditions . CONCLUSION: The intracellular level of RNA polymerase II stays constant, but the amount engaged in transcription cycle varies depending on the culture conditions, as estimated from the sites and levels of phosphorylation of Rpb1 CTD.

Nucleic Acids Res, 2002 Apr 1, 30(7), 1465 - 82
Transcriptional silencing in Saccharomyces cerevisiae and Schizosaccharomyces pombe; Huang Y; Transcriptional silencing is a heritable form of gene inactivation that involves the assembly of large regions of DNA into a specialized chromatin structure that inhibits transcription . This phenomenon is responsible for inhibiting transcription at silent mating-type loci, telomeres and rDNA repeats in both budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, as well as at centromeres in fission yeast . Although transcriptional silencing in both S.cerevisiae and S.pombe involves modification of chromatin, no apparent amino acid sequence similarities have been reported between the proteins involved in establishment and maintenance of silent chromatin in these two distantly related yeasts . Silencing in S.cerevisiae is mediated by Sir2p-containing complexes, whereas silencing in S.pombe is mediated primarily by Swi6-containing complexes . The Swi6 complexes of S.pombe contain proteins closely related to their counterparts in higher eukaryotes, but have no apparent orthologs in S.cerevisiae . Silencing proteins from both yeasts are also actively involved in other chromosome-related nuclear functions, including DNA repair and the regulation of chromatin structure.

DNA Seq, 2001 Dec, 12(5-6), 295 - 303
Identification and characterization of two genes encoding the eukaryotic initiation factor 4A in rice; Kato A et al.; A cDNA encoding eukaryotic translation initiation factor 4A (eIF4A) was isolated from a cDNA library of rice (Oryza sativa L.) . Based on this DNA sequence, a 414-amino acid protein exhibiting 67, 64 and 59% homology to the mouse, Schizosaccharomyces pombe and Saccharomyces cerevisiae eIF4A, respectively, was predicted . The deduced amino acid sequence contains the characteristic motifs shared by the DEAD box supergene family . Another cDNA of rice eIF4A was reported previously . Comparison of the coding sequences of the two rice eIF4As showed 85% homology in the nucleotide sequence and 90% homology in the amino acid sequence . The genomic clones corresponding to the two rice eIF4A cDNAs were also isolated from a genomic library of rice (Oryza sativa L.) . It was found that the two genes have common patterns of exon-intron boundaries . Their coding regions are split into four exons, and there is an additional exon in the 5'-non coding region.

Mol Cells, 2002 Feb 28, 13(1), 148 - 53
Development of a new xenoestrogen screening system using fission yeast Schizosaccharomyces pombe; Yoo EJ et al.; Endocrine disrupters refer to environmental or chemical compounds, which interfere with the endocrine system of organisms . In this study, our aim was to develop a screening method to detect xenoestrogen (an endocrine disrupter that is commonly encountered in our daily life) by using fission yeast Schizosaccharomyces pombe . Although the yeast (the simplest eukaryotic cell) has no endocrine system, estrogen receptors that are created to express in the yeast cell can be activated by estrogen in a similar manner to mammalian cells . First, in order to express the human estrogen receptor (hER) in the yeast cell, we constructed a yeast expression vector that contained hER (pREP42MHN-hER) . In the yeast cells that are transformed with the pREP42MHN-hER vector, estrogen receptors could recognize xenoestrogen, which allowed the determination of the presence of xenoestrogen in any given sample . Furthermore, we constructed a yeast strain that contained an estrogen responsive element (ERE) that fused with the Escherichia coli LacZ gene (pERE-LacZ) as a reporter for binding of xenoestrogen with the estrogen receptor . Since this vector system allows determination of the presence and level of xenoestrogen with simple procedures, it is expected that they can serve as an efficient assay system to detect xenoestrogen.

J Biol Chem, 2002 May 31, 277(22), 19817 - 22 Epub 2002 Mar 21.
The unique centromeric chromatin structure of Schizosaccharomyces pombe is maintained during meiosis; Smirnova JB et al.; In meiosis I sister centromeres are unified in their polarity on the spindle, and this unique behavior is known to require the function of meiosis-specific factors that set some intrinsic property of the centromeres . The fission yeast, Schizosaccharomyces pombe, possesses complex centromeres consisting of repetitive DNA elements, making it an excellent model in which to study the behavior of complex centromeres . In mitosis, during which sister centromeres mediate chromosome segregation by establishing bipolar chromosome attachments to the spindle, the central core of the S . pombe centromere chromatin has a unique irregular nucleosome pattern . Deletion of repeats flanking this core structure have no effect on mitotic chromosome segregation, but have profound effects during meiosis . While this demonstrates that the outer repeats are critical for normal meiotic sister centromere behavior, exactly how they function and how monopolarity is established remains unclear . In this study we provide the first analysis of the chromatin structure of a complex centromere during meiosis . We show that the nature and extent of the unique central core chromatin structure is maintained with no measurable expansion . This demonstrates that monopolarity of sister centromeres, and subsequent reversion to bipolarity, does not involve a global change to the centromeric chromatin structure.

Arch Microbiol, 2002 Mar, 177(3), 251 - 8 Epub 2002 Jan 09.
Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe; Voicu PM et al.; The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it . It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation . The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development . A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions . This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program . The events of the program that were affected by inositol starvation were investigated . Commitment to mating and production of pheromone M were shown not to be inositol-dependent . A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation . In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent . The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.

Mol Biol Cell, 2002 Mar, 13(3), 989 - 1000
The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe; Liu J et al.; Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring . Constriction of the actomyosin ring is accompanied by the centripetal addition of new membranes and cell wall material . In this article, we characterize the mechanism responsible for the localization of Cps1p, a septum-synthesizing 1,3-beta-glucan synthase, to the division site during cytokinesis . We show that Cps1p is an integral membrane protein that localizes to the cell division site late in anaphase . Neither F-actin nor microtubules are essential for the initial assembly of Cps1p to the medial division site . F-actin, but not microtubules, is however important for the eventual incorporation of Cps1p into the actomyosin ring . Assembly of Cps1p into the cell division ring is also dependent on the septation-inducing network (SIN) proteins that regulate division septum formation after assembly of the actomyosin ring . Fluorescence-recovery after-photobleaching experiments reveal that Cps1p does not diffuse appreciably within the plasma membrane and is retained at the division site by a mechanism that does not depend on an intact F-actin cytoskeleton . We conclude that the actomyosin ring serves as a spatial cue for Cps1p localization, whereas the maintenance of Cps1p at the division site occurs by a novel F-actin- and microtubule-independent mechanism . Furthermore, we propose that the SIN proteins ensure localization of Cps1p at the appropriate point in the cell cycle.

Mol Biol Cell, 2002 Mar, 13(3), 930 - 46
The 14-kDa dynein light chain-family protein Dlc1 is required for regular oscillatory nuclear movement and efficient recombination during meiotic prophase in fission yeast; Miki F et al.; A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family . Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle . During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1 . In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction . Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant . Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant . Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase . Strains mutated in both the dlc1 and dhc1 loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1 . S . pombe contains a homolog of the 8-kDa dynein light chain, Dlc2 . This class of dynein light chain, however, is not essential in either the vegetative or sexual phases.

Mol Biol Cell, 2002 Mar, 13(3), 805 - 16
Distinct regulatory proteins control the graded transcriptional response to increasing H(2)O(2) levels in fission yeast Schizosaccharomyces pombe; Quinn J et al.; The signaling pathways that sense adverse stimuli and communicate with the nucleus to initiate appropriate changes in gene expression are central to the cellular stress response . Herein, we have characterized the role of the Sty1 (Spc1) stress-activated mitogen-activated protein kinase pathway, and the Pap1 and Atf1 transcription factors, in regulating the response to H(2)O(2) in the fission yeast Schizosaccharomyces pombe . We find that H(2)O(2) activates the Sty1 pathway in a dose-dependent manner via at least two sensing mechanisms . At relatively low levels of H(2)O(2), a two component-signaling pathway, which feeds into either of the two stress-activated mitogen-activated protein kinase kinase kinases Wak1 or Win1, regulates Sty1 phosphorylation . In contrast, at high levels of H(2)O(2), Sty1 activation is controlled predominantly by a two-component independent mechanism and requires the function of both Wak1 and Win1 . Individual transcription factors were also found to function within a limited range of H(2)O(2) concentrations . Pap1 activates target genes primarily in response to low levels of H(2)O(2), whereas Atf1 primarily controls the transcriptional response to high concentrations of H(2)O(2) . Our results demonstrate that S . pombe uses a combination of stress-responsive regulatory proteins to gauge and effect the appropriate transcriptional response to increasing concentrations of H(2)O(2).

Genetics, 2002 Mar, 160(3), 891 - 908
UV irradiation causes the loss of viable mitotic recombinants in Schizosaccharomyces pombe cells lacking the G(2)/M DNA damage checkpoint; Osman F et al.; Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage . Cell cycle delays in response to DNA damage are mediated via checkpoint proteins . Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G(2)/M checkpoint stops cells passing from G(2) into mitosis . In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays . The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G(2)/M checkpoint . Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks . Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels . Mutants lacking the G(2)/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants . This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G(2)/M mutants after UV irradiation . Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G(2)/M checkpoint mutants.

Genome Biol . 2002;3(3):COMMENT2003 . Epub 2002 Feb 22.
The model unicellular eukaryote, Schizosaccharomyces pombe; Yanagida M; The fission yeast Schizosaccharomyces pombe has long been a model organism for studies of eukaryotic cells, winning renown especially for studies of the cell cycle . Now that its genome has been sequenced, S . pombe is ready to assume its rightful place in the pantheon of small eukaryotic giants.

Genes Cells, 2002 Feb, 7(2), 217 - 31
Meu10 is required for spore wall maturation in Schizosaccharomyces pombe; Tougan T et al.; BACKGROUND: Many genes are meiosis and/or sporulation-specifically transcribed during this process . Isolation and analysis of these genes might help us to understand how meiosis and sporulation are regulated . For this purpose, we have isolated a large number of cDNA clones from Schizosaccharomyces pombe whose expression is up-regulated during meiosis . RESULTS: We have isolated meu10+ gene, which encodes 416 amino acids and bears homology to SPS2 of Saccharomyces cerevisiae . A strain whose meu10+ gene has been deleted forms no viable spores . Thin-section electron micrographs showed that the meu10Delta strain has abnormally formed spore walls, and then they disrupt, allowing cytoplasmic material to escape . The Meu10-GFP fusion protein is localized to the spore periphery, thereafter returned to the cytoplasm after sporulation . Meu10-GFP localization to the spore wall was almost normal in the bgs2Delta or chs1Delta mutants that lack 1,3-beta-glucan or chitin, respectively . In contrast, 1,3-beta-glucan is abnormally localized in meu10Delta cells . Meu10 has an N-terminal domain with homology to the mammalian insulin receptor and a C-terminal domain with a transmembrane motif . Mutants whose N-terminal or C-terminal domain was truncated were severely defective for sporulation . CONCLUSIONS: Meu10 is a spore wall component and plays a pivotal role in the formation of the mature spore wall structure.

Genes Cells, 2002 Feb, 7(2), 199 - 215
Phosphatidylinositol 3-phosphate 5-kinase is required for the cellular response to nutritional starvation and mating pheromone signals in Schizosaccharomyces pombe; Morishita M et al.; BACKGROUND: Phosphatidylinositol (3,5) bisphosphate, which is converted from phosphatidylinositol 3-phosphate by phosphatidylinositol 3-phosphate 5-kinase, is implicated in vacuolar functions and the sorting of cell surface proteins within endosomes in the endocytic pathway of budding yeast . A homologous protein, SpFab1p, has been found in the fission yeast Schizosaccharomyces pombe, but its role is not known . RESULTS: Here we report that SpFab1p is encoded by ste12+ known as a fertility gene in S . pombe . The ste12 mutant grew normally under stress-free conditions, but was highly vacuolated and swelled at high temperatures and under starvation conditions . In nitrogen-free medium, ste12 cells were arrested in G1 phase, but partially defective in the expression of genes responsible for mating and meiosis . The ste12 mutant was defective both in the production of, and in the response to, mating pheromones . The amount of the pheromone receptor protein Map3p, was substantially decreased in ste12 cells . Map3p was transported to the cell surface, then internalized and eventually transported to the vacuolar lumen, even in the ste12 mutant . CONCLUSION: The results indicate that phosphatidylinositol(3,5)bisphosphate is essential for cellular responses to various stresses and for the mating pheromone signalling under starvation conditions.

Genes Cells, 2002 Feb, 7(2), 115 - 32
Isolation and characterization of a new nucleolar protein, Nrap, that is conserved from yeast to humans; Utama B et al.; BACKGROUND: The nucleolus is the site of rRNA synthesis and processing in eukaryotic cells, but its composition remains poorly understood . RESULTS: We have identified a novel nucleolar RNA-associated protein (Nrap) which is highly conserved from yeast (Saccharomyces cerevisiae) to human, with homologues in mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and other species . In the mouse, we have found that Nrap is ubiquitously expressed and is specifically localized in the nucleolus . We have also identified splice variants in human and mouse, and defined the intron-exon structure of the human Nrap gene . Nrap is inherited into daughter nuclei by associating with the condensed chromosomes during mitosis . RNase treatment of permeabilized cells indicated that the nucleolar localization of Nrap is RNA dependent . The effects of actinomycin D, cycloheximide and 5,6-dichloro-beta-d-ribofuranosyl-benzimidazole on Nrap expression and distribution in cultured cells suggest that Nrap is associated with the pre-rRNA transcript . CONCLUSIONS: Nrap is a large nucleolar protein (of more than 1000 amino acids), and is a new class of protein with new structural and functional motifs . Nrap appears to be associated with ribosome biogenesis by interacting with pre-rRNA primary transcript.

J Biol Chem, 2002 May 31, 277(22), 19639 - 48 Epub 2002 Mar 13.
Interactions between fission yeast mRNA capping enzymes and elongation factor Spt5; Pei Y et al.; Elongating RNA polymerase II is targeted by macromolecular assemblies that regulate mRNA synthesis and processing . The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA . Although recruitment of the capping enzymes to the transcription complex is dependent on phosphorylation of the C-terminal domain of the Rpb1 subunit of polymerase II (Pol-II), there may be additional levels of control that coordinate capping with elongation . Here we show that the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the fission yeast capping apparatus bind independently to the elongation factor Spt5 . The C-terminal domain of the 990-amino acid Schizosaccharomyces pombe Spt5 protein, composed of repeats of a nonapeptide motif (consensus sequence TPAWNSGSK), is necessary and sufficient for binding to the capping enzymes in vivo (in a two-hybrid assay) and in vitro . As few as four nonamer repeats suffice for Spt5 binding to Pct1 in vitro, whereas six repeats are required for Spt5 binding to Pce1 . A 116-amino acid fragment of the guanylyltransferase Pce1 suffices for binding to the Spt5 C-terminal domain (CTD) but not for binding to the Pol-II CTD . Pct1 and Pce1 can bind simultaneously to the Spt5 CTD in vitro . We find that Spt5 is essential for viability of S . pombe and that it interacts in vivo with S . pombe Spt4 via a central domain distinct from the Spt5 CTD . We suggest that Spt5-induced arrest of elongation at promoter proximal positions ensures a temporal window for recruitment of the capping enzymes.

Cancer Res, 2002 Mar 1, 62(5), 1246 - 50
Isolation of a novel gene, CABC1, encoding a mitochondrial protein that is highly homologous to Yyast activity of bc1 complex; Iiizumi M et al.; To search for p53 target genes throughout the human genome, we applied a cDNA microarray system using adenovirus-mediated transfer of p53 into p53-deficient U373MG (glioblastoma) cells . In this manner, we detected dozens of genes that appeared to be regulated by wild-type p53 . We describe here characterization of one such gene, termed CABC1 {chaperone-activity of bc1 complex in Schizosaccharomyces pombe (ABC1)-like}, which encodes a 647-amino acid peptide with significant sequence similarity to activity of bc1 complex (ABC1) in Arabidopsis thaliana and S . pombe . The CABC1 product was located in mitochondria, and colony-formation assays with cancer cell lines indicated its ability to suppress cell growth . Inhibition of CABC1 expression by transfection with antisense oligonucleotide significantly reduced the apoptotic response induced by wild-type p53 . These results suggest that CABC1 may play an important role in mediating p53-inducible apoptosis through the mitochondrial pathway.

J Biol Chem, 2002 May 17, 277(20), 18215 - 21 Epub 2002 Mar 08.
A transporter in the endoplasmic reticulum of Schizosaccharomyces pombe cells mediates zinc storage and differentially affects transition metal tolerance; Clemens S et al.; The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters . Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals . A deletion mutant is Zn(2+)/Co(2+)-hypersensitive yet displays significantly enhanced Cd(2+) and Ni(2+) tolerance . Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells . Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf(-) cells compared with wild-type cells in the presence of elevated Zn(2+) concentrations . The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway . Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted . Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf . Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis . Also, these observations indicate that Cd(2+) ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.

Nucleic Acids Res, 2002 Mar 15, 30(6), 1408 - 17
DAP-like kinase interacts with the rat homolog of Schizosaccharomyces pombe CDC5 protein, a factor involved in pre-mRNA splicing and required for G2/M phase transition; Engemann H et al.; DAP-like kinase (Dlk, also termed ZIP kinase) is a leucine zipper-containing serine/threonine-specific protein kinase with as yet unknown biological function(s) . Interaction partners so far identified are either transcription factors or proteins that can support or counteract apoptosis . Thus, Dlk might be involved in regulating transcription or, more generally, survival or apoptosis . Here we report on a new interaction partner, the rat homolog of Schizosaccharomyces pombe CDC5 protein, a presumptive transcription and splicing factor involved in the G(2)/M transition . In vitro, rat CDC5 forms complexes with, but is not phosphorylated by, Dlk . Rather, it was phosphorylated by an associated kinase which was identified as CK2 . The interaction domain of Dlk was mapped to the leucine zipper, while that of CDC5 was mapped to the C-terminal region between residues 500 and 802 . In vivo, both proteins co-localize perfectly in distinct speckle-like structures in the nucleus, some of which overlap with promyelocytic leukemia protein . Interestingly, splicing factor SC35, which also resides in speckles, was partially displaced upon overexpression of either CDC5 or Dlk, perhaps due to phosphorylation by Dlk . Together with previous data, these results suggest that Dlk might play a role in coordinating specific transcription and splicing events.

Nucleic Acids Res, 2002 Mar 15, 30(6), 1316 - 24
Differential expression and requirements for Schizosaccharomyces pombe RAD52 homologs in DNA repair and recombination; van den Bosch M et al.; In fission yeast two RAD52 homologs have been identified, rad22A(+) and rad22B(+) . Two-hybrid experiments and GST pull-down assays revealed physical interaction between Rad22A and Rad22B, which is dependent on the N-terminal regions . Interaction with Rhp51 is dependent on the C-terminal parts of either protein . Both Rad22A and Rad22B also interact with RPA . The expression of rad22B(+) in mitotically dividing cells is very low in comparison with rad22A(+) but is strongly enhanced after induction of meiosis, in contrast to rad22A(+) . Rad22B mutant cells are not hypersensitive to DNA-damaging agents (X-rays, UV and cisplatin) and display normal levels of recombination . In these respects the Schizosaccharomyces pombe rad22B mutant resembles the weak phenotype of vertebrate cells deficient for RAD52 . Mutation of rad22A(+) leads to severe sensitivity to DNA-damaging agents and to defects in recombination . In a rad22Arad22B double mutant a further increase in sensitivity to DNA-damaging agents and additional mitotic recombination defects were observed . The data presented here indicate that Rad22A and Rad22B have overlapping roles in repair and recombination, although specialized functions for each protein cannot be excluded.

Mol Cell Biol, 2002 Apr, 22(7), 2170 - 81
Functional divergence between histone deacetylases in fission yeast by distinct cellular localization and in vivo specificity; Bjerling P et al.; Histone deacetylases (HDACs) are important for gene regulation and the maintenance of heterochromatin in eukaryotes . Schizosaccharomyces pombe was used as a model system to investigate the functional divergence within this conserved enzyme family . S . pombe has three HDACs encoded by the hda1(+), clr3(+), and clr6(+) genes . Strains mutated in these genes have previously been shown to display strikingly different phenotypes when assayed for viability, chromosome loss, and silencing . Here, conserved differences in the substrate binding pocket identify Clr6 and Hda1 as class I HDACs, while Clr3 belongs in the class II family . Furthermore, these HDACs were shown to have strikingly different subcellular localization patterns . Hda1 was localized to the cytoplasm, while most of Clr3 resided throughout the nucleus . Finally, Clr6 was localized exclusively on the chromosomes in a spotted pattern . Interestingly, Clr3, the only HDAC present in the nucleolus, was required for ribosomal DNA (rDNA) silencing . Clr3 presumably acts directly on heterochromatin, since it colocalized with the centromere, mating-type region, and rDNA as visualized by in situ hybridization . In addition, Clr3 could be cross-linked to mat3 in chromatin immunoprecipitation experiments . Western analysis of bulk histone preparations indicated that Hda1 (class I) had a generally low level of activity in vivo and Clr6 (class I) had a high level of activity and broad in vivo substrate specificity, whereas Clr3 (class II) displayed its main activity on acetylated lysine 14 of histone H3 . Thus, the distinct functions of the S . pombe HDACs are likely explained by their distinct cellular localization and their different in vivo specificities.

Mol Cell Biol, 2002 Apr, 22(7), 2011 - 24
Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs; Ohi MD et al.; Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes . Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes . Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure . The components of the S . pombe Cdc5p/S . cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A . J . Link et al., Nat . Biotechnol . 17:676-682, 1999) . At least 26 proteins were detected in the Cdc5p/Cef1p complexes . Comparison of the polypeptides identified by S . pombe Cdc5p purification with those identified by S . cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition . The majority of S . pombe Cwf proteins and S . cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components . In addition, the complex contains the U2, U5, and U6 snRNAs . Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing . Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

Mol Cell Biol, 2002 Apr, 22(7), 1998 - 2010
Regulation of insulin-like growth factor type I (IGF-I) receptor kinase activity by protein tyrosine phosphatase 1B (PTP-1B) and enhanced IGF-I-mediated suppression of apoptosis and motility in PTP-1B-deficient fibroblasts; Buckley DA et al.; The insulin-like growth factor type I (IGF-I) receptor (IGF-IR), activated by its ligands IGF-I and IGF-II, can initiate several signal transduction pathways that mediate suppression of apoptosis, proliferation, differentiation, and transformation . Here we investigated the regulation of IGF-IR activation and function by protein tyrosine phosphatase 1B (PTP-1B) . Coexpression of PTP-1B with a beta-chain construct of the IGF-IR (betaWT) inhibited IGF-IR kinase activity in fission yeast Schizosaccharomyces pombe, in COS cells, and in IGF-IR-deficient fibroblasts . In both spontaneously immortalized and simian virus 40 T antigen-transformed embryonic fibroblast cell lines derived from PTP-1B knockout mice, IGF-I induced higher levels of IGF-IR autophosphorylation and kinase activity than were induced in PTP-1B-expressing control cells . PTP-1B-deficient cells exhibited enhanced IGF-I-mediated protection from apoptosis in response to serum withdrawal or etoposide killing, as well as enhanced plating efficiency and IGF-I-mediated motility . Reexpression of PTP-1B in spontaneously immortalized fibroblasts resulted in decreased IGF-IR and AKT activation, as well as decreased protection from apoptosis and decreased motility . These findings demonstrate that PTP-1B can regulate IGF-IR kinase activity and function and that loss of PTP-1B can enhance IGF-I-mediated cell survival, growth, and motility in transformed cells.

J Cell Sci, 2002 Mar 15, 115(Pt 6), 1113 - 22
Cell-cycle-dependent localisation of Ulp1, a Schizosaccharomyces pombe Pmt3 (SUMO)-specific protease; Taylor DL et al.; We report here on the characterisation of Ulp1, a component of the SUMO modification process in S . pombe . Recombinant S . pombe Ulp1 has de-sumoylating activity; it is involved in the processing of Pmt3 (S . pombe SUMO) and can, to a limited extent, remove Pmt3 from modified targets in S . pombe cell extracts . ulp1 is not essential for cell viability, but cells lacking the gene display severe cell and nuclear abnormalities . ulp1-null (ulp1.d) cells are sensitive to ultraviolet radiation in a manner similar to rad31.d and hus5.62, which have mutations in one subunit of the activator and the conjugator for the ubiquitin-like protein SUMO respectively . However ulp1.d cells are less sensitive to ionising radiation and hydroxyurea (HU) than are rad31.d and hus5.62 . ulp1-null cells are defective in processing precursor Pmt3 and display reduced levels of Pmt3 conjugates compared with wild-type cells . The slow growth phenotype of ulp1 null cells is not substantially rescued by over-expression of the mature form of Pmt3 (Pmt3-GG), suggesting that the de-conjugating activity of Ulp1 is required for normal cell cycle progression . During the S and G2 phases of the cell cycle the Ulp1 protein is localised to the nuclear periphery . However, during mitosis the pattern of staining alters, and during anaphase, Ulp1 is observed within the nucleus . Ulp1 localisation at the nuclear periphery is generally re-established by the time of septation (S phase).

Planta, 2002 Mar, 214(5), 783 - 91 Epub 2001 Nov 24.
Characterization of the ZAT1p zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes; Bloss T et al.; The ZAT1p zinc transporter from Arabidopsis thaliana (L.) Heynh . is a member of the cation diffusion facilitator (CDF) protein family . When heterologously expressed in Escherichia coli, ZAT1p bound zinc in a metal blot . Binding of zinc occurred mainly to the hydrophilic amino acid region from H182 to H232 . A ZAT1p/ZAT1p*Delta(M1-I25) protein mixture was purified and reconstituted into proteoliposomes . Uptake of zinc into the proteoliposomes did not require a proton gradient across the liposomal membrane . ZAT1p did not transport cobalt, and transported cadmium at only 1% of the zinc transport rate . ZAT1p functioned as an uptake system for 65Zn2+ in two strains of the Gram-negative bacterium Ralstonia metallidurans, which were different in their content of zinc-efflux systems . The ZAT1 gene did not rescue increased zinc sensitivity of a Delta ZRC1single-mutant strain or of a Delta ZRC1 Delta COT1 double-mutant strain of Saccharomyces cerevisiae, but ZAT1 complemented this phenotype in a Delta SpZRC1 mutant strain of Schizosaccharomyces pombe.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 147 - 56
Non-conventional yeasts; Spencer JF et al.; In the beginning there was yeast, and it raised bread, brewed beer, and made wine . After many not days but centuries and even millenia later, it was named Saccharomyces cerevisiae . After more years and centuries there was another yeast, and it was named Schizosaccharomyces pombe; now there were two stars in the yeast heaven . In only a few more years there were other yeasts, and then more, and more, and more . The era of the non-conventional yeasts had begun.

J Biochem (Tokyo), 2002 Mar, 131(3), 391 - 8
Genes for a nuclease and a protease are involved in the drastic decrease in cellular RNA amount in fission yeast cells during nitrogen starvation; Nakashima A et al.; Cellular RNA in Schizosaccharomyces pombe cells drastically decreases in amount during nitrogen starvation . Previously, we found and purified a soluble RNA-degrading enzyme whose activity drastically increased in the cells of S . pombe undergoing nitrogen starvation . The enzyme was a nuclease encoded by pnu1(+) . In this study, the increase in the RNA-degrading activity and the decrease in cellular RNA level are examined in a null-mutant of pnu1(+) (pnu1Delta) . During nitrogen starvation, wild-type cells show an apparent increase in RNA-degrading activity, whereas the pnu1Delta cells do not . The wild-type cells show a drastic decrease in cellular RNA amount, whereas the pnu1Delta cells show only a slight decrease . These results suggest that Pnu1 nuclease is implicated in the decrease in cellular RNA amount during nitrogen starvation, probably via the RNA-degrading activity . The increase in the RNA-degrading activity is independent of both the Wis1 stress-activated MAP kinase cascade and Tor1 signaling pathway, but it is strongly dependent on isp6(+), a gene for a possible protease, whose expression is induced during nitrogen starvation . A disruption mutant for isp6(+) (isp6Delta) is deficient in both the increase in the RNA-degrading activity and the drastic decrease in the cellular RNA amount during nitrogen starvation, which suggests that isp6(+) is involved in the RNA degradation via regulating the RNA-degrading activity of Pnu1.

J Cell Sci, 2002 Mar 1, 115(Pt 5), 887 - 98
F-actin ring formation and the role of F-actin cables in the fission yeast Schizosaccharomyces pombe; Arai R et al.; Cells of the fission yeast Schizosaccharomyces pombe divide by the contraction of the F-actin ring formed at the medial region of the cell . We investigated the process of F-actin ring formation in detail using optical sectioning and three-dimensional reconstruction fluorescence microscopy . In wild-type cells, formation of an aster-like structure composed of F-actin cables and accumulation of F-actin cables were recognized at the medial cortex of the cell during prophase to metaphase . The formation of the aster-like structure seemed to initiate from branching of the longitudinal F-actin cables at a site near the spindle pole bodies, which had been duplicated but not yet separated . A single cable extended from the aster and encircled the cell at the equator to form a primary F-actin ring during metaphase . During anaphase, the accumulated F-actin cables were linked to the primary F-actin ring, and then all of these structures seemed to be packed to form the F-actin ring . These observations suggest that formation of the aster-like structure and the accumulation of the F-actin cables at the medial region of the cell during metaphase may be required to initiate the F-actin ring formation . In the nda3 mutant, which has a mutation in ss-tubulin and has been thought to be arrested at prophase, an F-actin ring with accumulated F-actin cables similar to that of anaphase wild-type cells was formed at a restrictive temperature . Immediately after shifting to a permissive temperature, this structure changed into a tightly packed ring . This suggests that the F-actin ring formation progresses beyond prophase in the nda3 cells once the cells enter prophase . We further examined F-actin structures in both cdc12 and cdc15 early cytokinesis mutants . As a result, Cdc12 seemed to be required for the primary F-actin ring formation during prophase, whereas Cdc15 may be involved in both packing the F-actin cables to form the F-actin ring and rearrangement of the F-actin after anaphase . In spg1, cdc7 and sid2 septum initiation mutants, the F-actin ring seemed to be formed in order.

Biol Reprod, 2002 Mar, 66(3), 735 - 44
SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein; Hao Z et al.; To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114 . Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences . Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57 . SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256 . Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific . SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus . The SAMP32 locus consists of seven exons on chromosome 6q15-16.2 . Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel . This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence . Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome . By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis . Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic . Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.

EMBO J, 2002 Mar 1, 21(5), 1121 - 31
Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing; Schotta G et al.; Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing . Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9) . In Su(var)3-9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3-9 is the major heterochromatin-specific HMTase in Drosophila . SU (VAR)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3-7 . Notably, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins . In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome . By contrast, in HP1 null mutants SU(VAR)3-9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes . Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of HP1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV . Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects . Together, these data indicate a central role for the SU(VAR)3-9 HMTase in heterochromatin-induced gene silencing in Drosophila.

Cell Growth Differ, 2002 Feb, 13(2), 47 - 58
Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe; Flory MR et al.; In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p/Nuf1p facilitates mitotic spindle formation from the fungal centrosome or spindle pole body (SPB) . The human Spc110p orthologue kendrin is a centrosomal, calmodulin-binding pericentrin isoform that is specifically overexpressed in carcinoma cells . Here we establish an evolutionary and functional link between Spc110p and kendrin through identification and analysis of similar calmodulin-binding proteins in the fission yeast Schizosaccharomyces pombe (Pcp1p, pole target of calmodulin in S . pombe) and the filamentous fungus Aspergillus nidulans . Like Spc110p and kendrin, Pcp1p and the A . nidulans protein contain predicted coiled-coil secondary structure and a COOH-terminal calmodulin-binding region . Green fluorescent protein fusions of Pcp1p localize to the SPB as analyzed by fluorescence and immunoelectron microscopy . Pcp1p overexpression causes chromosome missegregation, multiple mitotic spindle fragments, and multiple abnormal SPB-like structures, a phenotype remarkably similar to that of many human carcinoma lines, which exhibit chromosome and spindle defects, and supernumerary centrosomes.

Mol Cell, 2002 Feb, 9(2), 433 - 7
RAC protein directs the complete removal of the 3' external transcribed spacer by the Pac1 nuclease; Spasov K et al.; In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein complex (RAC) which contains independent binding sites for each of the transcribed spacers . The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of the Pac1 nuclease, leading to the complete removal of the 3' ETS . Furthermore, the affinity of RAC protein for mutant 3' ETS correlates closely with in vivo effects on rRNA processing, and changes which disrupt RAC protein binding also inhibit Pac1 nuclease cleavage at the 3' end of the 25S rRNA sequence . The observations indicate that, in the presence of the RAC protein/3' ETS complex, cleavage by the RNase III-like homolog is not restricted to the known intermediate sites but also is directed at the 3' end of the 25S rRNA.

Mol Cell, 2002 Feb, 9(2), 253 - 63
Meiotic recombination remote from prominent DNA break sites in S . pombe; Young JA et al.; DNA breakage is intimately associated with meiotic recombination in the fission yeast Schizosaccharomyces pombe . Sites of prominent DNA breakage were found approximately 25 to approximately 200 kb apart in the genomic regions surveyed . We examined in detail a 501 kb region of chromosome I and found six sites, or tight clusters of sites, at which approximately 2%-11% of the DNA accumulated breaks in a rad50S mutant . In contrast to the discrete, widely spaced distribution of prominent break sites, recombination in this region was more uniformly distributed (0.7-1.6 cM/10 kb) whether the genetic interval tested contained no, one, or more such sites . We infer that although recombination depends upon DNA breakage, recombination often occurs remote from these sites (tens of kilobases away); we discuss mechanisms by which this may occur.

Nucleic Acids Res, 2002 Mar 1, 30(5), 1145 - 53
Glucose-inducible expression of rrg1+ in Schizosaccharomyces pombe: post-transcriptional regulation of mRNA stability mediated by the downstream region of the poly(A) site; Kim MJ et al.; rrg1+(rapid response to glucose) has been isolated previously as a UV-inducible gene in Schizosaccharomyces pombe, designated as uvi22+ . However, it was revealed that the transcript level of this gene was regulated by glucose, not by DNA-damaging agents . Glucose depletion led to a rapid decrease in the level of rrg1+ mRNA, by approximately 50% within 30 min . This effect was readily reversed upon re-introduction of glucose within 1 h . High concentrations (4 and 8%) of glucose showed similar effects on increasing the rrg1+ mRNA level compared with 2% glucose, while a low concentration (0.1%) was not effective in raising the rrg1+ mRNA level . In addition, sucrose and fructose could increase rrg1+ mRNA level . Interestingly, the rapid decline in mRNA level seen upon glucose deprivation resulted from precipitous reduction of mRNA half-life . Serial and internal deletions within the 3'-flanking region of rrg1+ revealed that a 210-nt region downstream of the distal poly(A) site was critical for glucose-regulated expression . Moreover, this downstream region participated in 3'-end formation of mRNA . Taken together, this is the first report on glucose-inducible expression regulated post-transcriptionally by control of mRNA stability in S.pombe.

J Cell Sci, 2002 Feb 1, 115(Pt 3), 587 - 98
Fission yeast Pds5 is required for accurate chromosome segregation and for survival after DNA damage or metaphase arrest; Wang SW et al.; Sister chromatid cohesion, which is established during the S phase of the eukaryotic cell cycle and persists until the onset of anaphase, is essential for the maintenance of genomic integrity . Cohesion requires the multi-protein complex cohesin, as well as a number of accessory proteins including Pds5/BIMD/Spo76 . In the budding yeast Saccharomyces cerevisiae Pds5 is an essential protein that localises to chromosomes in a cohesin-dependent manner . Here we describe the characterisation in the fission yeast Schizosaccharomyces pombe of pds5(+), a novel, non-essential orthologue of S . cerevisiae PDS5 . The S . pombe Pds5 protein was localised to punctate nuclear foci in a manner that was dependent on the Rad21 cohesin component . This, together with additional genetic evidence, points towards an involvement of S . pombe Pds5 in sister chromatid cohesion . S . pombe pds5 mutants were hypersensitive to DNA damage and to mitotic metaphase delay, but this sensitivity was apparently not due to precocious loss of sister chromatid cohesion . These cells also suffered increased spontaneous chromosome loss and meiotic defects and their viability was dependent on the spindle checkpoint protein Bub1 . Thus, while S . pombe Pds5 has an important cohesin-related role, this differs significantly from that of the equivalent budding yeast protein.

J Cell Sci, 2002 Feb 1, 115(Pt 3), 467 - 73
The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis; Bech-Otschir D et al.; Recently the COP9 signalosome (CSN) has become a focus of interest for many researchers, because of its function at the interface between signal transduction and ubiquitin-dependent proteolysis . It is required for the proper progression of the cell cycle in Schizosaccharomyces pombe and is essential for development in plants and DROSOPHILA: However, its function in mammalian cells remains obscure . Although the CSN shares structural similarities with the 26S proteasome lid complex (LID), its functions seem to be different from that of the LID . A variety of CSN-specific protein-protein interactions have been described in mammalian cells . However, it is currently unclear how many reflect true functions of the complex . Two activities associated with the CSN have been identified so far: a protein kinase and a deneddylase . The CSN-associated kinase phosphorylates transcription factors, which determines their stability towards the ubiquitin system . The associated deneddylase regulates the activity of specific SCF E3 ubiquitin ligases . The CSN thus appears to be a platform connecting signalling with proteolysis.

Genetics, 2002 Feb, 160(2), 445 - 56
Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis; Rajagopalan S et al.; The inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains . Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis . We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis . Here we further characterize S . pombe Bir1p using methods of cell biology and genetics . We show that Bir1p is dispersed throughout the nucleus during the cell cycle . In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis . Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A . Bir1p colocalizes with the S . pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone . Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis . In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation . We conclude that Bir1p is important for multiple processes that occur during mitosis in S . pombe.

Nature, 2002 Feb 21, 415(6874), 871 - 80
The genome sequence of Schizosaccharomyces pombe; Wood V et al.; We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824 . The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element . Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions . Some 43% of the genes contain introns, of which there are 4,730 . Fifty genes have significant similarity with human disease genes; half of these are cancer related . We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing . These genes may have originated with the appearance of eukaryotic life . Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.

Genes Cells, 2002 Jan, 7(1), 59 - 73
Polyanionic stretch-deleted histone chaperone cia1/Asf1p is functional both in vivo and in vitro; Umehara T et al.; BACKGROUND: CIA, an interactor of the CCG1 histone acetyltransferase subunit of TFIID, was identified as a human histone chaperone . The Saccharomyces cerevisiae orthologue ASF1, when it was over-expressed, was reported to cause de-repression of silent loci; however, the involvement of Asf1p in the alteration of nucleosomal structures remained unknown . Curiously, there is a polyanionic stretch, a structural motif characteristic of histone chaperones, in S . cerevisiae Asf1p, but not in human CIA . We investigated how CIA/Asf1p utilizes its domain(s) for the alteration of nucleosomal structure . RESULTS: To characterize the relationships between the domain structures and nuclear functions of CIA, we isolated the gene for the CIA counterpart in Schizosaccharomyces pombe, designated cia1+, whose putative product contains a polyanionic stretch . Gene disruption of cia1+ was lethal, which is the distinct phenotype of viable S . cerevisiae asf1 . The cia1- lethality was rescued by the introduction of S . cerevisiae ASF1, but not by the introduction of human CIA cDNA . To our surprise, the construct that produces Asf1p, lacking the polyanionic stretch, is capable of rescuing the lethality caused by the cia1+ deletion, while the highly conserved N-terminal region of Asf1p is essential for the complementation of cia1- growth defects . The polyanionic stretch-deleted Asf1p is sufficient both for interaction with histones H3/H4 and for nucleosome assembly in vitro, as well as for telomeric de-repression in vivo . CONCLUSION: These findings suggest that the areas responsible for both the conserved and species-specific functions of CIA/cia1/Asf1p are within their highly conserved regions and that the yeast-specific polyanionic stretch of cia1/Asf1p is not necessary for viability, histone binding, nucleosome assembly, or anti-silencing.

Eur J Biochem, 2002 Jan, 269(2), 519 - 26
Biosynthesis of riboflavin: 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe; Fischer M et al.; A cDNA sequence from Schizosaccharomyces pombe with similarity to 6,7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia coli strain . The recombinant protein is a homopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibrium centrifugation (it sediments at an apparent velocity of 5.0 S at 20 degrees C) . The protein has been crystallized in space group C2221 . The crystals diffract to a resolution of 2.4 A . The enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy- 2-butanone 4-phosphate . Steady-state kinetic analysis afforded a vmax value of 13 000 nmol.mg-1.h-1 and Km values of 5 and 67 microm for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, respectively . The enzyme binds riboflavin with a Kd of 1.2 microm . The fluorescence quantum yield of enzyme-bound riboflavin is < 2% as compared with that of free riboflavin . The protein/riboflavin complex displays an optical transition centered around 530 nm as shown by absorbance and CD spectrometry which may indicate a charge transfer complex . Replacement of tryptophan 27 by tyrosine or phenylalanine had only minor effects on the kinetic properties, but complexes of the mutant proteins did not show the anomalous long wavelength absorbance of the wild-type protein . The replacement of tryptophan 27 by aliphatic amino acids substantially reduced the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.

Arch Virol, 2002, 147(1), 103 - 17
Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling; Aberl HJ et al.; The movement proteins BC1 and BV1 of Abutilon mosaic geminivirus fused to glutathion-S-transferase (GST) and Flag-peptide were expressed in fission yeast (Schizosaccharomyces pombe) cells to analyse the fundamental intracellular distribution of these proteins in an eukaryotic cell in the absence of plant-specific factors . Most of BC1 protein sedimented rapidly after cell lysis and differential centrifugation . Using freeze-fracture immuno-labelling, the protein was detected in situ predominantly at plasma membranes and to a lower extent at cytoplasmic vesicles but not in the cytoplasm, the nuclei, or the mitochondria . Anti-BC1, anti-GST, and anti-Flag antibodies tagged smooth flecks only at the protoplasmic faces of the plasma membrane . The consequences of the BC1 behaviour for its use in two-hybrid analysis in yeast are discussed . In contrast, BV1 was detected mainly in the nucleus and partially in the cytoplasm but never associated with membranes.

Mol Biol Cell, 2002 Feb, 13(2), 515 - 29
The multiprotein exocyst complex is essential for cell separation in Schizosaccharomyces pombe; Wang H et al.; Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring . A mulitlayered division septum is assembled in concert with ring constriction . Finally, cleavage of the inner layer of the division septum results in the liberation of daughter cells . Although numerous studies have focused on actomyosin ring and division septum assembly, little information is available on the mechanism of cell separation . Here we describe a mutant, sec8-1, that is defective in cell separation but not in other aspects of cytokinesis . sec8-1 mutants accumulate about 100-nm vesicles and have reduced secretion of acid phosphatase, suggesting that they are defective in exocytosis . Sec8p is a component of the exocyst complex . Using biochemical methods, we show that Sec8p physically interacts with other members of the exocyst complex, including Sec6p, Sec10p, and Exo70p . These exocyst proteins localize to regions of active exocytosis-at the growing ends of interphase cells and in the medial region of cells undergoing cytokinesis-in an F-actin-dependent and exocytosis-independent manner . Analysis of a number of mutations in various exocyst components has established that these components are essential for cell viability . Interestingly, all exocyst mutants analyzed appear to be able to elongate and to assemble division septa but are defective for cell separation . We therefore propose that the fission yeast exocyst is involved in targeting of enzymes responsible for septum cleavage . We further propose that cell elongation and division septum assembly can continue with minimal levels of exocyst function.

Mol Biol Cell, 2002 Feb, 13(2), 493 - 502
Deletion mutants in COP9/signalosome subunits in fission yeast Schizosaccharomyces pombe display distinct phenotypes; Mundt KE et al.; The COP9/signalosome complex is highly conserved in evolution and possesses significant structural similarity to the 19S regulatory lid complex of the proteasome . It also shares limited similarity to the translation initiation factor eIF3 . The signalosome interacts with multiple cullins in mammalian cells . In the fission yeast Schizosaccharomyces pombe, the Csn1 subunit is required for the removal of covalently attached Nedd8 from Pcu1, one of three S . pombe cullins . It remains unclear whether this activity is required for all the functions ascribed to the signalosome . We previously identified Csn1 and Csn2 as signalosome subunits in S . pombe . csn1 and csn2 null mutants are DNA damage sensitive and exhibit slow DNA replication . Two further putative subunits, Csn4 and Csn5, were identified from the S . pombe genome database . Herein, we characterize null mutations of csn4 and csn5 and demonstrate that both genes are required for removal of Nedd8 from the S . pombe cullin Pcu1 and that their protein products associate with Csn1 and Csn2 . However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes . Our data suggest that the subunits of the signalosome cannot be considered as a distinct functional unit and imply that different subunits of the signalosome mediate distinct functions.

J Biol Chem, 2002 May 10, 277(19), 16920 - 7 Epub 2002 Feb 15.
Purification and characterization of the Schizosaccharomyces pombe origin recognition complex: interaction with origin DNA and Cdc18 protein; Chuang RY et al.; The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells . It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes . These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC . We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits . Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs . Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity . The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S . pombe are significantly less stringent than in S . cerevisiae . We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro . Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo . These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.

Nucleic Acids Res, 2002 Feb 15, 30(4), 894 - 902
The Schizosaccharomyces pombe mgU6-47 gene is required for 2'-O-methylation of U6 snRNA at A41; Zhou H et al.; Through a computer search of DNA databases, we have identified the homologs of the mgU6-47 snoRNA gene from the yeast Schizosaccharomyces pombe, the fly Drosophila melanogaster and human . The three box C/D-containing snoRNA genes showed no significant similarity in their sequences except for an 11 nt long complementarity to U6 snRNA, suggesting that the mechanism of snoRNA guided snRNA methylation is conserved from mammals to yeast . The corresponding snoRNAs have been positively detected by reverse transcription and northern blotting . Taking advantage of the fission yeast system, we have disrupted the yeast mgU6-47 gene and demonstrated that it is absolutely required for site-specific 2'-O-methylation of U6 at position A41 . No growth differences between mgU6-47 gene-disrupted and wild-type cells were observed, suggesting that the mgU6-47 gene, as for most rRNA methylation guides, is dispensable in yeast . Nevertheless, it was revealed by temperature shift assay that abolition of A41 methylation in yeast U6 snRNA might cause a small decrease in mRNA splicing efficiency . The timing of S.pombe U6 pre-RNA transport in the nucleus for splicing and methylation was also analyzed and is described.

Biochemistry, 2002 Feb 19, 41(7), 2311 - 21
Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal electron transfer protein, etp1; Bureik M et al.; Mitochondrial cytochrome P450 enzymes play a crucial role in the steroid biosynthesis in human adrenals, catalyzing regio- and stereospecific hydroxylations . In search of a new model system for the study of these enzymes, we expressed the human CYP11B2 (aldosterone synthase, P450(aldo)) in fission yeast Schizosaccharomyces pombe . Analysis of the subcellular localization of the P450 enzyme by Western blot analysis, fluorescence microscopy, and electron microscopy demonstrated that the mitochondrial localization signal of the human protein is functional in S . pombe . The transformed yeasts show the inducible ability to convert in vivo considerable amounts of 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone, respectively . Although in mammalian cells, mitochondrial steroid hydroxylases depend for their activity on an electron transport chain that consists of two proteins, adrenodoxin and adrenodoxin reductase, no coexpression of these proteins is needed for efficient substrate conversion by intact fission yeast cells . Searching the fission yeast genome for adrenodoxin homologues, a gene was identified that codes for a protein with an amino terminal domain homologous to COX15 of Saccharomyces cerevisiae and a carboxy terminal ferredoxin domain . It was found that overexpression of this gene significantly enhances steroid hydroxylase activity of CYP11B2 expressing fission yeast cells . Moreover, the bacterially expressed ferredoxin domain of this protein can replace adrenodoxin in a reconstituted steroid hydroxylation assay and transfer electrons from adrenodoxin reductase to a mammalian or a bacterial cytochrome P450 . Therefore, we suggest to name this protein etp1 (electron-transfer protein 1).

Mol Cell Biol, 2002 Mar, 22(5), 1577 - 88
Formation of a carboxy-terminal domain phosphatase (Fcp1)/TFIIF/RNA polymerase II (pol II) complex in Schizosaccharomyces pombe involves direct interaction between Fcp1 and the Rpb4 subunit of pol II; Kimura M et al.; In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors . To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3 . By immunoaffinity purification with anti-FLAG antibody-resin, a pol II complex containing the Rpb1 subunit with a nonphosphorylated carboxyl-terminal domain (CTD) was isolated . In addition to the pol II subunits, the complex was found to contain three subunits of a transcription factor TFIIF (TFIIF alpha, TFIIF beta, and Tfg3) and TFIIF-interacting CTD-phosphatase Fcp1 . The same type of pol II complex could also be purified from an Fcp1-tagged strain . The isolated Fcp1 showed CTD-phosphatase activity in vitro . The fcp1 gene is essential for cell viability . Fcp1 and pol II interacted directly in vitro . Furthermore, by chemical cross-linking, glutathione S-transferase pulldown, and affinity chromatography, the Fcp1-interacting subunit of pol II was identified as Rpb4, which plays regulatory roles in transcription . We also constructed an S . pombe thiamine-dependent rpb4 shut-off system . On repression of rpb4 expression, the cell produced more of the nonphosphorylated form of Rpb1, but the pol II complex isolated with the anti-FLAG antibody contained less Fcp1 and more of the phosphorylated form of Rpb1 with a concomitant reduction in Rpb4 . This result indicates the importance of Fcp1-Rpb4 interaction for formation of the Fcp1/TFIIF/pol II complex in vivo.

Mol Pathol, 2002 Feb, 55(1), 46 - 54
Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells; Buckley DA et al.; AIMS: To study the regulation of type 1 insulin like growth factor receptor (IGF-1R) tyrosine kinase activity using the fission yeast Schizosaccharomyces pombe and a green fluorescent protein (GFP) tagged, full length IGF-1R . METHODS: The beta chain of the IGF-1R (betawt) was expressed under inducible conditions in the fission yeast S . pombe . Western blot analysis with antiphosphotyrosine antibodies was used to assess the kinase activity of betawt . A GFP tagged IGF-1R (GFP-IGF-1R) was constructed to study the tyrosine kinase activity of the full length IGF-1R . The signalling capabilities of GFP-IGF-1R in response to IGF-1 stimulation were investigated in transiently transfected fibroblasts . Immunofluorescent staining for cellular phosphotyrosine content was used to assess the localisation and tyrosine kinase activity of GFP-IGF-1R . RESULTS: The betawt protein displayed functional tyrosine kinase activity in S pombe and phosphorylated endogenous yeast proteins . In response to IGF-1 stimulation, the GFP-IGF-1R became autophosphorylated and also activated the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways . Tyrosine phosphorylation and kinase activity of the GFP-IGF-1R could be visualised by immunofluorescence with antiphosphotyrosine antibodies . Coexpression of a mammalian tyrosine phosphatase PTP1B with betawt completely inhibited this tyrosine kinase activity in yeast and also reduced the tyrosine phosphorylation in COS cells transfected with the GFP-IGF-1R . CONCLUSIONS: Schizosaccharomyces pombe can be used to analyse the tyrosine kinase activity of the IGF-1R beta chain and its regulation by tyrosine phosphatases . In addition, the regulation of IGF-1R tyrosine kinase activity can be studied using a GFP tagged IGF-1R . Using both of these methods, IGF-1R kinase activity was shown to be inhibited by the protein tyrosine phosphatase, PTP1B.

Arch Biochem Biophys, 2002 Jan 15, 397(2), 312 - 8
Regulation of thioredoxin peroxidase activity by C-terminal truncation; Koo KH et al.; Thioredoxin peroxidase is a member of peroxiredoxin (Prx) family, which uses a thioredoxin (Trx) as an immediate electron donor for the reduction of peroxide . We have identified C-terminal truncated TPx from Schizosaccharomyces pombe and also have found the truncated form is significantly tenacious against the inactivation of H2O2 than the intact form . Peroxidase assay of a series of recombinant C-terminal truncation mutants (Delta192, Delta191, Delta188, Delta184, Delta176, and Delta165) revealed that TPx could be inactivated (Delta192), reactivated (Delta191-Delta176) and reinactivated (Delta165) by serial truncation from C-terminus . We did not find any significant kinetic difference among reactivated forms; however, distinctive loss of affinity to H2O2 (K(m) = 5 microM) than that of the intact form (<<5 microM, undeterminable) was monitored . Characterization of a series of Lys(191) point mutants manifested that the loss of affinity caused by a deprivation of positive charge born in Lys(191) and the loss of affinity resulted in the resistibility to H2O2 . Disk inhibition assay with S . pombe cells overexpressing wild-type, Delta192 and Delta191 mutants evidenced that the truncated forms functioning in vitro as well as in vivo . (c)2002 Elsevier Science.

FEBS Lett, 2002 Jan 30, 511(1-3), 85 - 9
A DMSO-sensitive conditional mutant of the fission yeast orthologue of the Saccharomyces cerevisiae SEC13 gene is defective in septation; Poloni D et al.; Dissection of complex processes using model organisms such as yeasts relies heavily upon the use of conditional mutants . We have generated a collection of fission yeast mutants sensitive to dimethylsulphoxide (DMSO) . Among these we have found a mutant in the Schizosaccharomyces pombe orthologue of the Saccharomyces cerevisiae SEC13 gene, which fails to cleave the division septum . Generation of a null allele demonstrates that the S . pombe sec13 gene is essential.

Biochem Biophys Res Commun, 2002 Feb 8, 290(5), 1399 - 407
Isolation and characterization of a novel F-box protein Pof10 in fission yeast; Ikebe C et al.; The SCF complex is a type of ubiquitin-protein ligase (E3) that consists of invariable components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins . Using a yeast two-hybrid system, we isolated six proteins that interact with Schizosaccharomyces pombe Skp1 . Among them, Pof10 is a novel F-box protein consisting of 662 amino acids, harboring the F-box domain required for the binding to Skp1 and followed by four WD40 repeats . Overexpression of Pof10 in fission yeast resulted in loss of viability with marked morphological changes that are similar to those in pop1 mutant yeast . Coexpression of Skp1 with Pof10 prevented the lethality, suggesting that the lethality from Pof10 overexpression results from the sequestration of Skp1 from other F-box proteins including Pop1 . Whereas most F-box proteins show rapid turnover, Pof10 has a remarkably long half-life in vivo and has been shown to be localized predominantly in cytoplasm . These results suggest that the stable F-box protein Pof10 might target abundant cytoplasmic proteins for degradation in fission yeast . (c)2002 Elsevier Science (USA).

Curr Biol, 2002 Jan 22, 12(2), 141 - 5
14-3-3 protein interferes with the binding of RNA to the phosphorylated form of fission yeast meiotic regulator Mei2p; Sato M et al.; The switch from mitosis to meiosis is controlled by the Pat1(Ran1) kinase-Mei2p system in Schizosaccharomyces pombe . Mei2p promotes both premeiotic DNA synthesis and meiosis I, and its RNA binding ability is essential for these two processes . Mei2p forms a dot structure in the nucleus prior to meiosis I, aided by a specific RNA species named "meiRNA" . Pat1 kinase phosphorylates Mei2p on two positions and downregulates its activity . Pat1 kinase undergoes inactivation under meiotic conditions, as a result of the production of a tethering pseudosubstrate Mei3p, and accumulation of the unphosphorylated form of Mei2p commits cells to meiosis . However, the mechanism of how phosphorylation of Mei2p suppresses its activity to induce meiosis remains largely unknown . Here we show that S . pombe Rad24p, a 14-3-3 protein, functions as a negative factor for meiosis by antagonizing the function of meiRNA to promote the formation of a nuclear Mei2p dot . Rad24p binds preferentially to Mei2p phosphorylated by Pat1 kinase . It inhibits association of meiRNA to the phosphorylated form of Mei2p but not to the unphosphorylated form in vitro . We speculate that Rad24p, bound tightly to the residues phosphorylated by Pat1 kinase, may mask the RNA recognition motifs on Mei2p . This model will explain, at least partly, why phosphorylation by Pat1 kinase inhibits the meiosis-inducing activity of Mei2p.

Front Biosci, 2002 Feb 01, 7, d349 - 57
HIV-1 VPR modulates cell cycle G2/M transition through an alternative cellular mechanism other than the classic mitotic checkpoints; Elder RT et al.; HIV-1 Vpr induces cell cycle G2/M arrest in both human and fission yeast cells, suggesting a highly conserved activity of this viral protein . In this review, we summarize the current understanding of Vpr-induced G2 arrest based on studies from both mammalian cells and the fission yeast (Schizosaccharomyces pombe) model system . Fission yeast has proven to be an excellent model system to investigate cell cycle G2/M control of eukaryotic cells . Similarly, fission yeast has also been instrumental in defining the molecular mechanism underlying the G2 arrest induced by Vpr . We have compared the classic DNA-damage and DNA-replication checkpoint controls of the cell cycle G2/M transition to the G2 arrest conferred by Vpr . Based on the current findings, we hypothesize that Vpr induces cell cycle G2 arrest through an alternative novel cellular pathway(s) rather than through the classic mitotic checkpoint controls . A number of cellular proteins which may be involved in this new cellular pathway(s) have been identified and are discussed.

J Biol Chem, 2002 Apr 5, 277(14), 12375 - 81 Epub 2002 Jan 25.
Solution NMR study of the monomeric form of p13suc1 protein sheds light on the hinge region determining the affinity for a phosphorylated substrate; Odaert B et al.; Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division . Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal beta-strand-exchanged conformation . With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer . Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13(suc1) CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the beta-hinge motif . Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping . However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer . To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S . pombe p13(suc1) and its hinge mutant P90A in solution by NMR spectroscopy . We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding . Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal beta-sheet involved in domain swapping and protein interactions.

Mol Genet Genomics, 2001 Dec, 266(4), 672 - 85 Epub 2001 Oct 05.
Aspergillus nidulans DigA, a potential homolog of Saccharomyces cerevisiae Pep3 (Vps18), is required for nuclear migration, mitochondrial morphology and polarized growth; Geissenhoner A et al.; The fungal vacuole is an acidic organelle that is involved in a variety of physiological processes, such as protein turnover, ion and pH homeostasis and osmoregulation . The function of the vacuole largely depends on vesicle transport providing the organelle with enzymes and substrates . The process of vesicle transportation has been studied best in Saccharomyces cerevisiae, where several proteins that are crucial for intracellular vesicle sorting have been identified . One such protein is Pep3 (Vps18) . In pep3 mutants vacuole function and vacuole morphology are affected . We cloned the gene for a potential homolog of Pep3 from the filamentous fungus Aspergillus nidulans . The gene, digA (for dichotomous growth), was identified in a screen for nuclear migration mutants . A . nidulans digA encodes a protein of 108.3 kDa, which includes a 122-amino acid clathrin repeat motif, two short coiled-coil regions, and a RING finger Zn-binding motif at the C-terminus . All three sequence motifs suggest interaction of DigA with other proteins . DigA is 25% identical to a homolog from Schizosaccharomyces pombe, 23% to a protein from human, 21% to the product of the Drosophila melanogaster gene deep orange (dor) and 18% to S . cerevisiae Pep3 (Vps18) . We localized DigA as a HA-epitope-tagged protein in the cytoplasm of A . nidulans vegetative cells, by secondary immunofluorescence . A digA mutant displays a pleiotropic phenotype with clustered mitochondria, clustered nuclei and a defect in polarization of the actin cytoskeleton . These results suggest that vacuolar functions are required for organelle positioning and polarized growth.

J Bacteriol, 2002 Feb, 184(4), 1003 - 9
Genetic evidence that the uvsE gene product of Deinococcus radiodurans R1 is a UV damage endonuclease; Earl AM et al.; An in vitro transposition system, developed to facilitate gene disruption in Deinococcus radiodurans R1, has been used to inactivate the gene designated dr1819 in uvrA-1(+) and uvrA-1 backgrounds . dr1819 encodes a protein with homology to a UV DNA damage endonuclease expressed by Schizosaccharomyces pombe . Interruption of dr1819 greatly sensitizes the uvrA-1 strain but not the uvrA-1(+) strain to UV light, indicating that the dr1819 gene product is a component in a DNA repair pathway that can compensate for the loss of nucleotide excision repair in this species . Clones of dr1819 will restore UV resistance to UVS78, a uvrA-1 uvsE strain, indicating that dr1819 and uvsE are the same locus.

J Biol Chem, 2002 Apr 5, 277(14), 11853 - 8 Epub 2002 Jan 22.
Functional interaction of MutY homolog with proliferating cell nuclear antigen in fission yeast, Schizosaccharomyces pombe; Chang DY et al.; The MutY homolog (MYH) is responsible for removing adenines misincorporated on a template DNA strand containing G or 7,8-dihydro-8-oxoguanine (8-oxoG) and thus preventing G:C to T:A mutations . Human MYH has been shown to interact physically with human proliferating cell nuclear antigen (hPCNA) . Here, we report that a similar interaction between SpMYH and SpPCNA occurs in the fission yeast Schizosaccharomyces pombe . Binding of SpMYH to SpPCNA was not observed when phenylalanine 444 in the PCNA binding motif of SpMYH was replaced with alanine . The F444A mutant of SpMYH expressed in yeast cells had normal adenine glycosylase and DNA binding activities . However, expression of this mutant form of SpMYH in a SpMYHDelta cell could not reduce the mutation frequency of the cell to the normal level . Moreover, SpMYH interacted with hPCNA, and SpPCNA interacted with hMYH but not with F518A/F519A mutant hMYH containing mutations in its PCNA binding motif . Although the SpMYHDelta cells expressing hMYH had partially reduced mutation frequency, the F518A/F519A mutant hMYH could not reduce the mutation frequency of SpMYHDelta cells . Thus, the interaction between SpMYH and SpPCNA is important for SpMYH biological function in mutation avoidance.

Biochemistry, 2002 Jan 29, 41(4), 1202 - 10
Folding and association of the human cell cycle regulatory proteins ckshs1 and ckshs2; Seeliger MA et al.; The two human proteins ckshs1 and ckshs2 are each 79 amino acids in length and consist of a four-stranded beta-sheet capped at one end by two alpha-helices . They are members of the cks family of essential cell cycle regulatory proteins that can adopt two native states, a monomer and a domain-swapped dimer formed by exchange of a C-terminal beta-strand . ckshs1 and ckshs2 both have marginal thermodynamic stability (the free energies of unfolding at 25 degrees C are 3.0 and 2.5 kcal/mol, respectively) and low kinetic stability (the rates of unfolding in water are approximately 1 s(-1)) . Refolding of their denatured states to the monomeric forms of the proteins is slowed by transient oligomerization that is likely to occur via domain swapping . The folding behavior of ckshs1 and ckshs2 is markedly different from that of suc1, the cks protein from Schizosaccharomyces pombe, but the domain swapping propensities are similar . The greater thermodynamic and kinetic stability of suc1 and the population of a folding intermediate are most likely a consequence of its larger size (113 residues) . The similarity in the domain swapping propensities, despite the contrast in other biophysical properties, may be attributable to the common double-proline motif in the hinge loop that connects the swapped domain to the rest of the protein . The motif was shown previously for suc1 to control the equilibrium between the monomer and the domain-swapped dimer . Finally, according to our model, the kinetic barrier separating the monomer and the domain-swapped dimer arises because the protein must unfold for beta-strand exchange to occur . Consistent with this, interconversion between the two states is much faster in the human proteins than it is for suc1, reflecting the faster unfolding rates of the former.

J Mol Biol, 2001 Oct 19, 313(2), 241 - 53
Role of the DNA repair nucleases Rad13, Rad2 and Uve1 of Schizosaccharomyces pombe in mismatch correction; Kunz C et al.; Repair of mismatched DNA occurs mainly by the long-patch mismatch repair (MMR) pathway, requiring Msh2 and Pms1 . In Schizosaccharomyces pombe mismatches can be repaired by a short-patch repair system, containing nucleotide excision repair (NER) factors . We studied mismatch correction efficiency in cells with inactivated DNA repair nucleases Rad13, Rad2 or Uve1 in MMR proficient and deficient background . Rad13 incises 3' of damaged DNA during NER . Rad2 has a function in the Uve1-dependent repair of DNA damages and in replication . Loss of Rad13 caused a strong reduction of short-patch processing of mismatches formed during meiotic recombination . Mitotic mutation rates were increased, but not to the same extent as in the NER mutant swi10, which is defective in 5' incision . The difference might be caused by an additional role of Rad13 in base excision repair or due to partial redundancy with other 3' endonucleases . Meiotic mismatch repair was not or only slightly affected in rad2 and uve1 mutants . In addition, inactivation of uve1 caused only weak effects on mutation avoidance . Mutation rates were elevated when rad2 was mutated, but not further increased in swi10 rad2 and rad13 rad2 double mutants, indicating an epistatic relationship . However, the mutation spectra of rad2 were different from that of swi10 and rad13 . Thus, the function of Rad2 in mutation avoidance is rather independent of NER . rad13, swi10 and rad2, but not uve1 mutants were sensitive to the DNA-damaging agent methyl methane sulphonate . Cell survival was further reduced in the double mutants swi10 rad2, rad13 rad2 and, surprisingly, swi10 rad13 . These data confirm that NER and Rad2 act in distinct damage repair pathways and further indicate that the function of Rad13 in repair of alkylated bases is partially independent of NER .

Chin Med J (Engl), 2001 Dec, 114(12), 1295 - 9
Expression of hHR21sp gene by peripheral blood and hematopoietic cells of normal subjects and Fanconi anemia patients; Deng Y et al.; OBJECTIVE: The radiation sensitive gene rad 21 of Schizosaccharomyces pombe is involved in the repair of double-stranded breaks in DNA and is essential for mitotic growth . The hHR21sp gene is its human homologue . In an attempt to investigate the role of hHR21sp in DNA repair, we studied the effects of UV and gamma-ray irradiation on hHR21sp gene expression in normal human peripheral blood cells, and non-iradiated peripheral and bone marrow cells from Fanconi anemia (FA) patients who have shown DNA repair deficiency . METHODS: Total steady state RNA was extracted from peripheral blood cells and bone marrow . RNA transcripts were quantified after RT-PCR and Southern blot, phosphoimmage and autoradiogram analysis . The results were compared with control groups . RESULTS: hHR21sp expression was significantly increased from 3 h to 9 h after UV irradiation in peripheral blood cells from normal subjects at doses of 40-80 j/m2 (P < 0.05) . hHR21sp was also up-regulated by gamma-ray irradiation at 6 h to 9 h at dose of 1 to 5 Gy (P < 0.01), which was more significant than the UV irradiation . In the non-irradiated FA patient group, hHR21sp expression was decreased in bone marrow hematopoietic cells (P < 0.05) . After activation by PHA and IL-2, there was still a significant depression in expression by the FA patients peripheral blood cells compared with control groups (P < 0.05) . CONCLUSION: hHR21sp was up-regulated at doses and times irradiated at the range tested in normal peripheral blood cells, and is more affected by gamma-ray irradiation than UV irradiation . FA patient bone marrow hematopoietic cells and peripheral blood mononuclear cells showed down-regulation of hHR21sp expression . The results imply that defects in DNA repair via hHR21sp expression may play an important role in the pathogenesis of FA syndrome.

J Cell Sci, 2001 Dec, 114(Pt 24), 4521 - 32
MTOC formation during mitotic exit in fission yeast; Heitz MJ et al.; Microtubules polymerise from nucleation templates containing gamma tubulin . These templates are generally concentrated in discrete structures called microtubule organising centres (MTOCs) . In Schizosaccharomyces pombe, an equatorial MTOC (EMTOC) forms mid-way through anaphase B and then disassembles during the final stages of cell separation . We show that the EMTOC was generated by recruiting gamma tubulin to the equatorial F-actin ring before it constricted to cleave the cell in two during cytokinesis . The EMTOC was not a continuous ring . It had a variable structure ranging from a horseshoe to a number of short bars . EMTOC integrity depended upon the integrity of the F-actin but not the microtubule cytoskeleton . EMTOC assembly required the activity of both the septation-inducing network (SIN) that regulates the onset of cytokinesis and the anaphase-promoting complex . Activation of the SIN in interphase cells induced F-actin ring formation and contraction and the synthesis of the primary septum but did not promote EMTOC assembly . In contrast, overproduction of the polo-like kinase, Plo1, which also induced multiple rounds of septation in interphase cells, induced EMTOC formation . Thus, the network governing EMTOC formation shared many of the regulatory elements that control cytokinesis but was more complex and revealed an additional function for Plo1 during mitotic exit.

Biosci Biotechnol Biochem, 2001 Nov, 65(11), 2528 - 34
Radicicol binding to Swo1/Hsp90 and inhibition of growth of specific temperature-sensitive cell cycle mutants of fission yeast; Ki SW et al.; A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature . The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation . Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not . A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S . pombe . Increased Swo1 expression partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature . On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature . These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.

Acta Microbiol Immunol Hung, 2001, 48(3-4), 519 - 31
Identification of Schizosaccharomyces pombe genes that encode putative homologues of Saccharomyces cerevisiae mediator complex subunits; Sipiczki M; The mediator complexes transduce regulatory information from upstream regulatory elements to the transcription machinery in organisms ranging from yeasts to humans . By a genome-wide search we identified 14 ORFs and genes in the genome of the fission yeast Schizosaccharomyces pombe that encode putative homologues of Saccharomyces cerevisiae mediator subunits . The Sch . pombe proteins are smaller and appear to form a mediator of lower complexity, which is consistent with the hypothesized ancient origin of fission yeasts.

Plant Physiol, 2002 Jan, 128(1), 38 - 51
Diversity of TITAN functions in Arabidopsis seed development; Tzafrir I et al.; The titan mutants of Arabidopsis exhibit striking defects in seed development . The defining feature is the presence of abnormal endosperm with giant polyploid nuclei . Several TTN genes encode structural maintenance of chromosome proteins (condensins and cohesins) required for chromosome function at mitosis . Another TTN gene product (TTN5) is related to the ARL2 class of GTP-binding proteins . Here, we identify four additional TTN genes and present a general model for the titan phenotype . TTN1 was cloned after two tagged alleles were identified through a large-scale screen of T-DNA insertion lines . The predicted gene product is related to tubulin-folding cofactor D, which interacts with ARL2 in fission yeast (Schizosaccharomyces pombe) and humans to regulate tubulin dynamics . We propose that TTN5 and TTN1 function in a similar manner to regulate microtubule function in seed development . The titan phenotype can therefore result from disruption of chromosome dynamics (ttn3, ttn7, and ttn8) or microtubule function (ttn1 and ttn5) . Three other genes have been identified that affect endosperm nuclear morphology . TTN4 and TTN9 appear to encode plant-specific proteins of unknown function . TTN6 is related to the isopeptidase T class of deubiquitinating enzymes that recycle polyubiquitin chains following protein degradation . Disruption of this gene may reduce the stability of the structural maintenance of chromosome complex . Further analysis of the TITAN network should help to elucidate the regulation of microtubule function and chromosome dynamics in seed development.

Plant Physiol, 2002 Jan, 128(1), 21 - 9
An oligopeptide transporter gene family in Arabidopsis; Koh S et al.; We have identified nine oligopeptide transporter (OPT) orthologs (AtOPT1 to AtOPT9) in Arabidopsis . These proteins show significant sequence similarity to OPTs of Candida albicans (CaOpt1p), Schizosaccharomyces pombe (Isp4p), and Saccharomyces cerevisiae (Opt1p and Opt2p) . Hydrophilicity plots of the OPTs suggest that they are integral membrane proteins with 12 to 14 transmembrane domains . Sequence comparisons showed that the AtOPTs form a distinct subfamily when compared with the fungal OPTs . Two highly conserved motifs (NPG and KIPPR) were found among all OPT members . The identification of multiple OPTs in Arabidopsis suggests that they may play different functional roles . This idea is supported by the fact that AtOPTs have a distinct, tissue-specific expression pattern . The cDNAs encoding seven of the AtOPTs were cloned into a yeast vector under the control of a constitutive promoter . AtOPT4 expressed in S . cerevisiae mediated the uptake of KLG-{3H}L . Similarly, expression of five of the seven AtOPT proteins expressed in yeast conferred the ability to uptake tetra- and pentapeptides as measured by growth . This study provides new evidence for multiple peptide transporter systems in Arabidopsis, suggesting an important physiological role for small peptides in plants.

Nucleic Acids Res, 2002 Jan 15, 30(2), 581 - 91
Involvement of rhp23, a Schizosaccharomyces pombe homolog of the human HHR23A and Saccharomyces cerevisiae RAD23 nucleotide excision repair genes, in cell cycle control and protein ubiquitination; Elder RT et al.; A functional homolog (rhp23) of human HHR23A and Saccharomyces cerevisiae RAD23 was cloned from the fission yeast Schizosaccharomyces pombe and characterized . Consistent with the role of Rad23 homologs in nucleotide excision repair, rhp23 mutant cells are moderately sensitive to UV light but demonstrate wild-type resistance to gamma-rays and hydroxyurea . Expression of the rhp23, RAD23 or HHR23A cDNA restores UV resistance to the mutant, indicating that rhp23 is a functional homolog of the human and S.cerevisiae genes . The rhp23::ura4 mutation also causes a delay in the G2 phase of the cell cycle which is corrected when rhp23, RAD23 or HHR23A cDNA is expressed . Rhp23 is present throughout the cell but is located predominantly in the nucleus, and the nuclear levels of Rhp23 decrease around the time of S phase in the cell cycle . Rhp23 is ubiquitinated at low levels, but overexpression of the rhp23 cDNA induces a large increase in ubiquitination of other proteins . Consistent with a role in protein ubiquitination, Rhp23 binds ubiquitin, as determined by two-hybrid analysis . Thus, the rhp23 gene plays a role not only in nucleotide excision repair but also in cell cycle regulation and the ubiquitination pathways.

Mol Cell Biol, 2002 Feb, 22(3), 801 - 15
Removal of a single alpha-tubulin gene intron suppresses cell cycle arrest phenotypes of splicing factor mutations in Saccharomyces cerevisiae; Burns CG et al.; Genetic and biochemical studies of Schizosaccharomyces pombe and Saccharomyces cerevisiae have identified gene products that play essential functions in both pre-mRNA splicing and cell cycle control . Among these are the conserved, Myb-related CDC5 (also known as Cef1p in S . cerevisiae) proteins . The mechanism by which loss of CDC5/Cef1p function causes both splicing and cell cycle defects has been unclear . Here we provide evidence that cell cycle arrest in a new temperature-sensitive CEF1 mutant, cef1-13, is an indirect consequence of defects in pre-mRNA splicing . Although cef1-13 cells harbor global defects in pre-mRNA splicing discovered through intron microarray analysis, inefficient splicing of the alpha-tubulin-encoding TUB1 mRNA was considered as a potential cause of the cef1-13 cell cycle arrest because cef1-13 cells arrest uniformly at G(2)/M with many hallmarks of a defective microtubule cytoskeleton . Consistent with this possibility, cef1-13 cells possess reduced levels of total TUB1 mRNA and alpha-tubulin protein . Removing the intron from TUB1 in cef1-13 cells boosts TUB1 mRNA and alpha-tubulin expression to near wild-type levels and restores microtubule stability in the cef1-13 mutant . As a result, cef1-13 tub1Deltai cells progress through mitosis and their cell cycle arrest phenotype is alleviated . Removing the TUB1 intron from two other splicing mutants that arrest at G(2)/M, prp17Delta and prp22-1 strains, permits nuclear division, but suppression of the cell cycle block is less efficient . Our data raise the possibility that although cell cycle arrest phenotypes in prp mutants can be explained by defects in pre-mRNA splicing, the transcript(s) whose inefficient splicing contributes to cell cycle arrest is likely to be prp mutant dependent.

Nat Cell Biol, 2001 Dec, 3(12), 1043 - 50
Feedback regulation of the MBF transcription factor by cyclin Cig2; Ayte J et al.; The Mlu1-binding factor (MBF) from the fission yeast Schizosaccharomyces pombe contains the proteins Res1p and Res2p and binds to the Mlu1 cell-cycle box (MCB) element in DNA, activating the transcription of genes required for S phase . We report here that the cell-cycle-regulated expression of the cyclin cig2 gene is dependent on MBF . Deletion of MCB elements in the cig2 promoter perturbed the expression not only of cig2 but also of other MBF-dependent genes, indicating that Cig2p could regulate MBF activity . Cig2p can bind to Res2p, promote the phosphorylation of Res1p and inhibit MBF-dependent gene transcription . Cig2p thus forms an autoregulating feedback-inhibition loop with MBF which is important for normal regulation of the cell cycle.

Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2347 - 52
Genetic analysis of the His-to-Asp phosphorelay implicated in mitotic cell cycle control: involvement of histidine-kinase genes of Schizosaccharomyces pombe; Aoyama K et al.; Common histidine-to-aspartate (His-to-Asp) phosphorelay signaling systems involve three types of signaling components: a sensor His-kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) protein . In the fission yeast Schizosaccharomyces pombe, two response regulators, Mcs4 and Prr1, have been identified, and it was shown that they are involved in signal transduction in stress responses . Furthermore, Mcs4 and Prr1 appear to be involved in mitotic cell-cycle control and meiosis, respectively . Recently we have identified Spy1 (also known as Mpr1), which encodes an HPt phosphotransmitter, and reported that Spy1, together with Mcs4, plays a role in cell cycle regulation . In this study, we identified and characterized three genes encoding histidine kinase, named Phk1, Phk2, and Phk3 (S . pombe histidine kinase) (also referred as Mak2, Mak3, and Mak1, respectively) . Deletion of individual kinase genes has no apparent phenotypes but multiple deletion of these kinases showed the same phenotype of Spyl (Mpr1)-deficient cells, indicating precocious entry into M phase . These results indicated that three histidine kinases that work upstream of the HPt-transmitter, Spy1 (Mpr1), have a redundant function in cell cycle control.

Yeast, 2002 Jan 15, 19(1), 55 - 67
The Candida albicans 14-3-3 gene, BMH1, is essential for growth; Cognetti D et al.; The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades . In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development . The Bmh proteins function in the cAMP-dependent RAS/MAPK and rapamycin-sensitive signalling cascades . Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions . Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses . In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable . The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR . Unlike other yeasts, C . albicans has only one 14-3-3 gene (BMH1) . It was not possible to construct double knockouts by routine methods . These results suggested that the C . albicans BMH1 gene is essential . The essentiality of C . albicans BMH1 was confirmed by a PCR disruption technique . The C . albicans bmh1 Delta/BMH1 heterozygotes exhibit growth and morphogenetic defects . Therefore, the BMH1 gene in C . albicans (Accession No . AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis .

Yeast, 2002 Jan 15, 19(1), 29 - 35
Sulphur amino acid synthesis in Schizosaccharomyces pombe represents a specific variant of sulphur metabolism in fungi; Brzywczy J et al.; Schizosaccharomyces pombe, in contrast to Saccharomyces cerevisiae and Aspergillus nidulans, lacks cystathionine beta-synthase and cystathionine gamma-lyase, two enzymes in the pathway from methionine to cysteine . As a consequence, methionine cannot serve as an efficient sulphur source for the fungus and does not bring about repression of sulphur assimilation, which is under control of the cysteine-mediated sulphur metabolite repression system . This system operates at the transcriptional level, as was shown for the homocysteine synthase encoding gene . Our results corroborate the growing evidence that cysteine is the major low-molecular-weight effector in the regulation of sulphur metabolism in bacteria, fungi and plants .

Sci STKE . 2001 Sep 04;2001(98):RE1.
Fungal histidine kinases; Santos JL et al.; Eukaryotic cells predominantly use serine, threonine, and tyrosine phosphorylation in various intracellular signal transduction pathways . In contrast, prokaryotic organisms employ numerous "two-component" systems, in which signaling is achieved by transferring a phosphoryl group from phosphohistidine in the "sensor kinase" component to aspartate in the "response regulator" component . In the last several years, genetic screens and genome projects have identified sensor kinases and response regulators in lower eukaryotes and plants, revealing that eukaryotic organisms also make use of His-Asp phosphotransfer in a limited number of signaling pathways . Extensive studies in yeasts have demonstrated that a variation of the two-component system, a multistep "phosphorelay," is the prevailing mechanism among distantly related yeast species . In the budding yeast Saccharomyces cerevisiae, a His-Asp-His-Asp phosphorelay transmits osmotic stress signals to a mitogen-activated protein kinase (MAPK) cascade to induce adaptive responses . A phosphorelay in the fission yeast Schizosaccharomyces pombe, analogous to the S . cerevisiae phosphorelay, is responsible for MAPK activation in response to peroxide stress . Mammalian cells do not have any two-component or phosphorelay systems, although protein histidine kinases unrelated to the sensor kinase may be involved in cellular signaling . Because some phosphorelay proteins are essential for virulence of microbial pathogens, including the yeast fungus Candida albicans, novel antibiotics targeted to phosphorelays may be effective against eukaryotic pathogens without causing host cell damage.

Sci STKE . 2000 Oct 31;2000(56):PL1.
Application of the chromatin immunoprecipitation method to identify in vivo protein-DNA associations in fission yeast; Takahashi K et al.; The chromatin immunoprecipitation (ChIP) method provides an ideal tool for detecting direct or indirect interactions between proteins of interest and DNAs with known sequences . Here, we introduce the ChIP protocol used in our laboratory to identify in vivo protein-DNA association in the fission yeast Schizosaccharomyces pombe . The cytological and genetic merits of the fission yeast for studying control of the eukaryotic cell cycle and chromosome dynamics are reinforced by application of this ChIP method.

J Biol Chem, 2002 Mar 22, 277(12), 10562 - 72 Epub 2001 Dec 20.
The fission yeast ES2 homologue, Bis1, interacts with the Ish1 stress-responsive nuclear envelope protein; Taricani L et al.; In fission yeast, nutrient starvation induces physiological, biochemical, and morphological changes that enable survival . Collectively these changes are referred to as stationary phase . We have used a green fluorescent protein random insertional mutagenesis system to isolate two novel stress-response proteins required in stationary phase . Ish1 is a nuclear envelope protein that is present throughout the cell cycle and whose expression is increased in response to stresses such as glucose and nitrogen starvation, as well as osmotic stress . Expression of Ish1 is regulated by the Spc1 MAPK pathway through the Atf1 transcription factor . Although overexpression of Ish1 is lethal, cells lacking ish1 exhibit reduced viability in stationary phase . Bis1 is a novel interacting partner of Ish1 . Bis1 is the Schizosaccharomyces pombe member of the ES2 nuclear protein family found in Mus musculus, Drosophila melanogaster, Homo sapiens, and Arabidopsis thaliana . Overexpression of Bis1 results in a cell elongation phenotype, whereas bis1(-) cells exhibit a reduced viability in stationary phase similar to that seen in ish1(-) cells.

Biochim Biophys Acta, 2001 Dec 5, 1568(2), 135 - 46
Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation; Kriegmaier M et al.; Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe . The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity . Due to their layered structure, living S . pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field . Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast . Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S . pombe cells (cell wall, plasma membrane and cytosol) . The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance . The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.

J Biol Chem, 2002 Apr 5, 277(14), 12388 - 95 Epub 2001 Dec 14.
Selective inhibition of MAPKK Wis1 in the stress-activated MAPK cascade of Schizosaccharomyces pombe by novel berberine derivatives; Jang MJ et al.; Intracellular molecular targets of novel berberine derivatives, HWY 289 and HWY 336, were identified by a screen of a variety of mutants in fission yeast Schizosaccharomyces pombe . HWY 289 and HWY 336 completely inhibited the proliferation of wild type as well as various mutant fission yeast cells (minimal inhibitory concentrations were 29.52 microm for HWY 289 and 11.83 microm for HWY 336), but did not affect the proliferation of Wis1 mitogen-activated protein kinase kinase (MAPKK) deletion mutants . In addition, HWY 289 with an IC(50) value of 7.3 microm or HWY 336 with IC(50) of 5.7 microm specifically inhibited in vitro kinase activities of purified Wis1, whereas either compound did not affect the activities of other kinases in the mitogen-activated protein kinase (MAPK) cascades of fission yeast . These genetic and biochemical results demonstrate the high degree of specificity of HWY 289 and HWY 336 to MAPKK Wis1 and suggest that the cytotoxicity of these compounds is not simply due to the inhibition of Wis1 kinase activity . High salt wash experiments have shown that strong noncovalent binding occurs between Wis1 and either HWY 289 or HWY 336 . The preincubation of Wis1 kinase with ATP did not affect the inhibition of Wis1 by HWY 289 and HWY 336, but when Wis1 was preincubated with MBP, a protein substrate, Wis1 kinase activity was no longer inhibited . These observations demonstrate that HWY 289/HWY 336 do inhibit Wis1 kinase, not by binding to the ATP-binding site but by disturbing the binding of substrate to the kinase . Target validation of the complex of HWY 289/HWY 336 and Wis1 kinase will provide important clues for the mechanism of specific cytotoxicity of these compounds in S . pombe . On a broader aspect, it would create an initiative to further modify and develop compounds that selectively inhibit kinases and cause cytotoxicity in various MAPK cascades including those of mammals.

J Mol Biol, 2001 Dec 14, 314(5), 1113 - 25
Crystal structure of the N-terminal domain of Oxytricha nova telomere end-binding protein alpha subunit both uncomplexed and complexed with telomeric ssDNA; Classen S et al.; Oxytricha nova telomere end-binding protein specifically recognizes and caps single strand (T(4)G(4))(n) telomeric DNA at the very 3'-ends of O . nova macronuclear chromosomes . Proteins homologous to the N-terminal domain of OnTEBP alpha subunit have now been identified in Oxytricha trifallax, Stylonychia mytilis, Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens, suggesting that this protein is widely distributed in eukaryotes . We describe here the crystal structures of the N-terminal single-stranded DNA (ssDNA)-binding domain of O . nova telomere end-binding protein alpha subunit both uncomplexed and complexed with single strand telomeric DNA . These structures show how the N-terminal domain of alpha alone, in the absence of the beta subunit and without alpha dimerization, can bind single-stranded telomeric DNA in a sequence-specific and 3'-end-specific manner . Furthermore, comparison of the uncomplexed and complexed forms of this protein shows that the ssDNA-binding site is largely pre-organized in the absence of ssDNA with modest, but interesting, rearrangements of amino acid side-chains that compose the ssDNA-binding site . The structures described here extend our understanding of structures of O . nova telomeric complexes by adding uncomplexed and complexed forms of monomeric alpha to previously described structures for (alpha 56/ssDNA)(2) dimer and alpha 56/beta 28/ssDNA ternary complexes . We believe that each of these four structures represent intermediates in an ordered assembly/disassembly pathway for O . nova telomeric complexes .

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 1237 - 42
Negative regulation of filamentous growth and flocculation by Lkh1, a fission yeast LAMMER kinase homolog; Kim KH et al.; We have isolated a full-length cDNA clone that encodes for a Schizosaccharomyces pombe homolog of the dual-specificity protein kinase of the LAMMER family, lkh1 (lammer kinase homolog) . The proposed Lkh1 protein contains 575 amino acids . The lkh1(+) null mutant is viable, but exhibits flocculation upon reaching stationary phase in liquid media and filamentous adhesion growth on solid media . Analysis of the flocculation activity of the lkh1(+) null mutant indicates that asexual aggregation of S . pombe cells into floccules is divalent cation-dependent and galactose-specific . We also demonstrate that the Saccharomyces cerevisiae LAMMER kinase homolog, Kns1, can substitute for the Lkh1 function in S . pombe.

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 987 - 92
Pleckstrin homology domain interacts with Rkp1/Cpc2, a RACK1 homolog, to modulate Pck2-mediated signaling process in Schizosaccharomyces pombe; Won M et al.; Rkp1/Cpc2, a fission yeast RACK1 homolog, interacts with Pck2, a PKC homolog, and is involved in the regulation of pck2-mediated signaling process . The N-terminal region of split pleckstrin homology domain (nPH) in human PLC-gamma1 bound to Rkp1/Cpc2 concomitantly with Pck2 . nPH inhibited kinase activity of GST-Pck2 purified from Schizosaccharomyces pombe in vitro . The lethality induced by pck2(+) overexpression was suppressed by coexpression of either rkp1(+) or nPH domain . This result suggests that Rkp1/Cpc2 interacts with PH domain-containing protein and regulates the Pck2-mediated signaling process in S . pombe.

Nat Genet, 2002 Jan, 30(1), 73 - 6 Epub 2001 Dec 10.
Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes; Boggs BA et al.; Studies of histone methylation have shown that H3 can be methylated at lysine 4 (Lys4) or lysine 9 (Lys9) . Whereas H3-Lys4 methylation has been correlated with active gene expression, H3-Lys9 methylation has been linked to gene silencing and assembly of heterochromatin in mouse and Schizosaccharomyces pombe . The chromodomain of mouse HP1 (and Swi6 in S . pombe) binds H3 methylated at Lys9, and methylation at this site is thought to mark and promote heterochromatin assembly . We have used a well-studied model of mammalian epigenetic silencing, the human inactive X chromosome, to show that enrichment for H3 methylated at Lys9 is also a distinguishing mark of facultative heterochromatin . In contrast, H3 methylated at Lys4 is depleted in the inactive X chromosome, except in three 'hot spots' of enrichment along its length . Chromatin immunoprecipitation analyses further show that Lys9 methylation is associated with promoters of inactive genes, whereas Lys4 methylation is associated with active genes on the X chromosome . These data demonstrate that differential methylation at two distinct sites of the H3 amino terminus correlates with contrasting gene activities and may be part of a 'histone code' involved in establishing and maintaining facultative heterochromatin.

Mol Biol Cell, 2001 Dec, 12(12), 3955 - 72
The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1(+)-encoding syntaxin-like protein; Nakamura T et al.; Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe . Here we report two novel proteins that localize to the forespore membrane . spo3(+) encodes a potential membrane protein, which was expressed only during sporulation . Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane . The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores . One of the spo3 alleles, spo3-KC51, was dose-dependently suppressed by psy1(+), which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex . psy1(+) was essential for vegetative growth, and its transcription was enhanced during sporulation . As expected, Psy1 localized to the plasma membrane during vegetative growth . Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated . In the spo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology . Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immature spo3Delta asci . These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein.

Mol Biol Cell, 2001 Dec, 12(12), 3892 - 903
G2/M arrest caused by actin disruption is a manifestation of the cell size checkpoint in fission yeast; Rupes I et al.; In budding yeast, actin disruption prevents nuclear division . This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton . The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, the budding yeast Cdc2 homolog . Wild-type Schizosaccharomyces pombe cells also arrest before mitosis after actin depolymerization . Oversized cells, however, enter mitosis uninhibited . We carried out a careful analysis of the kinetics of mitotic initiation after actin disruption in undersized and oversized cells . We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells . Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control . This represents a novel function of the Cdc25 phosphatase . Furthermore, we demonstrate that this cell size-monitoring system fulfills the formal criteria of a cell cycle checkpoint.

Mol Cell Biol, 2002 Jan, 22(1), 309 - 20
Novel fission yeast Cdc7-Dbf4-like kinase complex required for the initiation and progression of meiotic second division; Nakamura T et al.; Cdc7, a conserved serine/threonine protein kinase, controls initiation of DNA replication . A regulatory subunit, Dbf4, stimulates the kinase activity of Cdc7 and recruits it to the replication origins . Schizosaccharomyces pombe has a homologous kinase complex, composed of Hsk1 and Dfp1/Him1 . Here, we report a novel protein kinase of S . pombe, Spo4, which shares common structural features with the Cdc7 kinases . In spite of the structural similarities, Spo4 is dispensable for mitotic growth and premeiotic DNA replication . Intriguingly, spo4 null mutants are defective in initiation and progression of the second meiotic division . Spindles for meiosis II are often fragmented . Spo4 kinase activity is markedly enhanced when the enzyme is associated with its regulatory subunit, Spo6, a Dbf4-like protein . Expression of Spo4 is specifically induced during meiosis . Spo4 is preferentially present in nuclei, but this nuclear localization does not require Spo6 . These results suggest that Spo4 is a Cdc7 kinase whose primary role is in meiosis, not in DNA replication . This is the first report of an organism which has two Cdc7-related kinase complexes with different biological functions.

Mol Cell Biol, 2002 Jan, 22(1), 105 - 16
Mammalian Orc1 protein is selectively released from chromatin and ubiquitinated during the S-to-M transition in the cell division cycle; Li CJ et al.; Previous studies have shown that changes in the affinity of the hamster Orc1 protein for chromatin during the M-to-G(1) transition correlate with the activity of hamster origin recognition complexes (ORCs) and the appearance of prereplication complexes at specific sites . Here we show that Orc1 is selectively released from chromatin as cells enter S phase, converted into a mono- or diubiquitinated form, and then deubiquitinated and re-bound to chromatin during the M-to-G(1) transition . Orc1 is degraded by the 26S proteasome only when released into the cytosol, and peptide additions to Orc1 make it hypersensitive to polyubiquitination . In contrast, Orc2 remains tightly bound to chromatin throughout the cell cycle and is not a substrate for ubiquitination . Since the concentration of Orc1 remains constant throughout the cell cycle, and its half-life in vivo is the same as that of Orc2, ubiquitination of non-chromatin-bound Orc1 presumably facilitates the inactivation of ORCs by sequestering Orc1 during S phase . Thus, in contrast to yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin-bound ORCs.

BMC Microbiol . 2001;1(1):29 . Epub 2001 Nov 20.
RNA triphosphatase is essential in Schizosaccharomyces pombe and Candida albicans; Pei Y et al.; BACKGROUND: The first two steps in the capping of cellular mRNAs are catalyzed by the enzymes RNA triphosphatase and RNA guanylyltransferase . Although structural and mechanistic differences between fungal and mammalian RNA triphosphatases recommend this enzyme as a potential antifungal target, it has not been determined if RNA triphosphatase is essential for the growth of fungal species that cause human disease . RESULTS: We show by classical genetic methods that the triphosphatase (Pct1) and guanylyltransferase (Pce1) components of the capping apparatus in the fission yeast Schizosaccharomyces pombe are essential for growth . We were unable to disrupt both alleles of the Candida albicans RNA triphosphatase gene CaCET1, implying that the RNA triphosphatase enzyme is also essential for growth of C . albicans, a human fungal pathogen . CONCLUSIONS: Our results provide the first genetic evidence that cap synthesis is essential for growth of an organism other than Saccharomyces cerevisiae and they validate RNA triphosphatase as a target for antifungal drug discovery.

Genes Cells, 2001 Dec, 6(12), 1043 - 54
Cell death with predominant apoptotic features in Saccharomyces cerevisiae mediated by deletion of the histone chaperone ASF1/CIA1; Yamaki M et al.; BACKGROUND: Although no potential homologues of multicellular apoptotic genes (e.g . Bax, Bak, Bcl-2, caspases and p53) have been identified in a unicellular eukaryote, previous reports contain several implications of the apoptotic behaviour of yeasts (i.e . Saccharomyces cerevisiae and Schizosaccharomyces pombe) . Therefore, whether or not yeast undergoes apoptosis has been a topic of some debate . hCCG1, which is the largest subunit of TFIID and a histone acetyltransferase, appears to be involved in the regulation of apoptosis . The factor hCIA interacts with hCCG1 and functions as a histone chaperone in mammalian cells; its homologue in yeast is Asf1p/Cia1p . Therefore, we anticipated that a yeast mutant in Asf1p/Cia1p would be a valuable tool for studying apoptosis in yeast . RESULTS: We established a strain of S . cerevisiae lacking the histone chaperone ASF1/CIA1 . This disruptant, asf1/cia1, arrested preferentially at the G2/M-phase and died . We systematically analysed the phenotype associated with the death of this mutant yeast and identified many changes, such as fragmentation of the nuclei, condensation and fragmentation of chromatin, reduction of the mitochondrial membrane-potential, dysfunction of the mitochondrial proton pump, and a discernible release of cytochrome c to cytoplasm that resembles those in apoptotic multicellular organisms . Other changes potentially associated with the death in our mutant included a reduction in the vacuolar membrane potential, dysfunction of the vacuolar proton pump, reduction of endocytosis, and the presence of many autophagic bodies . However, these mutant yeast cells also showed cellular enlargement, which is characteristic of necrosis . CONCLUSIONS: Cell death in S . cerevisiae occurs with a phenotype that largely resembles apoptosis in multicellular organisms, but that has some features of necrosis . Therefore, we indicate that yeast undergoes a 'prototypal active cell death' that retains some characteristics of passive cell death (necrosis) . In addition, we think that active cell death is ubiquitously the essential attribute of life . Although such an active cell death system in yeast remains open to confirmation, we speculate that deletion of the histone chaperone Asf1p/Cia1p inhibits the normal assembly/disassembly of nucleosomes in yeast and thereby initiates the active cell death system.

Genes Cells, 2001 Dec, 6(12), 1031 - 42
Characterization of GTPase-activating proteins for the function of the Rho-family small GTPases in the fission yeast Schizosaccharomyces pombe; Nakano K et al.; BACKGROUND: The small GTPase Rho1 has been shown to regulate the organization of the actin cytoskeleton and formation of the cell wall in the fission yeast Schizosaccharomyces pombe . Activity of Rho1 must be precisely regulated in vivo, since both increases and decreases in its activity affect cell growth and shape . Thus, it is important to clarify the mechanism by which the activity of Rho1 is regulated in vivo . RESULTS: Seven genes encoding putative GAPs, GTPase-activating proteins, for the function of the Rho-family proteins were isolated from S . pombe . After disruption of these genes, rga1+ was found to play important roles in cell growth and morphogenesis . In rga1 null cells, delocalized F-actin patches and extraordinary thickening of the cell wall and the septum were observed . On the other hand, over-expression of Rga1 produced shrunken or dumpy cells . The phenotype of the rga1 null cells or the Rga1-over-expressing cells was similar to that of cells containing abnormally high or low Rho1 activity, respectively . Moreover, direct association of Rga1 with Rho1 was shown . Rga1 was localized to the cell ends and septum where Rho1 is known to function . CONCLUSIONS: In S . pombe, Rga1 is involved in the F-actin patch localization, cell morphogenesis, regulation of septation, and cell wall synthesis, probably functioning as a GAP for the function of Rho1.

Biochem Biophys Res Commun, 2001 Dec 14, 289(4), 807 - 12
A secretion signal is present in the Collybia velutipes oxalate decarboxylase gene; Azam M et al.; The oxalate decarboxylase (OXDC) gene from Collybia velutipes is overexpressed as an active form in Schizosaccharomyces pombe . The recombinant enzyme shows similar pH optima and stability, while substrate kinetic analysis shows a ninefold decrease in K(m) value with respect to native OXDC . Most of the expressed protein was present in periplasm and remained firmly bound to cell-wall materials . However, 20% of enzyme expressed was secreted out into the medium suggesting the presence of a secretion signal (C . velutipes) in the oxalate decarboxylase gene . This secretion signal is associated with the N-terminal of OXDC as is evident by secretion of nonsecretory genes AmA1 and beta-galactosidase . An expression vector using this signal is constructed for expression and secretion of heterologous proteins in S . pombe . (c)2001 Elsevier Science.

Curr Opin Microbiol, 2001 Dec, 4(6), 713 - 9
Cytokinesis and the contractile ring in fission yeast; Feierbach B et al.; The fission yeast Schizosaccharomyces pombe provides a genetic model system for the study of cytokinesis . As in many eukaryotes, cell division in the fission yeast requires an actin-myosin-based contractile ring . Numerous components of the contractile ring that function in ring assembly, positioning and contraction have been characterized . Many of these proteins are evolutionarily conserved, suggesting that common molecular mechanisms may govern aspects of eukaryotic cell division . Recent advances in the assembly and placement of the contractile ring are discussed . In particular, major findings have been made in the characterization of myosins in cytokinesis, and in how the cell division site may be positioned by the nucleus.

J Biol Chem, 2002 Feb 8, 277(6), 4050 - 5 Epub 2001 Nov 29.
Pnk1, a DNA kinase/phosphatase required for normal response to DNA damage by gamma-radiation or camptothecin in Schizosaccharomyces pombe; Meijer M et al.; We report the characterization of Pnk1, a 45-kDa homolog of the human polynucleotide kinase PNKP in Schizosaccharomyces pombe . Recombinant Pnk1 like human PNKP exhibits both 5'-DNA kinase and 3'-DNA phosphatase activities in vitro . Furthermore, we detected 3'-DNA phosphatase activity with a single-stranded substrate in extracts from wild-type yeast, but no activity was detected in pnk1delta strains . We have shown that GFP-tagged Pnk1 like mammalian PNKP localizes to the nucleus . Deletion of pnk1 does not affect cell growth under normal conditions but results in significant hypersensitivity to gamma-radiation or camptothecin, an inhibitor of topoisomerase I, suggesting that Pnk1 plays an important role in the repair of DNA strand breaks produced by these agents . The pnk1 deletion mutants were not hypersensitive to ethyl methanesulfonate, methyl methanesulfonate, or 4-nitroquinoline N-oxide . Expression of human PNKP in pnk1delta cells restores resistance to gamma-radiation or camptothecin, suggesting that the functions of yeast Pnk1 and human PNKP have been conserved.

EMBO J, 2001 Dec 3, 20(23), 6660 - 71
Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair; Hartsuiker E et al.; To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharomyces pombe RAD50 homologue . The deletion is sensitive to a range of DNA-damaging agents and shows dynamic epistatic interactions with other recombination-repair genes . We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow DNA replication . We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment . Interestingly, in rad50Delta cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination . We propose that Rad50, an SMC-like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair . In support of this, we found that Rad50 functions in the same pathway for the repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein . We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re-initiation of replication after replication fork collapse.

EMBO J, 2001 Dec 3, 20(23), 6601 - 11
Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2; Ceschini S et al.; Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange . Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria . We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA . This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes . Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.

Mol Pharmacol, 2001 Dec, 60(6), 1254 - 9
Anticancer agent E7070 inhibits amino acid and uracil transport in fission yeast; Tsukahara K et al.; E7070 is a novel sulfonamide anticancer agent that inhibits cell cycle progression in G1 in mammalian cells, but its action targets are not known . We recently employed the genetically amenable fission yeast Schizosaccharomyces pombe as a model organism to search for its targets . Here, we show that E7070 inhibits imports of amino acid and uracil into S . pombe cells . Unlike their prototrophic counterparts, leucine- and uracil-auxotrophic strains are sensitive to E7070 and are unable to proliferate with a delayed G1-S transition in low-glucose yeast extract-polypeptone medium containing this drug because this chemical markedly inhibits the uptake of leucine and uracil in low glucose medium . Furthermore, addition of leucine or uracil to the culture medium or overexpression of genes encoding an amino acid or uracil transporter suppresses the E7070-imposed growth inhibition of these auxotrophic strains . Thus, some of the molecular targets for E7070 action in S . pombe are likely to be leucine and uracil transporters.

RNA, 2001 Nov, 7(11), 1589 - 602
Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization; Long KS et al.; An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts . Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation . In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation . Experiments in which the human protein was expressed in S . pombe have suggested that phosphorylation of the La protein regulates tRNA maturation . To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S . cerevisiae La protein Lhp1p . Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p . Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p . Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth . Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs . Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location . Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S . cerevisiae protein.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13589 - 94
The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein; Lee JK et al.; The interaction between an origin sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication . In this report, we have examined the interaction between the Schizosaccharomyces pombe (sp) autonomously replicating sequence 1 (ars1) and the spORC . For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminal-deleted spOrc4 subunit (spORC(Delta N-Orc4)), and the spOrc4 subunit by using the baculovirus expression system . Wild-type spORC showed sequence-specific binding to ars1, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC . In contrast, the spORC(Delta N-Orc4) and the Delta N-spOrc4p alone did not bind significantly to ars1 . These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the ars1-binding activity . DNA-binding competition assays with fragments of ars1 and DNase I footprinting studies with full-length ars1 revealed that the spORC interacted with several AT-rich sequence regions of ars1 . These DNA-binding properties of spORC correlate with the previously determined sequence requirements of the S . pombe ars1 . These studies indicate that because of its unique Orc4 subunit, S . pombe uses a mechanism to recognize its origins different from that used by Saccharomyces cerevisiae.

J Bacteriol, 2001 Dec, 183(24), 7165 - 72
Conversion of pipecolic acid into lysine in Penicillium chrysogenum requires pipecolate oxidase and saccharopine reductase: characterization of the lys7 gene encoding saccharopine reductase; Naranjo L et al.; Pipecolic acid is a component of several secondary metabolites in plants and fungi . This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities . In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs . We have isolated two P . chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine . These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine . The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid . A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe . Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase . Our data suggest that in P . chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.

Nucleic Acids Res, 2001 Nov 15, 29(22), 4561 - 9
A second eIF4E protein in Schizosaccharomyces pombe has distinct eIF4G-binding properties; Ptushkina M et al.; The eukaryotic cap-binding proteins belonging to the eIF4E family are generally involved in mediating the recruitment of ribosomes to capped mRNA . We described previously a cap-binding protein (now called eIF4E1) in Schizosaccharomyces pombe that appears to have all of the usual structural and functional attributes of an eIF4E . We have now characterised a new type of cap-binding protein (eIF4E2) from this organism, which at the amino acid sequence level, is 52% identical and 59% similar to eIF4E1 . eIF4E2 is not essential in S.pombe but has some novel properties that may be related to a special function in the cell . The ratio of eIF4E2:eIF4E1 in the cell shifts in favour of eIF4E2 at higher temperatures . Despite having all of the dorsal face amino acids that have so far been associated with eIF4G binding to eIF4E1, eIF4E2 binds the eIF4E-binding domain of S.pombe eIF4G >10(2)-times weaker than eIF4E1 in vitro . The eIF4E2 cap-binding affinity is in the typical micromolar range . The results suggest that eIF4E2 is not active on the main pathway of translation initiation in fission yeast but might play a role in the adaptation strategy of this organism under specific growth conditions . Moreover, they provide insight into the molecular characteristics required for tight binding to eIF4G.

J Biol Chem, 2002 Jan 25, 277(4), 2637 - 43 Epub 2001 Nov 15.
The Ded1 DEAD box helicase interacts with Chk1 and Cdc2; Liu HY et al.; Ded1 is a fission yeast DEAD box protein involved in translation . We isolated Ded1 in a screen for multi-copy suppressors of a cold-sensitive, loss-of-function mutant of the cyclin-dependent kinase Cdc2 . The checkpoint protein kinase Chk1, required for cell cycle arrest in response to DNA damage, was also isolated in this screen . Ded1 interacts with Chk1 in a two-hybrid screen, and this physical interaction can be recapitulated in Schizosaccharomyces pombe . The Ded1 polypeptide is modified in response to heat shock and depletion of carbon source . These two stressors appear to cause different modifications . Thus, the Ded1 protein appears to respond to particular types of cellular stress and may influence the activity of Cdc2 as a result.

Philos Trans R Soc Lond B Biol Sci, 2001 Nov 29, 356(1415), 1725 - 33
Cellular signalling and the complexity of biological timing: insights from the ultradian clock of Schizosaccharomyces pombe; Kippert F; The molecular bases of circadian clocks are complex and cannot be sufficiently explained by the relatively simple feedback loops, based on transcription and translation, of current models . The existence of additional oscillators has been demonstrated experimentally, but their mechanism(s) have so far resisted elucidation and any universally conserved clock components have yet to be identified . The fission yeast, Schizosaccharomyces pombe, as a simple and well-characterized eukaryote, is a useful model organism in the investigation of many aspects of cell regulation . In fast-growing cells of the yeast an ultradian clock operates, which can serve as a model system to analyse clock complexity . This clock shares strict period homeostasis and efficient entrainment with circadian clocks but, because of its short period of 30 min, mechanisms other than a transcription/translation-based feedback loop must be working . An initial systematic screen involving over 200 deletion mutants has shown that major cellular signalling pathways (calcium/phosphoinositide, mitogen-activated protein kinase and cAMP/protein kinase A) are crucial for the normal functioning of this ultradian clock . A comparative examination of the role of cellular signalling pathways in the S.pombe ultradian clock and in the circadian timekeeping of different eukaryotes may indicate common principles in biological timing processes that are universally conserved amongst eukaryotes.

Structure (Camb), 2001 Nov, 9(11), 1043 - 50
The Ras-Byr2RBD complex: structural basis for Ras effector recognition in yeast; Scheffzek K et al.; BACKGROUND: The small GTP binding protein Ras has important roles in cellular growth and differentiation . Mutant Ras is permanently active and contributes to cancer development . In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade . In the lower eukaryotic Schizosaccharomyces pombe, Byr2 kinase represents a Ras target that in terms of signal-transduction hierarchy can be considered a homolog of mammalian Raf-kinase . The activation mechanism of protein kinases by Ras is not understood, and there is no detailed structural information about Ras binding domains (RBDs) in nonmammalian organisms . RESULTS: The crystal structure of the Ras-Byr2RBD complex at 3 A resolution shows a complex architecture similar to that observed in mammalian homologous systems, with an interprotein beta sheet stabilized by predominantly polar interactions between the interacting components . The C-terminal half of the Ras switch I region contains most of the contact anchors, while on the Byr2 side, a number of residues from topologically distinct regions are involved in complex stabilization . A C-terminal helical segment, which is not present in the known mammalian homologous systems and which is part of the auto-inhibitory region, has an additional binding site outside the switch I region . CONCLUSIONS: The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families . The extra helical segment might be involved in kinase activation.

Structure (Camb), 2001 Nov, 9(11), 1029 - 41
Solution structure of the Ras binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe; Gronwald W et al.; BACKGROUND: After activation, small GTPases such as Ras transfer the incoming signal to effectors by specifically interacting with the binding domain of these proteins . Structural details of the binding domain of different effectors determine which pathway is predominantly activated . Byr2 from fission yeast is a functional homolog of Raf, which is the direct downstream target of Ras in mammalians that initiates a protein kinase cascade . The amino acid sequence of Byr2's Ras binding domain is only weakly related to that of Raf, and Byr2's three-dimensional structure is unknown . RESULTS: We have solved the 3D structure of the Ras binding domain of Byr2 (Byr2RBD) from Schizosaccharomyces pombe in solution . The structure consists of three alpha helices and a mixed five-stranded beta pleated sheet arranged in the topology betabetaalphabetabetaalphabetaalpha with the first seven canonic secondary structure elements forming a ubiquitin superfold . 15N-(1)H-TROSY-HSQC spectroscopy of the complex of Byr2RBD with Ras*Mg(2+)*GppNHp reveals that the first and second beta strands and the first alpha helix of Byr2 are mainly involved in the protein-protein interaction as observed in other Ras binding domains . Although the putative interaction site of H-Ras from human and Ras1 from S . pombe are identical in sequence, binding to Byr2 leads to small but significant differences in the NMR spectra, indicating a slightly different binding mode . CONCLUSIONS: The ubiquitin superfold appears to be the general structural motif for Ras binding domains even in cases with vanishing sequence identity . However, details of the 3D structure and the interacting interface are different, thereby determining the specifity of the recognition of Ras and Ras-related proteins.

J Cell Sci, 2001 Oct, 114(Pt 20), 3779 - 88
Isolation and characterization of the Pin1/Ess1p homologue in Schizosaccharomyces pombe; Huang HK et al.; Pin1/Ess1p is a highly conserved WW domain-containing peptidyl-prolyl isomerase (PPIase); its WW domain binds specifically to phospho-Ser/Thr-Pro sequences and its catalytic domain isomerizes phospho-Ser/Thr-Pro bonds . Pin1 PPIase activity can alter protein conformation in a phosphorylation-dependent manner and/or promote protein dephosphorylation . Human Pin1 interacts with mitotic phosphoproteins, such as NIMA, Cdc25 and Wee1, and inhibits G(2)/M progression in Xenopus extracts . Depletion of Pin1 in HeLa cells and deletion of ESS1 in S . cerevisiae result in mitotic arrest . In addition, Pin1/Ess1p play roles in transcription in S . cerevisiae and in mammalian somatic cells . The S . pombe genome sequence has an open reading frame (ORF) that has 47% identity with Pin1 . Expression of this ORF rescued the growth defect caused by ess1 deletion in S . cerevisiae, indicating that S . pombe Pin1p is a functional Pin1 homologue . Overexpression of pin1(+) in S . pombe caused slow growth and a G(1) delay . Deletion of pin1(+) (pin1 Delta) did not affect cell cycle progression or cell growth, but increased sensitivity to the cyclophilin inhibitor, cyclosporin A, suggesting that cyclophilin family PPIases have overlapping functions with the Pin1p PPIase . Deletion of pin1(+) did not affect the DNA replication checkpoint, but conferred a modest increase in UV sensitivity . Furthermore, the pin1 Delta allele caused a synthetic growth defect when combined with either cdc25-22 or wee1-50 but not the cdc24-1 temperature-sensitive mutant . The pin1 Delta strain showed increased sensitivity to the PP1/PP2A family phosphatase inhibitor, okadaic acid, suggesting that Pin1p plays a role in protein dephosphorylation as a result of its ability to increase the population of phospho-Ser/Thr-Pro peptide bonds in the trans conformation that is required for PP2A-mediated dephosphorylation . Our genetic data also suggest that Pin1p might function as a positive regulator of Cdc25p and Wee1p.

FEBS Lett, 2001 Nov 9, 508(1), 136 - 42
The protein phosphatase 2A B'-regulatory subunit par1p is implicated in regulation of the S . pombe septation initiation network; Le Goff X et al.; In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway . One of these encodes the protein phosphatase 2A (PP2A) B'-regulatory subunit par1p . Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants . Our data suggest that PP2A-par1p acts as a negative regulator of SIN signalling.

Dev Cell, 2001 Aug, 1(2), 158 - 60
New insights into development from mitosis of a unicellular yeast; Hagan IM et al.; Studies in the fission yeast Schizosaccharomyces pombe have uncovered a new spindle checkpoint.

J Biol Chem, 2002 Jan 11, 277(2), 1190 - 4 Epub 2001 Nov 06.
Structure of the plant alternative oxidase . Site-directed mutagenesis provides new information on the active site and membrane topology; Albury MS et al.; All higher plants and many fungi contain an alternative oxidase (AOX), which branches from the cytochrome pathway at the level of the quinone pool . In an attempt, first, to distinguish between two proposed structural models of this di-iron protein, and, second, to examine the roles of two highly conserved tyrosine residues, we have expressed an array of site-specific mutants in Schizosaccharomyces pombe . Mitochondrial respiratory analysis reveals that S . pombe cells expressing AOX proteins in which Glu-217 or Glu-270 were mutated, no longer exhibit antimycin-resistant oxygen uptake, indicating that these residues are essential for AOX activity . Although such data corroborate a model that describes the AOX as an interfacial membrane protein, they are not in full agreement with the most recently proposed ligation sphere of its di-iron center . We furthermore show that upon mutation of Tyr-253 and Tyr-275 to phenylalanines, AOX activity is fully maintained or abolished, respectively . These data are discussed in reference to the importance of both residues in the catalytic cycle of the AOX.

Curr Biol, 2001 Oct 30, 11(21), 1656 - 65
Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division; Feierbach B et al.; BACKGROUND: Both symmetric and asymmetric cell divisions are required for the generation of appropriate cell lineages during development . Wild-type Schizosaccharomyces pombe cells divide in a symmetric fashion to produce two similar rod-shaped daughter cells . Formins are proteins with conserved roles in cell polarity, cytokinesis, and the regulation of actin and microtubule cytoskeletons . RESULTS: Here, we identify and characterize a new S . pombe formin, for3p . for3 Delta mutant cells divide in an asymmetric manner; a mother cell divides medially to produce one daughter cell that develops into a monopolar cell and one daughter that develops into a bipolar cell . Both daughter cells recapitulate similar asymmetric lineages themselves . Inheritance of the bipolar pattern correlates with inheritance of the recent birth scar, not with asymmetry in the spindle pole bodies . for3 Delta mutants lack interphase actin cables and have delocalized actin patch and myo52p (type V myosin) distributions . for3 Delta cells have normal microtubule dynamics and cortical interactions but have defects in microtubule organization and increased numbers of microtubule bundles . for3p-GFP is localized at both cell tips in an actin-dependent manner and at the cell division site . CONCLUSIONS: for3p is a cell polarity factor required for interphase actin cable formation and microtubule organization . The for3 Delta phenotype suggests that cells are able to grow in a polarized manner even in the absence of functional actin cables and polarized distribution of actin patches . for3p and possibly actin cables are part of a regulatory network that ensures that cell divisions are symmetric.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 175 - 81
Coflocculation of Escherichia coli and Schizosaccharomyces pombe; Peng X et al.; Several yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were found to coaggregate with Escherichia coli, but S . pombe showed much less coflocculation than the other yeasts (Peng et al . 2001)) . S . pombe is known to have galactose-rich cell walls and we investigated whether this might be responsible for its different behavior by studying the wild-type TP4-1D, with a mannose to galactose ratio of 1 to 1.2, and the glycosylation mutant gms1delta (Man:Gal=1:0) . The wild-type induced very low levels of coflocculation (3%) while gms1delta induced a remarkable amount of coflocculation (48%) . Coflocculation of the mutant was inhibited by mannose but not affected by galactose or glucose . The S . cerevisiae mnn2 mutant, with a mannan structure similar to gms1delta, also showed a high degree of coflocculation (40%) . However, S . cerevisiae mutant mnn9, with a mature core similar to S . pombe, showed decreased coflocculation (21.3%) . Both these S . cerevisae mutants were sensitive to mannose inhibition . Coflocculation of E . coli and gms1delta also could be inhibited by gms1delta mannan and plant lectins, such as HHA, GNA and NPA, specific to either alpha-1-3- or alpha-1-6-linked mannosyl units . From these results we conclude that the E . coli lectins may have specificity for alpha-1-6- and alpha-1-3-linked mannose residues either in the outer chain or in the core of S . pombe, but in wild-type strains these mannose residues are shielded by galactose residues.

Biochim Biophys Acta, 2001 Oct 31, 1521(1-3), 146 - 51
Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in Dictyostelium discoideum and Schizosaccharomyces pombe; Skruzny M et al.; We screened the Dictyostelium discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified a novel cyclophilin CypE . Independently, the Schizosaccharomyces pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found . Both cyclophilins bind the respective SNW protein in their autologous systems . The interaction was localized to the N-terminal part of SnwA as well as of Snw1 . CypE was confirmed in vitro to be a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase . Remarkably, both SNW proteins bind the cyclophilins in a cyclosporin A independent manner, possibly serving as adaptors for these novel isomerases . These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI-124/PPIL1 protein.

Mol Cell Biol, 2001 Dec, 21(23), 8095 - 103
Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit; Kong D et al.; The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe . Orc4p alone bound tightly and specifically to several sites within S . pombe replication origins that are genetically required for origin activity . These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences . Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences . ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p . Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle . These results reveal that the S . pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.

EMBO J, 2001 Nov 1, 20(21), 6115 - 26
Regulation of replication timing in fission yeast; Kim SM et al.; Here we report the first characterization of replication timing and its regulation in the fission yeast Schizosaccharomyces pombe . We used three different synchronization methods: centrifugal elutriation, cdc10 temperature-shift and release, and starvation for deoxyribonucleoside triphosphates (dNTPs) by treatment with hydroxyurea (HU) followed by removal of HU, to study the times when specific autonomously replicating sequence elements (ARS elements; potential replication origins) replicate during S phase . We found that individual ARS elements replicate at characteristic times, some early and some late, independently of synchronization method . In wild-type cells treated with HU, early ARS elements replicated but late ones did not . However, in HU-treated mutant cells lacking the Rad3 (similar to human ATR and ATM) or Cds1 (similar to human CHK2) checkpoint kinase, both early and late ARS elements were able to replicate . Thus under conditions of dNTP starvation the Rad3 and Cds1 kinases are needed to suppress the replication of normally late-replicating regions.

J Cell Sci, 2001 Aug, 114(Pt 16), 3013 - 23
XMog1, a nuclear ran-binding protein in Xenopus, is a functional homologue of Schizosaccharomyces pombe mog1p that co-operates with RanBP1 to control generation of Ran-GTP; Nicolas FJ et al.; Ran is a multifunctional small GTPase of the Ras superfamily that plays roles in nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation . By screening a Xenopus oocyte cDNA library for Ran-GTP-binding proteins using the two-hybrid system of co-expression in yeast, we identified XMog1, a 20.4 kDa polypeptide related to Mog1p in Saccharomyces cerevisiae and similar gene products in Schizosaccharomyces pombe, Arabidopsis and mammals . We show that cDNAs encoding XMog1 and S . cerevisiae Mog1p rescue the growth defect of S . pombe cells lacking mog1, demonstrating conservation of their functions . In Xenopus somatic cells and transfected mammalian cells, XMogl is localised to the nucleus . XMog1 alone does not stimulate Ran GTPase activity or nucleotide exchange, but causes nucleotide release from Ran-GTP and forms a complex with nucleotide-free Ran . However, in combination with Ran-binding protein 1 (RanBP1), XMog1 promotes the release of GDP and the selective binding of GTP to Ran . XMog1 and RanBP1 also promote selective GTP loading onto Ran catalysed by the nuclear guanine nucleotide exchange factor, RCC1 . We propose that Mog1-related proteins, together with RanBP1, facilitate the generation of Ran-GTP from Ran-GDP in the nucleus.

J Cell Sci, 2001 Aug, 114(Pt 16), 2929 - 41
Intracellular pH homeostasis during cell-cycle progression and growth state transition in Schizosaccharomyces pombe; Karagiannis J et al.; Accurate measurement of intracellular pH in unperturbed cells is fraught with difficulty . Nevertheless, using a variety of methods, intracellular pH oscillations have been reported to play a regulatory role in the control of the cell cycle in several eukaryotic systems . Here, we examine pH homeostasis in Schizosaccharomyces pombe using a non-perturbing ratiometric pH sensitive GFP reporter . This method allows for accurate intracellular pH measurements in living, entirely undisturbed, logarithmically growing cells . In addition, the use of a flow cell allows internal pH to be monitored in real time during nutritional, or growth state transition . We can find no evidence for cell-cycle-related changes in intracellular pH . By contrast, all data are consistent with a very tight homeostatic regulation of intracellular pH near 7.3 at all points in the cell cycle . Interestingly, pH set point changes are associated with growth state . Spores, as well as vegetative cells starved of either nitrogen, or a carbon source, show a marked reduction in their internal pH compared with logarithmically growing vegetative cells . However, in both cases, homeostatic regulation is maintained.

Chromosoma, 2001 Sep, 110(5), 322 - 34
A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint; Nabetani A et al.; The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells . We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast . Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest . Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact . Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2 . Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body . We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

FEBS Lett, 2001 Oct 26, 507(2), 215 - 9
Arabidopsis thaliana expresses a second functional phytochelatin synthase; Cazale AC et al.; Phytochelatins represent a major detoxifying pathway for heavy metals in plants and many other organisms . The Arabidopsis thaliana CAD1 (=AtPCS1) gene encodes a phytochelatin synthase and cad1 mutants are phytochelatin deficient and cadmium hypersensitive . The Arabidopsis genome contains a highly homologous gene, AtPCS2, of which expression and function were studied in order to understand the apparent non-redundancy of the two genes . Low constitutive AtPCS2 expression is detected in all plant organs analyzed . The AtPCS2 gene encodes a functional phytochelatin synthase as shown by expression in Saccharomyces cerevisiae and the complementation of a Schizosaccharomyces pombe phytochelatin synthase knockout strain.

Differentiation, 2001 Jun, 67(4-5), 98 - 106
Bob1, a Gim5/MM-1/Pfd5 homolog, interacts with the MAP kinase kinase Byr1 to regulate sexual differentiation in the fission yeast, Schizosaccharomyces pombe; Henkel J et al.; The MAPKK Byr1 is an essential component of a Ras-dependent MAPK module required for sexual differentiation in the fission yeast, Schizosaccharomyces pombe . Here we describe the genetic and molecular characterization of a highly conserved protein, Bob1, which was identified from a two-hybrid screen for Byr1-interacting proteins . Byrl and Bobl proteins coprecipitate from S . pombe cell lysates, and both proteins localize to the tips and septa of S . pombe cells . S . pombe bob1 null (bob1delta) mutants lack obvious growth defects but exhibit a significant mating deficiency, which can be suppressed by overexpression of Byrl . Overexpression of Bob1 also leads to inhibition of mating in S . pombe, and this defect is likewise suppressed by Byrl overexpression . Bob1 is highly homologous in structure to the mammalian MM-1/Pfd5 and budding yeast Gim5/Pfd5-Sc proteins, which have been implicated as regulators of actin and tubulins . Similar to budding yeast gim5/pfd5-Sc mutants, S . pombe bob1delta cells have cytoskeletal defects, as judged by hypersensitivity to cytoskeletal disrupting drugs . byr1delta mutants do not share this characteristic with bob1delta mutants, and byr1delta bob1delta mutants are not significantly more sensitive to cytoskeletal disrupting drugs than cells carrying only the bob1delta mutation . Taken together, our results suggest that Bob1 has Byr1-related function(s) required for proper mating response of S . pombe cells and Byrl-independent function(s) required for normal cytoskeletal control . We show that the human MM-1/Pfd5 protein can substitute for its counterpart in fission yeast, providing evidence that the functions of Bob1-related proteins have been highly conserved through evolution . Our results lead us to propose that Bob1-related proteins may play diverse roles in eukaryotic organisms.

J Cell Sci, 2001 Jul, 114(Pt 14), 2649 - 64
Flp1, a fission yeast orthologue of the s . cerevisiae CDC14 gene, is not required for cyclin degradation or rum1p stabilisation at the end of mitosis; Cueille N et al.; In Saccharomyces cerevisiae, the phosphoprotein phosphatase Cdc14p plays a central role in exit from mitosis, by promoting B-type cyclin degradation and allowing accumulation of the cyclin-dependent kinase inhibitor Sic1p . Cdc14p is sequestered in the nucleolus during interphase, from where it is released at the end of mitosis, dependent upon mitotic exit network function . The CDC14 gene is essential and loss-of-function mutants arrest at the end of mitosis . We have identified a fission yeast orthologue of CDC14 through database searches . A Schizosaccharomyces pombe flp1 (cdc fourteen-like-phosphatase) null mutant is viable, divides at a reduced size and shows defects in septation . flp1p is not the essential effector of the S . pombe septation initiation network, but may potentiate signalling of the onset of septation . In contrast to S . cerevisiae Cdc14p, flp1p is not required for the accumulation or destruction of the B-type cyclin cdc13p, the cyclin-dependent kinase inhibitor rum1p, or for dephosphorylation of the APC/C specificity factor ste9p in G1 . Like its budding yeast counterpart, flp1p is restricted to the nucleolus until mitosis, when it is dispersed through the nucleus . In contrast to S . cerevisiae Cdc14p, flp1p is also present on the mitotic spindle and contractile ring . The potential roles of flp1p in cell cycle control are discussed.

Mol Genet Genomics, 2001 Oct, 266(2), 336 - 42
Fission yeast (Schizosaccharomyces pombe) cells defective in the MutY-homologous glycosylase activity have a mutator phenotype and are sensitive to hydrogen peroxide; Chang DY et al.; The modified base 7,8-dihydro-8-oxo-guanine (8-oxoG) is one of the most stable deleterious products of oxidative DNA damage because it mispairs with adenine during DNA replication . In the fission yeast Schizosaccharomyces pombe, the MutY homolog (SpMYH) is responsible for removing misincorporated adenines from A/8-oxoG or A/G mismatches and thus preventing G:C to T:A mutations . In order to study the functional role of SpMYH, an SpMYH knockout strain was constructed . The SpMYH knockout strain, which does not express SpMYH and has no A/8-oxoG glycosylase activity, displays a 36-fold higher frequency of spontaneous mutations than the wild type strain . Disruption of SpMYH causes increased sensitivity to H2O2 but not to UV-irradiation . Expression of SpMYH in the mutant cells restores the adenine glycosylase activity, reduces the mutation frequency, and elevates the resistance to H2O2 . Asp172 of SpMYH is conserved in a helix-hairpin-helix superfamily of glycosylases . The SpMYHA strain expressing D172N SpMYH retained the mutator phenotype . Moreover, when D172N mutant SpMYH was expressed in the wild-type cells, the mutation frequency observed was even higher than that of the parental strains . Thus, a mutant SpMYH that retains substrate-binding activity but is defective in glycosylase activity exhibits a dominant negative effect . This is the first demonstration that a MutY homolog plays an important role in protecting cells against oxidative DNA damage in eukaryotes.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 169 - 74
Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae; Juvvadi PR et al.; Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi . Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae . The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids . Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain . The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%) . Further, A . oryzae cnaA cDNA complemented S . cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.

Curr Genet, 2001 Sep, 40(2), 110 - 8
HBP2: a new mammalian protein that complements the fission yeast MBF transcription complex; Sanchez-Diaz A et al.; The mammalian HBP2 gene has been isolated by functional complementation of cells unable to undergo DNA replication in fission yeast . HBP2 is a protein with a high mobility group (HMG) domain that belongs to the Sox (Sry-related HMG box) family of transcription factors . As in other members of the family, the HMG box in HBP2 is a DNA-binding domain that is essential for its function in Schizosaccharomyces pombe . Expression of HBP2 in fission yeast activates CdclO-dependent transcription at G1/S and allows growth of cdc10-129, resldelta and rep2delta mutant cells at the restrictive temperature . The mammalian HBP2 activates transcription at G1/S in the fission yeast Sch . pombe.

Mol Microbiol, 2001 Oct, 42(1), 257 - 67
KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs) conserved from fission yeast to man; Krzywicka A et al.; In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization . This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines . As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein . We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus . No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.

Mol Microbiol, 2001 Oct, 42(1), 29 - 36
Accumulation of metal-binding peptides in fission yeast requires hmt2+; Vande Weghe JG et al.; The fission yeast Schizosaccharomyces pombe detoxifies cadmium by synthesizing phytochelatins, peptides of the structure (gamma-GluCys)nGly, which bind cadmium and mediate its sequestration into the vacuole . The fission yeast protein HMT2, a mitochondrial enzyme that can oxidize sulphide, appears to be essential for tolerance to multiple forms of stress, including exposure to cadmium . We found that the hmt2- mutant is unable to accumulate normal levels of phytochelatins in response to cadmium, although the cells possess a phytochelatin synthase that is active in vitro . Radioactive pulse-chase experiments demonstrated that the defect lies in two steps: the synthesis of phytochelations and the upregulation of glutathione production . Phytochelatins, once formed, are stable . hmt2- cells accumulate high levels of sulphide and, when exposed to cadmium, display bright fluorescent bodies consistent with cadmium sulphide . We propose that the precipitation of free cadmium blocks phytochelatin synthesis in vivo, by preventing upregulation of glutathione production and formation of the cadmium-glutathione thiolate required as a substrate by phytochelatin synthase . Thus, although sulphide is required for phytochelatin-mediated metal tolerance, aberrantly high sulphide levels can inhibit this pathway . Precise regulation of sulphur metabolism, mediated in part by HMT2, is essential for metal tolerance in fission yeast.

Curr Biol, 2001 Oct 16, 11(20), 1618 - 23
Telomere binding of the Rap1 protein is required for meiosis in fission yeast; Chikashige Y et al.; Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation . Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae . Here we report that S . pombe Rap1 is a telomeric protein essential for meiosis . While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S . cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism . In S . pombe, unlike in S . cerevisiae, an ortholog of human TRF has been identified . This ortholog, Taz1, binds directly to telomere repeats {18} and is necessary for telomere clustering in meiotic prophase . Our results demonstrate that S . pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis . Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.

Curr Biol, 2001 Oct 16, 11(20), 1559 - 68
S . pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body; Krapp A et al.; BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN) . Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered . Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic . RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant . Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation . Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase . It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB . Cdc11p and sid4p can be coimmunoprecipitated from cell extracts . Finally, like its S . cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis . CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.

J Mol Evol, 2001 Oct-Nov, 53(4-5), 290 - 8
Codon usage and tRNA genes in eukaryotes: correlation of codon usage diversity with translation efficiency and with CG-dinucleotide usage as assessed by multivariate analysis; Kanaya S et al.; The species-specific diversity of codon usage in five eukaryotes (Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and Homo sapiens) was investigated with principal component analysis . Optimal codons for translation were predicted on the basis of tRNA-gene copy numbers . Highly expressed genes, such as those encoding ribosomal proteins and histones in S . pombe, C . elegans, and D . melanogaster, have biased patterns of codon usage which have been observed in a wide range of unicellular organisms . In S . pombe and C . elegans, codons contributing positively to the principal component with the largest variance (Z1-parameter) corresponded to the optimal codons which were predicted on the basis of tRNA gene numbers . In D . melanogaster, this correlation was less evident, and the codons contributing positively to the Z1-parameter corresponded primarily to codons with a C or G in the codon third position . In X . laevis and H . sapiens, codon usage in the genes encoding ribosomal proteins and histones was not significantly biased, suggesting that the primary factor influencing codon-usage diversity in these species is not translation efficiency . Codon-usage diversity in these species is known to reflect primarily isochore structures . In the present study, the second additional factor was explained by the level of use of codons containing CG-dinucleotides, and this is discussed with respect to transcription regulation via methylation of CG-dinucleotides, which is observed in mammalian genomes.

Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2155 - 9
Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged protein by a single metal-affinity chromatography step; Loddenkotter B et al.; The mature part of the chloroplast triose phosphate-phosphate translocator was cloned into the yeast expression vector pEVP11 . This construct was used to transform cells from both Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe . The chloroplast translocator protein was functionally expressed in the transformed yeast cells and represented about 1-2% of the Sch . pombe cell membrane protein . It was localized to mitochondrial membranes and/or membranes of the rough endoplasmic reticulum . In order to purify the recombinant translocator protein, a sequence encoding a C-terminal tag of six histidine residues was introduced into the corresponding cDNA . The expressed histidine-tagged translocator protein was purified from the transformed yeast cells under nondenaturing conditions to apparent homogeneity by a single-step affinity chromatography using a Ni2+ . nitrilotriacetic acid resin . Both the expressed triose phosphate translocator and the recombinant histidine-tagged protein possess substrate specificities identical to those of the authentic chloroplast protein, providing definitive evidence for its identity as the triose phosphate translocator and further disproving its assignment as the receptor for chloroplast protein import . The yeast expression system in combination with the Ni2+ . nitrilotriacetic acid chromatography thus provides a valuable tool for the production of purified membrane proteins in a functional state.

Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 7949 - 52
Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe; Sauer N et al.; Schizosaccharomyces pombe cells were transformed with an S . pombe expression vector containing a full-length cDNA of the Chlorella hexose transporter . The transformed cells accumulated 3-O-methylglucose up to 10-fold, whereas wild-type S . pombe and control transformants could only equilibrate this sugar analogue . In a pH-jump experiment, in which extracellular pH was lowered by 1.9 units, the accumulation ratio was increased in transformed cells but not in control cells . This result indicates that the gene product, Chlorella H+/glucose-symporter protein, and a pH gradient suffice for active sugar uptake . Km values for glucose, 6-deoxyglucose, and 3-O-methylglucose of 1.5 x 10(-5) M, 2.7 x 10(-4) M, and 1.0 x 10(-3) M, respectively, were identical in Chlorella and in S . pombe cells transformed with Chlorella cDNA and approximately 100-fold lower than those of the endogenous transport system of S . pombe.

Genetics, 2001 Oct, 159(2), 471 - 86
Characterization of Schizosaccharomyces pombe mcm7(+) and cdc23(+) (MCM10) and interactions with replication checkpoints; Liang DT et al.; MCM proteins are required for the proper regulation of DNA replication . We cloned fission yeast mcm7(+) and showed it is essential for viability; spores lacking mcm7(+) begin S phase later than wild-type cells and arrest with an apparent 2C DNA content . We isolated a novel temperature-sensitive allele, mcm7-98, and also characterized two temperature-sensitive alleles of the fission yeast homolog of MCM10, cdc23(+) . mcm7-98 and both cdc23ts alleles arrest with damaged chromosomes and an S phase delay . We find that mcm7-98 is synthetically lethal with the other mcmts mutants but does not interact genetically with either cdc23ts allele . However, cdc23-M36 interacts with mcm4ts . Unlike other mcm mutants or cdc23, mcm7-98 is synthetically lethal with checkpoint mutants Deltacds1, Deltachk1, or Deltarad3, suggesting chromosomal defects even at permissive temperature . Mcm7p is a nuclear protein throughout the cell cycle, and its localization is dependent on the other MCM proteins . Our data suggest that the Mcm3p-Mcm5p dimer interacts with the Mcm4p-Mcm6p-Mcm7p core complex through Mcm7p.

Cancer Res, 2001 Oct 15, 61(20), 7417 - 21
Human Rad17 is phosphorylated upon DNA damage and also overexpressed in primary non-small cell lung cancer tissues; Wang X et al.; The spRAD17 gene is an essential component of the DNA damage and replication checkpoints in the fission yeast Schizosaccharomyces pombe . Cloning of the human homologue of spRAD17, hRAD17, indicated that it exhibits structural similarity with the replication accessory protein family, which include subunits of the Replication factor C complex . We have analyzed the phosphorylation status of hRad17 in response to DNA damaging agents . Our results showed that phosphorylation of hRad17 occurred immediately after UV and ionizing radiation treatment and reached peak level at approximately 3 h, suggesting that hRad17 may be a component of the DNA damage checkpoint . When primary tumor samples were analyzed, we observed that the majority (74%) of non-small cell lung carcinoma samples exhibited a significantly higher level of hRad17 expression compared with matched normal tissue controls . In contrast, hRad17 protein levels in a panel of primary colon carcinoma samples did not show an elevated level of expression compared with normal colon tissues . This observation suggests that the function of the hRAD17 gene may be involved in lung cancer development and may serve as a potential tumor marker.

Mol Biol (Mosk), 2001 Sep-Oct, 35(5), 750 - 63
{Recombinational repair in Schizosaccharomyces pombe: role in maintaining genomic integrity}; Khasanov FK et al.; Recombinational repair was first detected in budding yeast Saccharomyces cerevisiae and was also studied in fission yeast Schizosaccharomyces pombe over the recent decade . The discovery of Sch . pombe homologs of the S . cerevisiae RAD52 genes made it possible not only to identify and to clone their vertebrate counterparts, but also to study in detail the role of DNA recombination in certain cell processes . For instance, recombinational repair was shown to play a greater role in maintaining genome integrity in fission yeast and in vertebrates compared with S . cerevisiae . The present state of the problem of recombinational double-strand break repair in fission yeast is considered with a focus on comparisons between Sch . pombe and higher eukaryotes . The role of double-strand break repair in maintaining genome stability is discussed.

FEBS Lett, 2001 Oct 12, 506(3), 262 - 6
Elements from the cAMP signaling pathway are involved in the control of expression of the yeast gluconeogenic gene FBP1; Zaragoza O et al.; cAMP represses the transcription of some Saccharomyces cerevisiae genes sensitive to catabolite repression . The effect of cAMP on the expression of FBP1, encoding fructose-1,6-bisphosphatase (FbPase), has been further investigated . In yeast cells shifted to a derepressing medium, synthesis of FbPase was delayed if the strong decrease in intracellular cAMP, which occurs during the shift, was prevented . A similar delay occurred in a RAS2val19 strain, while in a tpk1w strain, with weak protein kinase A activity, induction of FbPase occurred earlier than in a TPK1 strain . In the tpk1w strain, proteins which bind the UAS1 element of FBP1 were present during growth on glucose but they were only weakly operative . Expression of CAT8 and SIP4, encoding proteins which bind the UAS2 element, was blocked by a high concentration of cAMP, but catabolite repression of these genes was not much relieved in a tpk1w strain . We conclude that in S . cerevisiae, as reported for Schizosaccharomyces pombe, control of FBP1 requires both cAMP-dependent and independent pathways; however, the mechanisms operating in the two yeasts are different.

Nucleic Acids Res, 2001 Oct 15, 29(20), 4179 - 86
SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52; Ho JC et al.; The Schizosaccharomyces pombe rad31 and hus5 genes are required for the DNA damage response, as mutants defective in these genes are sensitive to DNA damaging agents, such as UV and ionising radiation and to the DNA synthesis inhibitor hydroxyurea (HU) . Sequence analysis has suggested that rad31 and hus5 encode components of the Pmt3 (SUMO) modification process in S.pombe . We show here that the rad31 null and hus5.62 mutants display reduced levels of Pmt3 modification . We have initiated a search for proteins required for the DNA damage response, which may be modified by Pmt3 and have identified Rad22, the fission yeast homologue of the recombination protein Rad52 . Purification of myc + His-tagged Rad22 protein from cells expressing HA-tagged Pmt3 identifies an 83 kDa species which cross-reacts with anti-HA antisera . We show here that Rad22 interacts with Rhp51 and Rpa70 (the fission yeast homologues of Rad51 and the large subunit of RPA, respectively), but that neither of these proteins appears to be responsible for the 83 kDa species . The 83 kDa species is observed when extracts are prepared under both native and denaturing conditions, and is also observed when myc + His-tagged Rad22 and Pmt3 are expressed at wild type levels, suggesting that Rad22 is modified by Pmt3 in vivo . We have established an S.pombe in vitro Pmt3 modification system and have shown that Rad22 and Rhp51 are modified in vitro, but that Rpa70 is not.

Mol Biol Cell, 2001 Oct, 12(10), 3161 - 74
Gamma-tubulin and the C-terminal motor domain kinesin-like protein, KLPA, function in the establishment of spindle bipolarity in Aspergillus nidulans; Prigozhina NL et al.; Previous research has found that a gamma-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction . We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, gamma-tubulin gene . We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles . In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity . At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A . In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar . These results indicate, surprisingly, that gamma-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity . We propose a model to account for these data.

Mol Biol Cell, 2001 Oct, 12(10), 2907 - 20
Adaptins: the final recount; Boehm M et al.; Adaptins are subunits of adaptor protein (AP) complexes involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles . In this article, we report the results of a survey for adaptins from sequenced genomes including those of man, mouse, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the plant Arabidopsis thaliana, and the yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe . We find that humans, mice, and Arabidopsis thaliana have four AP complexes (AP-1, AP-2, AP-3, and AP-4), whereas D . melanogaster, C . elegans, S . cerevisiae, and S . pombe have only three (AP-1, AP-2, and AP-3) . Additional diversification of AP complexes arises from the existence of adaptin isoforms encoded by distinct genes or resulting from alternative splicing of mRNAs . We complete the assignment of adaptins to AP complexes and provide information on the chromosomal localization, exon-intron structure, and pseudogenes for the different adaptins . In addition, we discuss the structural and evolutionary relationships of the adaptins and the genetic analyses of their function . Finally, we extend our survey to adaptin-related proteins such as the GGAs and stonins, which contain domains homologous to the adaptins.

Gene, 2001 Aug 8, 273(2), 191 - 8
A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe; Bellemare DR et al.; The increasing use of the fission yeast Schizosaccharomyces pombe as a model organism for elucidating the mechanisms of critical biological processes such as cell-cycle control, DNA replication, and stress-mediated signal transduction has fostered the development and utilization of expression systems for gene function analysis . Using the promoter of the ctr4(+) copper transporter gene from S . pombe, we created a series of vectors, named pctr4(+)-X, which regulate the expression of heterologous genes as a function of copper availability . In this system, the addition of copper ions at levels that are non-toxic to yeast cells represses gene expression, while copper deprivation strongly induces gene expression . Conveniently, changes of growth medium or carbon sources are not required to shut down or induce gene expression . The Cu-starvation-mediated inducible expression system is rapid, producing heterologous proteins within 3 h, with sustained expression of proteins that persists for several hours . The pctr4(+)-X expression vectors harbor unique restriction sites constructed in-frame to DNA sequences encoding for epitope tags, which facilitate the detection or purification of the heterologous proteins using commercially available antibodies and affinity columns . Furthermore, the pctr4(+)-X copper-regulatable protein expression vectors have been constructed with three different selectable markers, offering more versatility for studying gene function in fission yeast.

Oncogene, 2001 Sep 20, 20(42), 6001 - 8
A "no-hybrids" screen for functional antagonizers of human p53 transactivator function: dominant negativity in fission yeast; Waddell S et al.; We have developed a functional "no-hybrids" screen in the fission yeast Schizosaccharomyces pombe based on the transcription transactivator activity of human p53 . The screen can be used to identify antagonizers and modulators of p53 activity . Expression of functional full-length human p53 is conditionally lethal to the screen reporter strains . Co-expression of specific inhibitory proteins promotes cell survival and growth . We have validated the "no-hybrids" system by (a) successful modeling of human wild-type p53 interaction with SV40 large T antigen, Mdm2 and a panel of tumor-derived human p53 mutants, (b) demonstrating the screening system's efficiency through identification of a dominant negative fragment of p53 itself in a library screen context and (c) using Drosophila p53 to demonstrate that the system can detect evolutionarily distant p53 homologues based on their transactivator activity . The "no-hybrids" screen will be of utility in searches for p53 function-modulators of both cellular and viral origin.

J Chemother, 2001 Aug, 13(4), 377 - 83
In vitro activity of tea tree oil against Candida albicans mycelial conversion and other pathogenic fungi; D'Auria FD et al.; The antifungal activity of Melaleuca alternifolia Maiden (Myrtaceae) essential oil against yeasts (Candida spp., Schizosaccharomyces pombe, Debaryomyces hansenii) and dermatophytes (Microsporum spp . and Tricophyton spp.) is reported . We focused on the ability of tea tree oil to inhibit Candida albicans conversion from the yeast to the pathogenic mycelial form . Moreover we carried out broth microdilution test and contact tests to evaluate the killing time . M . alternifolia essential oil inhibited the conversion of C . albicans from yeast to the mycelial form at a concentration of 0.16% (v/v) . The minimum inhibitory concentrations (MICs) ranged from 0.12% to 0.50% (v/v) for yeasts and 0.12% to 1% (v/v) for dermatophytes; the cytocidal activity was generally expressed at the same concentration . These results, if considered along with the lipophilic nature of the oil which enables it to penetrate the skin, suggest it may be suitable for topical therapeutic use in the treatment of fungal mucosal and cutaneous infections.

J Biol Chem, 2001 Dec 21, 276(51), 47814 - 21 Epub 2001 Oct 01.
Chromodomain protein Swi6-mediated role of DNA polymerase alpha in establishment of silencing in fission Yeast; Ahmed S et al.; Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication . In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers . The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S . pombe . To investigate the connection between DNA replication and silencing in S . pombe, we analyzed several temperature-sensitive mutants of DNA polymerase alpha . We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci . This effect is independent of the checkpoint and replication defects of the mutant . Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer . The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase alpha in vivo and in vitro in wild type cells . However, it does not interact with the mutant pol alpha and is delocalized away from the silent mat loci in the mutant . Our results demonstrate a role of DNA polymerase alpha in the establishment of silencing . We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.

Mol Microbiol, 2001 Sep, 41(6), 1339 - 47
Failure to farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in the Schizosaccharomyces pombe farnesyltransferase mutant; Yang W et al.; Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology . The Schizosaccharomyces pombe mutant, cpp1-, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine . In this work, we report a novel phenotype of this mutant, enrichment of G0/G1 phase cells . This phenotype results mainly from the inability to farnesylate the Rheb G-protein, as normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb-CVIL) that can bypass farnesylation . In contrast, a farnesylation-defective mutant of SpRheb (SpRheb-SVIA) is incapable of restoring the normal cell cycle profile to the cpp1- mutant . Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle . This growth arrest phenotype of the sprheb- disruption can be complemented by the introduction of wild-type sprheb+ . The complementation is dependent on farnesylation, as the farnesylation-defective SpRheb-SVIA mutant is incapable of complementing the sprheb- disruption . Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb- disruption . Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb . Our findings suggest that protein farnesylation is important for cell cycle progression of S . pombe cells and that farnesylated SpRheb is critical in this process.

Biosci Biotechnol Biochem, 2001 Aug, 65(8), 1789 - 95
Production of pyridoxal phosphate by a mutant strain of Schizosaccharomyces pombe; Chumnantana R et al.; Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leul strain were examined . The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP . The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively . D-Glucose (1%, w/v) among other sugars gave the best productivity . Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP . Superoxide anion producers, menadione and plumbagin, and H202 increased the productivity of PLP . Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.

Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11985 - 90 Epub 2001 Sep 25.
Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells; Spahr H et al.; With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme . The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S . cerevisiae components encoded by nonessential genes . S . pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types . Presently, pmc2(+) and pmc3(+) have been shown to be nonessential genes . The data suggest that S . pombe and S . cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits . In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32 . Therefore, 8 of 10 essential genes conserved between S . pombe and S . cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells . Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated.

Mol Cell Biol, 2001 Oct, 21(20), 6870 - 81
Widespread use of TATA elements in the core promoters for RNA polymerases III, II, and I in fission yeast; Hamada M et al.; In addition to directing transcription initiation, core promoters integrate input from distal regulatory elements . Except for rare exceptions, it has been generally found that eukaryotic tRNA and rRNA genes do not contain TATA promoter elements and instead use protein-protein interactions to bring the TATA-binding protein (TBP), to the core promoter . Genomewide analysis revealed TATA elements in the core promoters of tRNA and 5S rRNA (Pol III), U1 to U5 snRNA (Pol II), and 37S rRNA (Pol I) genes in Schizosaccharomyces pombe . Using tRNA-dependent suppression and other in vivo assays, as well as in vitro transcription, we demonstrated an obligatory requirement for upstream TATA elements for tRNA and 5S rRNA expression in S . pombe . The Pol III initiation factor Brf is found in complexes with TFIIIC and Pol III in S . pombe, while TBP is not, consistent with independent recruitment of TBP by TATA . Template commitment assays are consistent with this and confirm that the mechanisms of transcription complex assembly and initiation by Pol III in S . pombe differ substantially from those in other model organisms . The results were extended to large-rRNA synthesis, as mutation of the TATA element in the Pol I promoter also abolishes rRNA expression in fission yeast . A survey of other organisms' genomes reveals that a substantial number of eukaryotes may use widespread TATAs for transcription . These results indicate the presence of TATA-unified transcription systems in contemporary eukaryotes and provide insight into the residual need for TBP by all three Pols in other eukaryotes despite a lack of TATA elements in their promoters.

Biochemistry (Mosc), 2001 Jul, 66(7), 753 - 62
Putative DNA-(amino)methyltransferases in eucaryotes; Shorning BY et al.; By computer analysis of the known data bases, we have established that the open reading frames (ORF) coding for proteins that possess high degree of homology with procaryotic DNA-(amino)methyltransferases are present in the genomes of Leishmania major, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens . Conservative motifs typical for bacterial DNA-(amino)methyltransferases are detected in the amino acid sequences of these putative proteins . The ORF of all putative eucaryotic DNA-(amino)methyltransferases found are encoded in nuclear DNA . In mitochondrial genomes including a few fully sequenced higher plant mtDNA, nucleotide sequences significantly homologous to genes of procaryotic DNA-(amino)methyltransferases are not found . Thus, ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclei of protozoa, yeasts, insects, nematodes, vertebrates, higher plants, and other eucaryotes . A special search for corresponding proteins and, in particular, adenine DNA-methyltransferases in these organisms and a study of their functions are quite promising.

Genetics, 2001 Sep, 159(1), 91 - 105
Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe; Tsutsui Y et al.; Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair . Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p . Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation . However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants . Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S . pombe proteins involved in recombinational DNA repair . Similar to S . cerevisiae Rad51p-Rad54p, S . pombe Rhp51p and Rhp54p were found to interact . Both putative Rad52 homologs in S . pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein . Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions . In contrast to the S . cerevisiae interacting pair Rad51p-Rad55p, S . pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein . In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S . pombe RPA . Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S . cerevisiae DSB repair are evolutionarily conserved.

Lung Cancer, 2001 Oct, 34(1), 47 - 52
Overexpression of Hrad17 gene in non-small cell lung cancers correlated with lymph node metastasis; Sasaki H et al.; We used palindromic PCR-driven cDNA differential display technique to identify and isolate a gene, human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, from colon cancer tissues . The loss of checkpoint control in mammalian cells results in genomic instability, leading to the amplification, rearrangement, or loss of chromosomes, events associated with tumor progression . We hypothesized that the Hrad17 may be expressed in non-small cell lung cancer (NSCLC) . We attempted to determine the influence of Hrad17 expression on clinicopathological features for patients with NSCLC who had undergone surgery . Expression of Hrad17 messenger RNA was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in 102 non-small cell lung carcinomas and adjacent histologically normal lung samples from patients for whom follow up data were available . Hrad17 transcripts were detected in 26 (25.5%) of the tumor samples, although some of the paired normal lung samples showed weak expression . There was no relationship between Hrad17 gene expression and age, gender or T-status . About 13 of 31 (41.9%) NSCLC patients with Hrad17 overexpressions were node positive, on the other hand, 13 of 76 (18.3%) cases without Hrad17 overexpressions were node positive . Thus the expression of Hrad17 mRNA correlated with lymph node metastasis (P=0.0231) from NSCLC . Hrad17 protein was highly expressed at the advancing margin of the tumor of lung cancer tissue but not within the normal lung tissue by immunohistochemistry . Thus the expression of Hrad17 might correlate with more advanced NSCLC.

Clin Cancer Res, 2001 Sep, 7(9), 2815 - 20
Overexpression of HRad17 mRNA in human breast cancer: correlation with lymph node metastasis; Kataoka A et al.; PURPOSE: A novel human gene, designated HRad17, was identified as the human homologue of the Rad17 of Schizosaccharomyces pombe and Rad24 of Saccharomyces cerevisiae . In yeast, these genes play a critical role in maintaining genomic stability . The aim of this study was to evaluate the expression of HRad17 in human breast cancer . EXPERIMENTAL DESIGN: We investigated HRad17 mRNA expression in 64 cases of human breast cancer by means of reverse-transcription-PCR, in situ hybridization, and immunohistochemistry . RESULTS: The HRad17 mRNA was overexpressed in 35 cases (54.7%) . Twenty-four (68.6%) of 35 cases with HRad17 overexpression in cancer tissues were node-positive, whereas only 8 (27.6%) of 29 cases without HRad17 overexpressions were node-positive . The expression of HRad17 mRNA correlated with both lymph node metastasis (P = 0.001) and high Ki67 labeling index (P = 0.006) . Although not significantly different, expression of HRad17 mRNA tended to correlate with tumor size (P = 0.06) and expression of mutant p53 protein (P = 0.10) . Furthermore, expression of HRad17 mRNA was an independent predictor of axillary lymph node metastasis as well as of lymphatic permeation by multivariate analysis (P < 0.0001) . CONCLUSIONS: Our study demonstrates that HRad17 might be related to the development of lymph node metastasis in human breast cancers . Although its function still remains unclear, the expression of HRad17 mRNA could open up a new window for the diagnostic staging and treatment of human breast cancers.

Genes Cells, 2001 Sep, 6(9), 789 - 802
The RGS domain-containing fission yeast protein, Rgs1p, regulates pheromone signalling and is required for mating; Pereira PS et al.; BACKGROUND: When nutritionally starved, the fission yeast Schizosaccharomyces pombe enters a cell differentiation process which leads to mating and meiosis . The Ste11 protein is a key regulator of this differentiation pathway, activating the transcription of mating and meiotic genes upon starvation . RESULTS: Here, we describe rgs1, a member of the Regulator of G-protein Signalling (RGS) family, as a novel Ste11 target gene . rgs1 expression requires both an Ste11-mediated nitrogen starvation signal and the pheromone-induced activation of the Byr2/Byr1/Spk1 MAPK pathway . We show that rgs1 deletion results in a sensitivity to pheromone and in a mating defect . Deltargs1 cells initiate the mating pathway normally, undergoing sexual agglutination and G1 arrest, while inducing pheromone-dependent transcription, but then fail to fuse with a mating partner while elongating abnormal conjugation tubes . Endogenous Rgs1 tagged with GFP localizes to the nucleus and cytoplasm, and this localization pattern is not altered during pheromone treatment . Importantly, Rgs1 function requires its C-terminal RGS domain, as well as a central DEP domain and a novel homology domain present in its N-terminal region (Fungal-DR domain) . CONCLUSIONS: These results demonstrate that rgs1 expression requires nutritional starvation and pheromone signalling . Rgs1 negatively regulates pheromone signalling during mating, acting in a negative feedback loop that is essential for the mating process.

J Biol Chem, 2001 Nov 16, 276(46), 43294 - 9 Epub 2001 Sep 11.
GERp95 belongs to a family of signal-transducing proteins and requires Hsp90 activity for stability and Golgi localization; Tahbaz N et al.; GERp95 (Golgi-endoplasmic reticulum protein 95 kDa) is part of a large family of highly conserved proteins found in all metazoans and the fission yeast Schizosaccharomyces pombe . Genetic studies suggest that homologs of GERp95 are components of signaling pathways that regulate cellular differentiation, development, and RNA interference . However, the precise molecular functions of these proteins remain unknown . Genetic analysis of GERp95 homologs has been complicated by the presence of multiple genes with overlapping functions in most organisms . Binding partners for members of this protein family have not been identified . The purpose of this study was to identify proteins that associate with GERp95 . Glutathione S-transferase-GERp95 fusions were expressed in transfected cells, and proteins that bound to GERp95 were co-purified using glutathione-agarose beads . The amino-terminal region of GERp95 was found to interact with the specialized chaperone Hsp90 and a number of its cognate binding proteins . Inhibition of Hsp90 activity with geldanamycin or radicicol resulted in rapid degradation of newly synthesized GERp95 . The membrane-associated pool of GERp95 was not bound to Hsp90, although activity of this chaperone was required for stable association of GERp95 with the Golgi in normal rat kidney cells . These results indicate that GERp95 engages an Hsp90 chaperone complex prior to association with intracellular membranes.

J Biol Chem, 2001 Nov 16, 276(46), 42857 - 62 Epub 2001 Sep 10.
Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7; Madril AC et al.; DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Poleta in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV) . Both Saccharomyces cerevisiae and human Poleta efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer . Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1(+) encoded protein is comprised of two domains, wherein the NH(2) terminus is highly homologous to Poleta, and the COOH terminus is highly homologous to the S . cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase . Here we characterize the DNA polymerase activity of S . pombe GST-Eso1 fusion protein and a truncated version containing only the Poleta domain . Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of approximately 10(-2) to 10(-3) . We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well . Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Poleta . The possible role of Ctf7 fusion with Poleta in the replication of Cohesin-bound DNA sequences is discussed.

J Biol Chem, 2001 Nov 9, 276(45), 41898 - 905 Epub 2001 Sep 10.
DNA damage-dependent and -independent phosphorylation of the hRad9 checkpoint protein; St Onge RP et al.; Cell cycle checkpoints are regulatory mechanisms that maintain genomic integrity by preventing cell cycle progression when genetic anomalies are present . The hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a checkpoint protein required for preventing the onset of mitosis if DNA damage is present or if DNA replication is incomplete . Genetic and biochemical analyses indicate that hRad9 is a component of the checkpoint response in humans and has possible roles in regulating the cell cycle, apoptosis, and DNA repair . Previous studies indicate that hRad9 is modified by phosphorylation, both in the absence of exogenous stress and in response to various genotoxins . In this study, we report the mapping of several sites of constitutive phosphorylation of hRad9 to (S/T)PX(R/P) sequences near the C terminus of the protein . We also demonstrate that a serine to alanine mutation at residue 272 abrogates an ionizing radiation (IR)-induced phosphorylation of hRad9 and further show that phosphorylation at (S/T)P sites is not a prerequisite for IR-induced phosphorylation of serine 272 . Finally, we report that hRad9 undergoes cell cycle-regulated hyper-phosphorylation in G(2)/M that is enhanced by IR but distinct from that on serine 272 . Unlike the IR-induced phosphorylation at serine 272, this event is dependent on serine 277 and threonine 292, two C-terminal (S/T)P sites in hRad9.

Yeast, 2001 Sep 15, 18(12), 1111 - 6
Analysis of 41 kb of the DNA sequence from the right arm of chromosome II of Schizosaccharomyces pombe; Sanchez M et al.; We report the complete sequence of cosmid c18A7 (41 046 bp insert), located on the right arm of chromosome II of the Schizosaccharomyces pombe genome . The sequence, which partially overlaps with cosmids SPBC4F6 and SPBC336, contains 16 open reading frames (ORFs) capable of coding for proteins of at least 100 amino acid residues in length (one partial) and one small nucleolar RNA (snoRNA) . Four known genes were found: swi10 (encoding a mating-type switching protein also involved in nucleotide excision repair); dim1 (encoding a dimethyladenosine transferase); arf1 (encoding ADP-ribosylation factor 1); and pol3 (cdc6) the partial fragment, encoding the 125 kDa catalytic subunit of the DNA polymerase type B . Six ORFs similar to known proteins were found . They include a transporter of the major facilitator superfamily class, a vacuolar sorting protein, an asparagine synthase, a nuclear protein, a reticulum oxidoreductin and a heat shock protein . Each protein product of the other six ORFs has conserved domains and can be assigned a molecular, but not a biological, function . The sequence has been submitted to the EMBL database under Accession No . AL080287 .

Nat Rev Mol Cell Biol, 2001 Sep, 2(9), 647 - 56
A journey into space; Hayles J et al.; The fission yeast, Schizosaccharomyces pombe, has been used as a model eukaryote to study processes such as the cell cycle and cell morphology . In this single-celled organism, growing in a straight line and maintaining the nucleus in the centre of the cell depend on intracellular positional information . Microtubules and microtubular transport are important for generating positional information within the fission yeast cell, and these molecular mechanisms are also probably relevant for generating positional information in other eukaryotic cells.

Mol Cell Biol, 2001 Oct, 21(19), 6681 - 94
Study of cyclin proteolysis in anaphase-promoting complex (APC) mutant cells reveals the requirement for APC function in the final steps of the fission yeast septation initiation network; Chang L et al.; Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes . An apparent exception to this relationship is found in Schizosaccharomyces pombe mutants with mutations of the anaphase-promoting complex (APC) . These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p . Although failing at nuclear division, these mutants septate and divide . Since septation requires Cdc2p inactivation in wild-type S . pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation . In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APC cut mutants due to Cdc13/cyclin B destruction . In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable . We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation . Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cut mutants and the normal reorganization of septation initiation network components during anaphase.

Virology, 2001 Sep 1, 287(2), 359 - 70
HIV-1 Vpr induces cell cycle G2 arrest in fission yeast (Schizosaccharomyces pombe) through a pathway involving regulatory and catalytic subunits of PP2A and acting on both Wee1 and Cdc25; Elder RT et al.; Viral protein R (Vpr) of human immunodeficiency virus type 1 induces G2 arrest in cells from distantly related eukaryotes including human and fission yeast through inhibitory phosphorylation of tyrosine 15 (Tyr15) on Cdc2 . Since the DNA damage and DNA replication checkpoints also induce G2 arrest through phosphorylation of Tyr15, it seemed possible that Vpr induces G2 arrest through the checkpoint pathways . However, Vpr does not use either the early or the late checkpoint genes that are required for G2 arrest in response to DNA damage or inhibition of DNA synthesis indicating that Vpr induces G2 arrest by an alternative pathway . It was found that protein phosphatase 2A (PP2A) plays an important role in the induction of G2 arrest by Vpr since mutations in genes coding for a regulatory or catalytic subunit of PP2A reduce Vpr-induced G2 arrest . Vpr was also found to upregulate PP2A, supporting a model in which Vpr activates the PP2A holoenzyme to induce G2 arrest . PP2A is known to interact genetically in fission yeast with the Wee1 kinase and Cdc25 phosphatase that act on Tyr15 of Cdc2 . Both Wee1 and Cdc25 play a role in Vpr-induced G2 arrest since a wee1 deletion reduces Vpr-induced G2 arrest and a direct in vivo assay shows that Vpr inhibits Cdc25 . Additional support for both Wee1 and Cdc25 playing a role in Vpr-induced G2 arrest comes from a genetic screen, which identified genes whose overexpression affects Vpr-induced G2 arrest . For this genetic screen, a strain was constructed in which cell killing by Vpr was nearly eliminated while the effect of Vpr on the cell cycle was clearly indicated by an increase in cell length . Overexpression of the wos2 gene, an inhibitor of Wee1, suppresses Vpr-induced G2 arrest while overexpression of rad25, an inhibitor of Cdc25, enhances Vpr-induced G2 arrest . These two genes may be part of the uncharacterized pathway for Vpr-induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25 .

Biophys Chem, 2001 Aug 30, 92(1-2), 1 - 15
A stochastic, molecular model of the fission yeast cell cycle: role of the nucleocytoplasmic ratio in cycle time regulation; Sveiczer A et al.; We propose a stochastic version of a recently published, deterministic model of the molecular mechanism regulating the mitotic cell cycle of fission yeast, Schizosaccharomyces pombe . Stochasticity is introduced in two ways: (i) by considering the known asymmetry of cell division, which produces daughter cells of slightly different sizes; and (ii) by assuming that the nuclear volumes of the two newborn cells may also differ . In this model, the accumulation of cyclins in the nucleus is proportional to the ratio of cytoplasmic to nuclear volumes . We have simulated the cell-cycle statistics of populations of wild-type cells and of wee1(-) mutant cells . Our results are consistent with well known experimental observations.

Appl Environ Microbiol, 2001 Sep, 67(9), 4144 - 51
Characterization of Schizosaccharomyces pombe malate permease by expression in Saccharomyces cerevisiae; Camarasa C et al.; In Saccharomyces cerevisiae, L-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid . Expression in S . cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased L-malic acid uptake in this yeast . In this strain, at pH 3.5 (encountered in industrial processes), L-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: Vmax = 8.7 nmol/mg/min; Km = 1.6 mM) and some simple diffusion of the undissociated L-malic acid (Kd = 0.057 min(-1)) . As total L-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain . L-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the DeltapH component of the proton motive force . Dicarboxylic acids with stearic occupation closely related to L-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited L-malic acid uptake, suggesting that these compounds use the same carrier . We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation . In S . pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed . Surprisingly, no proton fluxes were observed during Mae1p-mediated L-malic acid import in S . cerevisiae, and intracellular pH remained constant . This suggests that, in S . cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.

Mol Genet Genomics, 2001 Jul, 265(5), 837 - 50
The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants; Ui A et al.; The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome . Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles . SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ . A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region) . In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end . These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe . In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants . In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes . Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes . Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.

Mol Genet Genomics, 2001 Aug, 265(6), 993 - 1003
A homologue of the Rad18 postreplication repair gene is required for DNA damage responses throughout the fission yeast cell cycle; Verkade HM et al.; Cells activate DNA repair pathways and cell cycle checkpoints when they suffer damage to their genome . They also activate tolerance pathways that facilitate survival . In Escherichia coli, a mechanism known as postreplication repair (PRR) is used to bypass lesions that would otherwise present a physical block to DNA polymerase . PRR has also been proposed to occur in eukaryotic cells, although the partitioning of DNA synthesis to a discrete S-phase would suggest that it is only operative within a defined period of the cell cycle . Eukaryotic PRR has been most extensively studied in the budding yeast Saccharomyces cerevisiae . Two important genes for components of this repair pathway are RAD6, which encodes an ubiquitin-conjugating enzyme, and RAD18, which encodes a RING-finger protein and forms a heterodimer with Rad6p . Rad18p can also bind to DNA . We report here the identification of the Schizosaccharomyces pombe homologue of RAD18, which we have denoted rhp18 . rhp18 mutants are hypersensitive to DNA-damaging agents, but show this hypersensitivity throughout the cell cycle . rhp18 mutants are characterised by a longer than usual DNA damage checkpoint arrest that is required for their residual viability following irradiation . Genetic analyses show that rhp18 controls a unique DNA damage repair/tolerance pathway that extends beyond the requirement to tolerate damage during S-phase, suggesting a broader definition of the function of this eukaryotic PRR protein.

Mol Genet Genomics, 2001 Aug, 265(6), 1031 - 8
Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system; Thomason LC et al.; The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe . A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene . High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed . Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site . Sequence analysis showed that the attBxattP recombination was precise . In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S . pombe cells bearing an attP duplication . The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement . The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines . The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily . Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

Tsitologiia, 2001, 43(5), 491 - 500
{Internal symmetry in nucleotide sequences of genes encoding the dolichol cycle enzymes}; Shpakov AO; In genes alg5, alg8 and swp1 of Saccharomyces cerevisiae, gpt of Schizosaccharomyces pombe and human gene alg6, encoding the dolichol cycle enzymes, a mirror type internal symmetry was found . The symmetry was detected in both complete nucleotide sequences and sequences of the first, second and third nucleotide bases of codons . In the encoding gene regions the density of single- and double-point centres of the internal symmetry for sequences of the second bases was higher in comparison with the sequences of the first and third bases of codons, whereas in the noncoding regions degrees of symmetry of the first, second and third bases sequences did not differ significantly . A clear positive correlation was revealed in the internal symmetry distribution in the second base sequences of codons in genes, on the one hand, and in the gene encoded amino acid sequences, on the other hand . The maximum internal symmetry of gene segments encoding the functionally important regions of proteins was found at the level of the second base sequences . The obtained results corroborate a hypothesis about the determining role of the second bases of codons in encoding amino acid residues . The investigation of internal symmetry in nucleotide sequences has first shown the existence of internal symmetry at the level of gene primary structure.

Curr Biol, 2001 Aug 7, 11(15), 1192 - 6
Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells; d'Adda di Fagagna F et al.; DNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man . In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions . Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents . We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages . By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components . We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells . This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ.

Curr Biol, 2001 Jun 5, 11(11), 836 - 45
Role of bud6p and tea1p in the interaction between actin and microtubules for the establishment of cell polarity in fission yeast; Glynn JM et al.; BACKGROUND: In many cell types, microtubules are thought to direct the spatial distribution of F-actin in cell polarity . Schizosaccharomyces pombe cells exhibit a regulated program of polarized cell growth: after cell division, they grow first in a monopolar manner at the old end, and in G2 phase, initiate growth at the previous cell division site (the new end) . The role of microtubule ends in cell polarity is highlighted by the finding that the cell polarity factor, tea1p, is present on microtubule plus ends and cell tips {1} . RESULTS: Here, we characterize S . pombe bud6p/fat1p, a homolog of S . cerevisiae Bud6/Aip3 . bud6Delta mutant cells have a specific defect in the efficient initiation of growth at the new end and like tea1Delta cells, form T-shaped cells in a cdc11 background . Bud6-GFP localizes to both cell tips and the cytokinesis ring . Maintenance of cell tip localization is dependent upon actin but not microtubules . Bud6-GFP localization is tea1p dependent, and tea1p localization is not bud6p dependent . tea1Delta and bud6Delta cells generally grow in a monopolar manner but exhibit different growth patterns . tea1(Delta)bud6Delta mutants resemble tea1Delta mutants . Tea1p and bud6p coimmunoprecipitate and comigrate in large complexes . CONCLUSIONS: Our studies show that tea1p (a microtubule end-associated factor) and bud6p (an actin-associated factor) function in a common pathway, with bud6p downstream of tea1p . To our knowledge, bud6p is the first protein shown to interact physically with tea1p . These studies delineate a pathway for how microtubule plus ends function to polarize the actin cytoskeleton through actin-associated polarity factors.

Mol Biol Cell, 2001 Aug, 12(8), 2469 - 81
Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis; Hendrickson TW et al.; gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast . For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe . To understand the structural/functional domains of gamma-tubulin, we performed a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) and studied phenotypes of each mutant allele in S . pombe . Our screen, both in the presence and absence of the endogenous S . pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations . Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice . As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle . Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis . These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.

Genetics, 2001 Aug, 158(4), 1413 - 29
Correct regulation of the septation initiation network in Schizosaccharomyces pombe requires the activities of par1 and par2; Jiang W et al.; In Schizosaccharomyces pombe, the initiation of cytokinesis is regulated by a septation initiation network (SIN) . We previously reported that deletion of par1 and par2, two S . pombe genes encoding B' regulatory subunits of protein phosphatase 2A, causes a multiseptation phenotype, very similar to that seen in hyperactive SIN mutants . In this study, we examined the genetic interactions between par deletions and mutations in the genes encoding components of SIN and found that deletion of par1 and par2 suppressed the morphological and viability defects caused by overproduction of Byr4p and rescued a loss-of-function allele of spg1 . However, par deletions could not suppress any mutations in genes downstream of spg1 in the SIN pathway . We showed further that, in suppressing the lethality of a spg1 loss-of-function allele, the correct localization of Cdc7p to the spindle pole body (SPB), which is normally lost in spg1 mutant cells, was restored . The fact that par mutant cells themselves exhibited a symmetric localization of Cdc7p to SPBs indicated a hyperactivity of SIN in such cells . On the basis of our epistasis analyses and cytological studies, we concluded that par genes normally negatively regulate SIN at or upstream of cdc7, ensuring that multiple rounds of septation do not occur.

Genetics, 2001 Aug, 158(4), 1397 - 411
Coordination between fission yeast glucan formation and growth requires a sphingolipase activity; Feoktistova A et al.; css1 mutants display a novel defect in Schizosaccharomyces pombe cell wall formation . The mutant cells are temperature-sensitive and accumulate large deposits of material that stain with calcofluor and aniline blue in their periplasmic space . Biochemical analyses of this material indicate that it consists of alpha- and beta-glucans in the same ratio as found in cell walls of wild-type S . pombe . Strikingly, the glucan deposits in css1 mutant cells do not affect their overall morphology . The cells remain rod shaped, and the thickness of their walls is unaltered . Css1p is an essential protein related to mammalian neutral sphingomyelinase and is responsible for the inositolphosphosphingolipid-phospholipase C activity observed in S . pombe membranes . Furthermore, expression of css1(+) can compensate for loss of ISC1, the enzyme responsible for this activity in Saccharomyces cerevisiae membranes . Css1p localizes to the entire plasma membrane and secretory pathway; a C-terminal fragment of Css1p, predicted to encode a single membrane-spanning segment, is sufficient to direct membrane localization of the heterologous protein, GFP . Our results predict the existence of an enzyme(s) or process(es) essential for the coordination of S . pombe cell wall formation and division that is, in turn, regulated by a sphingolipid metabolite.

Biochim Biophys Acta, 2001 Aug 30, 1520(2), 179 - 85
Characterization and regulation of glutathione S-transferase gene from Schizosaccharomyces pombe; Kim HG et al.; A glutathione S-transferase (GST) gene has been cloned from Schizosaccharomyces pombe for the first time . The nucleotide sequence determined was found to contain 2030 base pairs including an open reading frame of 229 amino acids that would encode a protein of a molecular mass of 27017 Da . The cloned GST gene was expressed and was found to function in S . pombe, Saccharomyces cerevisiae, and Escherichia coli . The plasmid pGT207 encoding the S . pombe GST gene appeared to be able to accelerate the growth of a wild type S . pombe culture . In a culture of S . pombe containing plasmid pGT207, the growth was inhibited less by mercuric chloride than in a culture with vector alone . The 1088 bp region upstream from the GST gene as well as the region encoding the N-terminal 14 amino acids was transferred into the promoterless beta-galactosidase gene of plasmid YEp357R to yield the fusion plasmid pYSH2000 . beta-Galactosidase synthesis was induced by cadmium chloride, mercuric chloride, hydrogen peroxide, and menadione . It was also induced by high temperature . These results suggest that the cloned S . pombe GST gene is involved in the oxidative stress response.

FEBS Lett, 2001 Aug 17, 503(2-3), 131 - 4
mik1(+) G1-S transcription regulates mitotic entry in fission yeast; Ng SS et al.; In the fission yeast Schizosaccharomyces pombe Mik1p, in combination with Wee1p, is an important inhibitor of mitosis through direct phosphorylation of Cdc2p . Here we present the observation that mik1(+) is transcribed during G1- and S-phase in normally dividing cells . mik1(+) transcription is regulated by the MCB-DSC1 system, which controls expression of other genes at the G1-S interval . mik1(+) is shown to be an important target of MCB-DSC1 as it is epistatic for the mitotic delay phenotype displayed in cdc10-C4 cells, which are mutated in a component of DSC1 . The mitotic delay in cdc10-C4 cells is bypassed by cdc2-1w, suggesting that mik1(+) acts directly on cdc2(+), with no checkpoint function involved . Thus, mik1(+) represents a new type of MCB-DSC1 regulated gene in fission yeast, whose gene product is exclusively expressed during G1- and S-phase to prevent premature mitosis during this cell cycle stage.

Genes Dev, 2001 Aug 15, 15(16), 2060 - 8
A DNA replication-arrest site RTS1 regulates imprinting by determining the direction of replication at mat1 in S . pombe; Dalgaard JZ et al.; Mating-type switching in Schizosaccharomyces pombe involves a strand-specific, alkali-labile imprint at the mat1 (mating-type) locus . The imprint is synthesized during replication in a swi1, swi3, and polymerase alpha (swi7) dependent manner and is dependent on mat1 being replicated in a specific direction . Here we show that the direction of replication at mat1 is controlled by a cis-acting polar terminator of replication (RTS1) . Two-dimensional gel analysis of replication intermediates reveals that RTS1 only terminates replication forks moving in the centromere-distal direction . A genetic analysis shows that RTS1 optimizes the imprinting process . Transposing the RTS1 element to the distal side of mat1 abolishes imprinting of the native mat1 allele but restores imprinting of an otherwise unimprinted inverted mat1 allele . These data provide conclusive evidence for the "direction of replication model" that explains the asymmetrical switching pattern of S . pombe, and identify a DNA replication-arrest element implicated in a developmental process . Such elements could play a more general role during development and differentiation in higher eukaryotes by regulating the direction of DNA replication at key loci.

Biochimie, 2001 Jun, 83(6), 481 - 6
New subtilisin-like collagenase from leaves of common plantain; Bogacheva AM et al.; A new subtilisin-like proteinase hydrolyzing chromogenic peptide substrate Glp-Ala-Ala-Leu-p-nitroanilide optimally at pH 8.1 was found in common plantain leaves . The protease named plantagolisin was isolated by ammonium sulfate precipitation of the leaves' extract followed by affinity chromatography on bacitracin-Sepharose and ion-exchange chromatography on Mono Q in FPLC regime . Its molecular mass is 19000 Da and pI 5.0 . pH-stability range is 7-10 in the presence of 2 mM Ca(2+), temperature optimum is 40 degrees C . The substrate specificity of subtilase towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases: proteinase from leaves of the sunflower and taraxalisin . Besides, the proteinase is able to hydrolyze substrates with Pro in P(1) position . The enzyme hydrolyzes collagen . alpha and beta chains are hydrolyzed simultaneously in parallel; there are only low-molecular-mass hydrolysis products in the sample after 2 h of incubation . Pure serine proteinase was inactivated by specific serine proteinases inhibitors: diisopropylfluorophosphate, phenylmethylsulfonyl fluoride and Hg(2+) . The plantagolisin N-terminal sequence ESNSEQETQTESGPGTAFL-, traced for 19 residues, revealed 37% homology with that of subtilisin from yeast Schizosaccharomyces pombe.

Pharmacogenetics, 2001 Aug, 11(6), 511 - 20
Functional characterization of nucleotide polymorphisms in the coding region of N-acetyltransferase 1; Fretland AJ et al.; N-acetyltransferase 1 (NAT1) catalyses the activation and/or deactivation of aromatic and heterocyclic amine carcinogens . A genetic polymorphism in NAT1 is associated with an increased risk of various cancers and drug toxicities, but epidemiological investigations are severely compromised by a poor understanding of the relationship between NAT1 genotype and phenotype . Human reference NAT1*4 and 12 known human NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe) . Large reductions in N- and O-acetyltransferase catalytic activities were observed for recombinant NAT1 allozymes encoded by NAT1*14B, NAT1*15, NAT1*17, NAT1*19 and NAT1*22 . Each of these alleles exhibited NAT1 protein expression levels below the limit of detection as measured by Western blot . No differences between high and low activity NAT1 alleles were observed in relative mRNA expression or relative transformation efficiency . The recombinant NAT1 17 and NAT1 22 allozymes showed reduced intrinsic stability when compared with NAT1 4 . 2-Amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) N-acetylation was not catalysed by any of the NAT1 allozymes . Large differences in the metabolic activation via O-acetylation of 2-hydroxyamino-1-methyl-6-phenylimidazo{4,5-b}pyridine (N-hydroxy-PhIP) were noted for NAT1 allelic variants . The results of these studies suggest an important role for the NAT1 genetic polymorphism in metabolism of aromatic and heterocyclic amine carcinogens . Furthermore, these results suggest that low NAT1 phenotype results from NAT1 allelic variants that encode reduced expression of NAT1 and/or less-stable NAT1 protein.

Cell Biol Toxicol, 2001, 17(1), 51 - 63
Interaction of benzo{c}phenanthridine and protoberberine alkaloids with animal and yeast cells; Slaninova I et al.; We compared the effects of four quaternary benzo{c}phenanthridine alkaloids--chelerythrine, chelilutine, sanguinarine, and sanguilutine--and two quaternary protoberberine alkaloids-berberine and coptisine--on the human cell line HeLa (cervix carcinoma cells) and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces japonicus var . versatilis . The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells . Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied . In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 microg/ml . The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 microg/ml . IC50 values for individual alkaloids were: sanguinarine IC50 = 0.8 microg/ml, sanguilutine IC50 = 8.3 microg/ml, chelerythrine IC50 = 6.2 microg/ml, chelilutine IC50 = 5.2 microg/ml, coptisine IC50 = 2.6 microg/ml and berberine IC50 > 10.0 microg/ml . In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 microg/ml . The other alkaloids showed a similar effect but at higher concentrations (5-50 microg/ml) . The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity.

Appl Microbiol Biotechnol, 2001 Jul, 56(1-2), 165 - 72
Expression, processing and high level secretion of a virus toxin in fission yeast; Heintel T et al.; The virally encoded K28 toxin of Saccharomyces cerevisiae kills sensitive yeast cells in a multi-step receptor-mediated fashion by cell cycle arrest and inhibition of DNA synthesis . In vivo, the toxin is translated as a 38 kDa preprotoxin (pptox) which is enzymatically processed to the biologically active alpha/beta heterodimer during passage through the yeast secretory pathway . Here, we demonstrate that Schizosaccharomyces pombe, a yeast from which no natural toxin-secreting killer strains are known, is perfectly capable of expressing a killer phenotype . Episomal as well as integrating K28 pptox gene cassettes were constructed that allowed a tightly thiamine-regulated killer phenotype expression under transcriptional control of the Sch . pombe nmt1 promotor . Northern analysis of the toxin-coding transcript as well as Western analysis of the secreted toxin indicated that fission yeast is capable of expressing a correctly processed and fully functional virus toxin . Moreover, toxin secretion in recombinant Sch . pombe was at least ten-fold higher than in any natural and/or recombinant Sac . cerevisiae killer strain, indicating that pptox-derived vectors might be attractive in the fast growing field of heterologous protein expression and secretion in yeast.

Plant J, 2001 Jun, 26(6), 637 - 49
The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S . pombe cdc7p and may be involved in cell division; Jouannic S et al.; We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively . This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans . The B . napus protein is able to partially complement a cdc7 loss of function mutation in S . pombe . RT-PCR and in situ hybridisation revealed that the A . thaliana and B . napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells . Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases . Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S . pombe.

Mol Cell Biol, 2001 Sep, 21(17), 5767 - 77
Control of DNA rereplication via Cdc2 phosphorylation sites in the origin recognition complex; Vas A et al.; Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe . Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC) . Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle . Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation . Phosphorylation of Orp2 appears first in S phase and becomes maximal in G(2) and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication . A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated . Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis . Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

Protein Expr Purif, 2001 Aug, 22(3), 479 - 83
Expression of a VEGF-like protein from Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe; Kettner K et al.; We report on the expression of a VEGF-like protein encoded by Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . We show that a lysine residue at amino acid position 2 (K2) is an important determinant for the stability of this protein in S . cerevisiae . Replacement of K2 by an arginine results in stabilization of the protein . This observation suggests that this lysine may be a target for ubiquitinylation, which is a prerequisite for proteasome-mediated protein degradation . Interestingly, in S . pombe the lysine (K2) has no influence on the stability of the protein . This result indicates that the two yeast species exhibit significant differences in their protein degradation pathways .

Yeast, 2001 Aug, 18(11), 1015 - 21
High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation; Suga M et al.; A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed . Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells . Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment . A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30 degrees C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure . It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency . The optimal cell concentration and amount of DNA during the electric pulse were 1x10(9) cells/ml and 10 ng, respectively . The maximum transformation efficiency, 1.2x10(7) transformants/microg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms . Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol .

Arch Microbiol, 2001 Jul, 176(1-2), 106 - 13
Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase; De Lucas JR et al.; The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described . The system is based on the sC gene encoding ATP sulfurylase . Several A . fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance . The coding region plus upstream and downstream regulatory sequences of the A . fumigatus sC gene were cloned by inverse PCR and then sequenced . Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene . The A . fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum . By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues . A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM . Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system . Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus . This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A . fumigatus sC locus, facilitating studies of gene regulation and function.

J Biol Chem, 2001 Oct 5, 276(40), 37133 - 40 Epub 2001 Jul 30.
Plasmodium falciparum possesses a classical glutaredoxin and a second, glutaredoxin-like protein with a PICOT homology domain; Rahlfs S et al.; The genes coding for two different proteins with homologies to glutaredoxins have been identified in the genome of the malarial parasite Plasmodium falciparum . Both genes were amplified from a gametocytic cDNA and overexpressed in Escherichia coli . The smaller protein (named PfGrx-1) with 12.4 kDa in size exhibits the typical glutaredoxin active site motif "CPYC," shows glutathione-dependent glutaredoxin activity in the beta-hydroxyethyl disulfide (HEDS) assay, and reduces Trypanosoma brucei ribonucleotide reductase . Glutathione:HEDS transhydrogenase activity (approximately 60 milliunits/mg of protein) was clearly detectable in trophozoite extracts from eight different P . falciparum strains and did not differ between chloroquine-resistant and -sensitive parasites . Five different antimalarial drugs at 100 microm did not significantly influence isolated PfGrx-1 activity . In contrast, the second protein (deduced mass 19.9 kDa) with homology to glutaredoxins (31% identity to Schizosaccharomyces pombe in a 140-amino acid overlap) was not active in the HEDS assay; however, its general dithiol reducing activity was demonstrated in the insulin assay in the presence of dithiothreitol . Interestingly, the sequence contains a PICOT (for protein kinase C-interacting cousin of thioredoxin) homology domain, which might suggest regulatory functions of the protein . We named this protein PfGLP-1, for P . falciparum 1-Cys-glutaredoxin-like protein-1 . In contrast to glutaredoxins, PfGLP-1 could not be reduced by glutathione . This is the first report on glutaredoxin-like proteins in the family of Plasmodia.

Nat Struct Biol, 2001 Aug, 8(8), 701 - 4
Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones; Thaw P et al.; The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive . We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family . We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs) . Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF) . We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family . This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.

Biochim Biophys Acta, 2001 Jun 29, 1532(3), 223 - 33
Very-long-chain fatty acid-containing phospholipids accumulate in fatty acid synthase temperature-sensitive mutant strains of the fission yeast Schizosaccharomyces pombe fas2/lsd1; Yokoyama K et al.; Fission yeast lsd1 strains show aberrant mitosis with a lsd phenotype, large and small daughter nuclei, and a very thick septum, the phenotypic expression being temperature-sensitive . The lsd1(+) gene is the homologue of the budding yeast FAS2 gene encoding the fatty acid synthase alpha-subunit as reported previously (S . Saitoh, K . Takahashi, K . Nabeshima, Y . Yamashita, Y . Nakaseko, A . Hirata, M . Yanagida, J . Cell Biol . 134 (1996) 949--961) . In this paper, lsd1 is considered to represent fas2 . Here, three fas2 strains were investigated and found to have missense point mutations at different sites in the gene encoding the alpha-subunit of fatty acid synthase . The mutation affected only slightly the enzymatic activities monitored in vitro . Unexpectedly, abnormal phospholipids, phosphatidylcholine and phosphatidylethanolamine, both of which contain a very-long-chain fatty acyl residue (1-melissoyl-2-oleolyl-sn-glycero-3-phosphocholine and 1-melissoyl-2-oleolyl-sn-glycero-3-phosphoethanolamine), accumulated in fas2 strains in a temperature-sensitive manner . Rescue of the fas2 strains by addition of palmitate to the medium at restrictive temperature was accompanied by disappearance of these abnormal phospholipids . Accumulation of these lipids in membranes may cause alteration of various cellular functions.

J Biol Chem, 2001 Sep 28, 276(39), 36116 - 24 Epub 2001 Jul 19.
An essential function of Saccharomyces cerevisiae RNA triphosphatase Cet1 is to stabilize RNA guanylyltransferase Ceg1 against thermal inactivation; Hausmann S et al.; Saccharomyces cerevisiae RNA triphosphatase (Cet1) and RNA guanylyltransferase (Ceg1) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex . Here we show that the guanylyltransferase activity of Ceg1 is highly thermolabile in vitro (98% loss of activity after treatment for 10 min at 35 degrees C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures . Candida albicans guanylyltransferase Cgt1 is also thermolabile and is stabilized by binding to Cet1(232-265) . In contrast, Schizosaccharomyces pombe and mammalian guanylyltransferases are intrinsically thermostable in vitro and they are unaffected by Cet1(232-265) . We show that the requirement for the Ceg1-binding domain of Cet1 for yeast cell growth can be circumvented by overexpression in high gene dosage of a catalytically active mutant lacking the Ceg1-binding site (Cet1(269-549)) provided that Ceg1 is also overexpressed . However, such cells are unable to grow at 37 degrees C . In contrast, cells overexpressing Cet1(269-549) in single copy grow at all temperatures if they express either the S . pombe or mammalian guanylyltransferase in lieu of Ceg1 . Thus, the cell growth phenotype correlates with the inherent thermal stability of the guanylyltransferase . We propose that an essential function of the Cet1-Ceg1 interaction is to stabilize Ceg1 guanylyltransferase activity rather than to allosterically regulate its activity . We used protein-affinity chromatography to identify the COOH-terminal segment of Ceg1 (from amino acids 245-459) as an autonomous Cet1-binding domain . Genetic experiments implicate two peptide segments, (287)KPVSLYVW(295) and (337)WQNLKNLEQPLN(348), as likely constituents of the Cet1-binding site on Ceg1.

J Biol Chem, 2001 Oct 26, 276(43), 40190 - 201 Epub 2001 Jul 18.
Isolation of a novel gene from Schizosaccharomyces pombe: stm1+ encoding a seven-transmembrane loop protein that may couple with the heterotrimeric Galpha 2 protein, Gpa2; Chung KS et al.; A putative seven transmembrane protein gene, stm1(+), which is required for proper recognition of nitrogen starvation signals, was isolated as a multicopy suppressor of a ras1 synthetic lethal mutant in Schizosaccharomyces pombe . Under nitrogen-deficient conditions, transcription of the stm1 gene was induced; deletion of stm1 was associated with early entry into G(1) arrest . Under nutritionally sufficient conditions, overexpression of Stm1 inhibited vegetative cell growth, resulted in decreased intracellular cAMP levels, increased the expression of the meiosis-specific genes ste11, mei2, and mam2, and facilitated sexual development in homothallic cells . However inhibition of vegetative cell growth and reduction of cAMP levels were not observed in a deletion mutant of the heterotrimeric G protein Galpha2 gene, gpa2, that is responsible for regulating intracellular cAMP levels, a key factor in determining the sexual development in S . pombe . Stm1 protein was shown to interact with Gpa2 through its C-terminal transmembrane domains 5-7 . Mutation at Lys(199) in the C-terminal domain (stm1(K199A)) abolished the Stm1 overexpression effect on lowering cAMP levels . Induction of ste11, a meiosis-specific gene transcription factor, by Stm1 overexpression was enhanced in gpa2-deleted cells but was absent in a deletion mutant of sty1, a key protein kinase that links mitotic control with environmental signals and induces stress-responsive genes . Moreover, deletion of both stm1 and ras1 caused delayed entry into G(1) arrest in S . pombe when the cells were grown in a nitrogen-deficient medium . Thus we consider that the stm1 gene can function through Gpa2-dependent and/or -independent pathways and may play a role in providing the prerequisite state for entering the pheromone-dependent differentiation cycle in which heterotrimeric Galpha1 protein, Gpa1, and Ras1 play major roles . Stm1 could function as a sentinel molecule sensing the nutritional state of the cells, stopping the proliferative cell cycle, and preparing the cell to enter meiosis under nutritionally deficient conditions.

Nature, 2001 Jul 19, 412(6844), 352 - 5
A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast; Gachet Y et al.; The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid . The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment . Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented . This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8395 - 402
Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe; Davis L et al.; In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors . This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms . Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes . Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.

Mol Genet Genomics, 2001 Jun, 265(4), 673 - 82
The 5' terminal region of the Schizosaccharomyces pombe mes1 mRNA is crucial for its meiosis-specific splicing; Shimoseki M et al.; The mes1+ gene of Schizosaccharomyces pombe is required for the second meiotic division . The single 75-nt intron in mes1 is spliced out only in meiotic cells . Here we report a cis-acting element which is responsible for meiosis-specific splicing . Both 5' and 3' splice sites of the mes1 intron deviate from the consensus sequence . Point mutations which altered these sites so that they conformed to the consensus, however, did not affect the splicing pattern of mes1 . Neither replacement of the mes1 intron with the constitutively spliced intron of the nda3 gene, nor replacement of the 3' exon with E . coli lacZ changed the splicing pattern . In contrast, deletion of the 5' terminal 125 nt from the 5' exon derepressed splicing in vegetative cells, implying that this 5' terminal sequence, named SRE (mes1 splicing repression element), inhibits splicing of the downstream intron . A potential stem-loop structure in the SRE is predicted . Disruption of this stem structure by mutation abolished the repression of mes1 splicing in vegetative cells . Overexpression of the SRE sequence on a multicopy plasmid also relieved the repression of splicing of the authentic mes1 transcripts . These results suggest that as yet unknown trans-acting factors inhibit splicing of the mes1 transcript in vegetative cells by interacting with the cis-element SRE.

J Biol Chem, 2001 Sep 14, 276(37), 34948 - 57 Epub 2001 Jul 16.
Saccharomyces cerevisiae protein Pci8p and human protein eIF3e/Int-6 interact with the eIF3 core complex by binding to cognate eIF3b subunits; Shalev A et al.; Mammalian, plant, and Schizosaccharomyces pombe eukaryotic initiation factor-3 (eIF3) contains a protein homologous to the product of int-6 (eIF3e), a frequent integration site of mouse mammary tumor viruses . By contrast, Saccharomyces cerevisiae does not encode a protein closely related to eIF3e/Int-6 . Here, we characterize a novel S . cerevisiae protein (Pci8p, Yil071cp) that contains a PCI (proteasome-COP9 signalosome-eIF3) domain conserved in eIF3e/Int-6 . We show that both Pci8p and human eIF3e/Int-6 expressed in budding yeast interact with the yeast eIF3 complex in vivo and in vitro by binding to a discrete segment of its eIF3b subunit Prt1p and that human eIF3e/Int-6 interacts with the human eIF3b segment homologous to the Pci8p-binding site of yeast Prt1p . These results refine our understanding of subunit interactions in the eIF3 complex and suggest structural similarity between human eIF3e/Int-6 and yeast Pci8p . However, deletion of PCI8 had no discernible effect on cell growth or translation initiation as judged by polysome analysis, suggesting that Pci8p is not required for the essential function of eIF3 in translation initiation . Motivated by the involvement of Int-6 in transcriptional control, we investigated the effects of deleting PCI8 on the total mRNA expression profile by oligonucleotide microarray analysis and found reduced mRNA levels for a subset of heat shock proteins in the pci8Delta mutant . We discuss possible dual functions of Pci8p and Int-6 in transcriptional and translational control.

Curr Genet, 2001 Jun, 39(4), 222 - 30
Factors involved in the regulation of the Schizosaccharomyces pombe malic enzyme gene; Groenewald M et al.; Transcription of the Schizosaccharomyces pombe malic enzyme gene, mae2, is induced when cells are grown on high glucose concentrations or under nonaerated conditions . Two cis-acting elements in the mae2 promoter, upstream activator sequences UAS1 and UAS2, are required for basal expression, whilst three negative-acting, upstream repressor sequences are involved in general derepression of mae2 . Both the Pka1 and Sty1 signal transduction pathways are involved in the induced expression of mae2 under fermentative conditions . Expression of mae2 seems to be regulated in response to the carbon source, lack of oxygen and osmotic stress conditions, probably to assist in maintaining the intracellular redox balance.

Curr Genet, 2001 Jun, 39(4), 210 - 21
Functional analysis of RNA polymerase II Rpb3 mutants of the fission yeast Schizosaccharomyces pombe; Mitobe J et al.; The RNA polymerase II (Pol II) of Schizosaccharomyces pombe is composed of 12 subunits . Subunit Rpb3 has sequence homology with the N-terminal domain of the prokaryotic alpha subunit, which plays a key role in RNA polymerase assembly . Together with the Rpb2 (the beta homologue) and Rpb11 (the second alpha homologue) subunits, Rpb3 constitutes a core subassembly (Rpb2-Rpb3-Rpb11) which corresponds to the the alpha2beta assembly intermediate of prokaryotic RNA polymerase . For the functional mapping of Rpb3, we made a collection of 12 heat-sensitive (Ts) or cold-sensitive (Cs) S . pombe mutants, each carrying a single mutation in one of the four conserved regions of Rpb3 . The altered functions of six representative Pol II mutants containing the mutant Rpb3 were analyzed in vitro using an improved version of the GAL4-VP16 activator-dependent transcription system catalyzed by S . pombe cell extracts . The transcription activity by the extracts from Rpb3 mutants decreased to varying extents after heat treatment; but the extracts from Rpb3 mutants which had mutations in the eukaryote-specific conserved regions B and C regained their activity by the addition of GAL4-VP16, to a larger extent than those from the region A and D mutants . We propose that both terminal regions (A and D) play important roles in RNA polymerase assembly, while the central portion (regions B and C) is involved in activated transcription.

Curr Genet, 2001 Jun, 39(4), 205 - 9
The Schizosaccharomyces pombe Cdc42p GTPase signals through Pak2p and the Mkh1p-Pek1p-Spm1p MAP kinase pathway; Merla A et al.; The Cdc42p GTPase is involved in many aspects of growth and cell-cycle regulation, including actin cytoskeletal rearrangements and activation of signal transduction pathways . To further investigate these functions, genetic interactions were examined between Schizosaccharomyces pombe Cdc42p, its effectors Pak1p and Pak2p, and the Mkh1p-Pek1p-Spm1p signal transduction pathway, which functions in cytokinesis and cell division . Expression of a truncated version of Pak2p lacking its N-terminal autoinhibitory domain led to a growth defect that was suppressed by deltamkh1 and deltaspm1 null mutations and an elongated cell phenotype indicative of a cell division defect that was suppressed by the deltamkh1 mutation . In addition, expression of the constitutively activated cdc42G12V mutant allele led to a growth defect that was rescued by the deltapak2 and deltamkh1 mutations . The deltapak2 mutation did not suppress the growth defect conferred by plasmid expression of Mkh1p, suggesting that Pak2p functions upstream of Mkh1p in this pathway . A two-hybrid protein interaction was observed between Pak2p and Mkh1p, but not between Pak1p and Mkh1p . These results are consistent with Cdc42p interacting with Pak2p to signal through the Mkh1p-Pek1p-Spm1p pathway.

Nucleic Acids Res, 2001 Jul 15, 29(14), 3030 - 40
Expression of hsp16 in response to nucleotide depletion is regulated via the spc1 MAPK pathway in Schizosaccharomyces pombe; Taricani L et al.; A universal response to elevated temperature and other forms of physiological stress is the induction of heat shock proteins (HSPs) . Hsp16 in Schizosaccharomyces pombe encodes a polypeptide of predicted molecular weight 16 kDa that belongs to the HSP20/alpha-crystallin family whose members range in size from 12 to 43 kDa . Heat shock treatment increases expression of the hsp16 gene by 64-fold in wild-type cells and 141-fold in cdc22-M45 (ribonucleotide reductase) mutant cells . Hsp16 expression is mediated by the spc1 MAPK signaling pathway through the transcription factor atf1 and in addition through the HSF pathway . Nucleotide depletion or DNA damage as occurs in cdc22-M45 mutant cells, or during hydroxyurea or camptothecin treatment, is sufficient to activate hsp16 expression through atf1 . Our findings suggest a novel role for small HSPs in the stress response following nucleotide depletion and DNA damage . This extends the types of damage that are sensed by the spc1 MAPK pathway via atf1.

Nucleic Acids Res, 2001 Jul 15, 29(14), 2963 - 72
Involvement of Schizosaccharomyces pombe Srs2 in cellular responses to DNA damage; Wang SW et al.; In the budding yeast Saccharomyces cerevisiae the Srs2/RadH DNA helicase promotes survival after ultraviolet (UV) irradiation, and has been implicated in DNA repair, recombination and checkpoint signalling following DNA damage . A second helicase, Sgs1, is the S.cerevisiae homologue of the human BLM and WRN proteins, which are defective in cancer predisposition and/or premature ageing syndromes . Saccharomyces cerevisiae cells lacking both Srs2 and Sgs1 exhibit a severe growth defect . We have identified an Srs2 orthologue in the fission yeast Schizosaccharomyces pombe, and have investigated its role in responses to UV irradiation and inhibition of DNA replication . Deletion of fission yeast srs2 caused spontaneous hyper-recombination and UV sensitivity, and simultaneous deletion of the SGS1 homologue rqh1 caused a severe growth defect reminiscent of that seen in the equivalent S.cerevisiae mutant . However, unlike in budding yeast, inactivation of the homologous recombination pathway did not suppress this growth defect . Indeed, the homologous recombination pathway was required for maintenance of normal fission yeast viability in the absence of Srs2, and loss of homologous recombination and loss of Srs2 contributed additively to UV sensitivity . We conclude that Srs2 plays related, but not identical, roles in the two yeast species.

Nucleic Acids Res, 2001 Jul 15, 29(14), 2938 - 49
Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae; Wu K et al.; A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae . NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1 . Database mining revealed that Nop12p was similar to a related protein, Nop13p . Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM) . Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein . Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe . Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15 degrees C and slow growth at 20 and 25 degrees C . Growth of a nop12Delta strain at 15 and 20 degrees C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA . A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined . Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy . Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm . Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.

Curr Biol, 2001 Jun 26, 11(12), 931 - 40
Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression; Trautmann S et al.; Background: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus . This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk) . Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S . cerevisiae . In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete.Results: We have identified the S . pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle . As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division . Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis . Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle . In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry . Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine.Conclusions: S . pombe Clp1p affects cell cycle progression in a markedly different manner than its S . cerevisiae homolog, Cdc14p . This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.

Yeast, 2001 Jul, 18(10), 963 - 70
Malo-ethanolic fermentation in grape must by recombinant strains of Saccharomyces cerevisiae; Volschenk H et al.; Recombinant strains of Saccharomyces cerevisiae with the ability to reduce wine acidity could have a significant influence on the future production of quality wines, especially in cool climate regions . L-Malic acid and L-tartaric acid contribute largely to the acid content of grapes and wine . The wine yeast S . cerevisiae is unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of a malo-ethanolic fermentation . However, strains of Sz . pombe are not suitable for vinification due to the production of undesirable off-flavours . Heterologous expression of the Sz . pombe malate permease (mae1) and malic enzyme (mae2) genes on plasmids in S . cerevisiae resulted in a recombinant strain of S . cerevisiae that efficiently degraded up to 8 g/l L-malic acid in synthetic grape must and 6.75 g/l L-malic acid in Chardonnay grape must . Furthermore, a strain of S . cerevisiae containing the mae1 and mae2 genes integrated in the genome efficiently degraded 5 g/l of L-malic acid in synthetic and Chenin Blanc grape musts . Furthermore, the malo-alcoholic strains produced higher levels of ethanol during fermentation, which is important for the production of distilled beverages .

Yeast, 2001 Jul, 18(10), 903 - 14
Characterization of a Schizosaccharomyces pombe mutant deficient in UDP-galactose transport activity; Tanaka N et al.; In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae . We previously found that the disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz . pombe . The Deltagms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz . pombe . The deletion strain of gms1+ was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with beta-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white . A reduction of galactomannan layers of the cell wall in the Deltagms1 strain was observed by scanning and transmission electron microscopic analyses . The addition of osmotic stabilizer suppressed the morphologic defect of the Deltagms1 cells, while other phenotypes were weakly suppressed . The Deltagms1 (h90) strain was incapable of sexual conjugation during nutritional starvation . These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz . pombe .

EMBO J, 2001 Jul 16, 20(14), 3871 - 81
A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination; Nabeshima K et al.; Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation . We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination . Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe . Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not . We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion . Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe . Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.

Plant Mol Biol, 2001 May, 46(2), 205 - 13
An upstream region of the Arabidopsis thaliana CDKA;1 (CDC2aAt) gene directs transcription during trichome development; Imajuku Y et al.; The cell cycle of eukaryotes is tightly regulated through the activity of cyclin-dependent kinases . The Arabidopsis thaliana CDKA;1 (CDC2aAt) gene is thought to encode such a protein kinase, since it is actively transcribed in proliferating tissues and can complement defects in the Schizosaccharomyces pombe cdc2 gene . We analyzed the functional structure of the CDKA;1 promoter, using fusion genes between various upstream regions of CDKA;1 and the Escherichia coli beta-glucuronidase (GUS) gene . A 595 bp DNA fragment upstream from the transcription start site conferred GUS activity on developing trichomes, but not on proliferating tissues . On the other hand, another upstream fragment extending to the 5' non-coding transcribed region gave GUS activity to both proliferating tissues and developing trichomes . Against the gl2 mutant background, GUS activity directed by the 595 bp fragment was detected in single-stalk cells, but not in giant cells without obvious polar extension growth . These results revealed that the 595 bp fragment lacks cis element(s) essential for proliferating-cell-specific promoter activity, but can direct transcription in a specific period during trichome development, which does not include cell division . This suggests that CDKA;1 functions during cell morphogenesis as well as cell proliferation.

Somat Cell Mol Genet, 1999 May, 25(3), 159 - 71
Cloning and characterization of Chinese hamster CDC7 (ChCDC7); Guo B et al.; The Cdc7 serine/threonine kinase is essential for entry into and to traverse through S phase . We have cloned the putative Chinese hamster CDC7 (ChCDC7) cDNA that is capable of encoding a protein of 572 amino acids with predicted molecular mass of 62.6 kDa . The ChCdc7 protein includes all 11 kinase domains that are conserved among the Cdc7-related protein kinases . In addition, the ChCdc7 protein kinase contains at least two kinase inserts that show substantial identity to those of huCdc7p . Overall, ChCdc7p shares 81, 56, 30, and 27% amino acid sequence identity with the Cdc7-related proteins of human, Xenopus, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, respectively . Although the levels of ChCDC7 mRNA and protein are relatively constant throughout the cell cycle in the cycling cells, they are extremely low in the cells synchronized in the quiescent stage (i.e., G0) . When cells in G0 are released into the cell cycle, the levels of ChCDC7 mRNA and protein increase slowly until the cells reach the G1/S border, at which time the increase is rapid . This suggests that a number of signal transduction pathways may have to be activated prior to CDC7 gene expression . Interestingly, the ChCdc7-GFP fusion protein formed discrete granules in the nuclei of cells arrested in early S phase by aphidicolin, raising the possibility that ChCdc7p is part of the "replication factory."

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8756 - 61 Epub 2001 Jul 03.
The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia; Fuchs U et al.; We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17 . It maps to chromosome 9q34 centromeric to ABL . The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23) . The rearrangement resulted in a 5'-MLL/FBP17-3' fusion mRNA . We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability . In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay . Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype . FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe . Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin . We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay . None of the various Rho proteins tested, however, interacted with FBP17 . We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17 . These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Biochemistry, 2001 Jul 10, 40(27), 8030 - 42
Structural role of the proline residues of the beta-hinge region of p13suc1 as revealed by site-directed mutagenesis and fluorescence studies; Simeoni F et al.; Site-directed mutagenesis, gel filtration, and fluorescence spectroscopy approaches were used to study the molecular hinge mechanism involved in the beta-strand-exchanged dimer formation of the cyclin-dependent protein kinase regulatory subunit p13(suc1) from Schizosaccharomyces pombe . Single and double mutants of residues Pro-90 and Pro-92 (P90V, P92V, and P90V/P92V) were prepared and assayed . Substitution of Pro-90 prevented dimer formation by arm exchange . However, single point mutations did not affect the two-state unfolding transition of wild-type p13(suc1) at equilibrium (i.e., wild type, DeltaG degrees (0,un) = 7.38 +/- 0.35 kcal mol(-1), vs P90V, DeltaG degrees (0,un) = 6.71 +/- 0.18 kcal mol(-1)) . On the contrary, the double mutant unfolded with a complex transition, and the reaction was best described by a three-state model (N <==> I <==> U) . Resolution of the state-dependent (native vs denatured) intrinsic fluorescence decay amplitudes of p13(suc1) showed that with P90V/P92V these parameters were affected at {GuHCl} significantly less than with wild-type and single mutant proteins . Moreover, with the latter products, fluorescence quenching measurements at 1 M GuHCl revealed linear Stern-Volmer plots with quenching constants typical of tryptophan residues located in a native environment (1.6 M(-1) < K(SV) < 2.3 M(-1)) . Dissimilarly, with P90V/P92V a significant deviation from linearity of the Stern-Volmer plot was obtained . Nonlinear least-squares analysis of these data resolved the significant contribution of highly solvent-accessible emitting species (K(SV) = 26 M(-1)) consistent with large exposure of the tryptophan residues . These results are compatible with the existence of an intermediate unfolding state of the double mutation product . Thus, while single residue substitution studies give support to the primary role of Pro-90 in the p13(suc1) dimer formation by domain swapping, double residue substitution studies indicate the important role of the conserved repeat, Pro-x-Pro, for the proper beta-strand spatial organization and stability.

Nucleic Acids Res, 2001 Jul 1, 29(13), 2675 - 90
Comparison of the RNA polymerase III transcription machinery in Schizosaccharomyces pombe, Saccharomyces cerevisiae and human; Huang Y et al.; Multi-subunit transcription factors (TF) direct RNA polymerase (pol) III to synthesize a variety of essential small transcripts such as tRNAs, 5S rRNA and U6 snRNA . Use by pol III of both TATA-less and TATA-containing promoters, together with progress in the Saccharomyces cerevisiae and human systems towards elucidating the mechanisms of actions of the pol III TFs, provides a paradigm for eukaryotic gene transcription . Human and S.cerevisiae pol III components reveal good general agreement in the arrangement of orthologous TFs that are distributed along tRNA gene control elements, beginning upstream of the transcription initiation site and extending through the 3' terminator element, although some TF subunits have diverged beyond recognition . For this review we have surveyed the Schizosaccharomyces pombe database and identified 26 subunits of pol III and associated TFs that would appear to represent the complete core set of the pol III machinery . We also compile data that indicate in vivo expression and/or function of 18 of the fission yeast proteins . A high degree of homology occurs in pol III, TFIIIB, TFIIIA and the three initiation-related subunits of TFIIIC that are associated with the proximal promoter element, while markedly less homology is apparent in the downstream TFIIIC subunits . The idea that the divergence in downstream TFIIIC subunits is associated with differences in pol III termination-related mechanisms that have been noted in the yeast and human systems but not reviewed previously is also considered.

Eur J Biochem, 2001 Jul, 268(13), 3640 - 3
Caenorhabditis elegans expresses a functional phytochelatin synthase; Clemens S et al.; The formation of phytochelatins, small metal-binding glutathione-derived peptides, is one of the well-studied responses of plants to toxic metal exposure . Phytochelatins have also been detected in some fungi and some marine diatoms . Genes encoding phytochelatin synthases (PCS) have recently been cloned from Arabidopsis, wheat and Schizosaccharomyces pombe . Surprisingly, database searches revealed the presence of a homologous gene in the Caenorhabditis elegans genome, DDBJ/EMBL/GenBank accession no . 266513 . Here we show that C . elegans indeed expresses a gene coding for a functional phytochelatin synthase . CePCS complements the Cd2+ sensitivity of a Schizosaccharomyces pombe PCS knock-out strain and confers phytochelatin synthase activity to these cells . Thus, phytochelatins may play a role for metal homeostasis also in certain animals.

Nat Genet, 2001 Jul, 28(3), 290 - 3
Regulation of premeiotic S phase and recombination-related double-strand DNA breaks during meiosis in fission yeast; Murakami H et al.; The meiotic cell cycle is characterized by high levels of recombination induced by DNA double-strand breaks (DSBs), which appear after completion of premeiotic S phase, leading to the view that initiation of recombination depends on meiotic DNA replication . It has also been indicated that DNA replication initiation proteins may differ between the meiotic and mitotic cell cycles, giving rise to an altered S phase, which could contribute to the high level of recombination during meiosis . We have investigated these possibilities in the fission yeast Schizosaccharomyces pombe and found that core DNA replication initiation proteins used during the mitotic cell cycle, including Cdc18p (budding yeast Cdc6p), Cdc19p (Mcm2p), Cdc21p (Mcm4p) and Orp1p (Orc1p), are also required for premeiotic S phase . Reduced activity of these proteins prevents completion of DNA replication but not formation of DSBs . We conclude that recombination-related DSB formation does not depend on the completion of meiotic DNA replication and we propose two parallel developmental sequences during the meiotic cell cycle: one for premeiotic S phase and the other for initiating recombination.

Yeast, 2001 Jun 30, 18(9), 849 - 58
A single-copy suppressor of the Saccharomyces cerevisae late-mitotic mutants cdc15 and dbf2 is encoded by the Candida albicans CDC14 gene; Jimenez J et al.; The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle . By complementation of a S . cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14 . The sequence analysis of the C . albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5' region . Analysis of C . albicans cDNA proved that the intron is processed in vivo . The CaCDC14 gene shares 49% of amino acid sequence identity with the S . cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively . As expected, the C . albicans CDC14 gene complemented a S . cerevisiae cdc14-1 mutant . We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant . Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S . cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny . The sequence of CaCDC14 was deposited in the EMBL database under Accession No . AJ243449 .

Yeast, 2001 Jun 30, 18(9), 827 - 40
Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway; Martin-Yken H et al.; The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a growth defect and increased release of beta-1,6-glucan and beta-glucosylated proteins into the growth medium at increased temperatures . The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-16 transmembrane segments containing several putative phosphorylation and glycosylation sites . The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster . The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D . melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA . A Cwh43-GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells . Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant . This allele-specific phenotype appears to be due to a G-R substitution at position 57 in a highly conserved region of the protein . Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G(1), no growth defect was observed in a double cwh43 cln3 mutants . Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes .

Acta Biol Hung, 2001, 52(2-3), 315 - 23
Multifunctional cytokinesis genes in Schizosaccharomyces pombe; Sipiczki M et al.; The proper division of cells is essential for the production of viable daughter cells . In plants and fungi, the dividing cell produces a cross-wall or septum that bisects the cytoplasm . For separation of the daughter cells, the septum has to be cleaved . To study the regulation of this process, we isolated mutants defective in septum cleavage . The mutants showed highly pleiotropic phenotypes and defined 17 novel genes . The deduced amino acid sequences of the products of the cloned genes exhibited homologies to various transcription regulators of other organisms . The homologies and the pleiotropic effects of the mutations on sexual development, stress response, mitotic stability, septum initiation and septum placement indicated that these genes affect cell separation indirectly, through multifunctional regulatory modules.

Acta Biol Hung, 2001, 52(2-3), 231 - 9
Ethanol-induced cell aggregation (flocculation) and its physiological background in Schizosaccharomyces pombe rive 4-2-1; Maraz A et al.; Cell aggregation (flocculation) of the yeast Schizosaccharomyces pombe strain RIVE 4-2-1 developed in glucose-containing medium, but only in the presence of ethanol . Cell surface proteins which participated in cell to cell interactions were characterised by the susceptibility of flocculation to different proteolytic enzymes, heat treatment, denaturing and thiol compounds and by the inhibition of flocculation by sugars and derivatives . It was shown that a galactose-specific lectin was involved in this new type of flocculation.

FEBS Lett, 2001 Jun 22, 499(3), 251 - 5
The fission yeast meiotic regulator Mei2p undergoes nucleocytoplasmic shuttling; Sato M et al.; Schizosaccharomyces pombe Mei2p is an RNA-binding protein that switches the cell cycle from mitotic to meiotic . Mei2p forms a unique dot in the nucleus prior to meiosis I, aided by a non-coding RNA molecule termed meiRNA . Here we show that Mei2p intrinsically undergoes nucleocytoplasmic shuttling . Artificial acceleration of nuclear migration of Mei2p advances nuclear dot formation, but meiRNA does not appear to promote the dot formation by modulating the migration rate of Mei2p into the nucleus . Rather, this RNA is likely to facilitate the assembly of Mei2p into a dot structure and trap the protein as such in the nucleus.

Res Microbiol, 2001 Apr-May, 152(3-4), 375 - 89
Fungal ABC proteins: pleiotropic drug resistance, stress response and cellular detoxification; Wolfger H et al.; A number of prominent genetic diseases are caused by mutations in genes encoding ATP-binding cassette (ABC) proteins (Ambudkar, Gottesmann, 1998) . Moreover, several mammalian ABC proteins such as P-glycoprotein (P-gp) (Gottesman et al., 1995) and multidrug-resistance-associated proteins (MRPs) (Cole, Deeley, 1998) have been implicated in multidrug resistance (MDR) phenotypes of tumor cells highly resistant to many different anticancer drugs . The characteristics of MDR phenomena include the initial resistance to a single anticancer drug, followed by the development of cross-resistance to many structurally and functionally unrelated drugs . Similar mechanisms of MDR exist in pathogenic fungi, including Candida and Aspergillus (Vanden Bossche et al., 1998), and also in parasites such as Plasmodium and Leishmania (Ambudkar, Gottesmann, 1998), as well as in many bacterial pathogens (Nikaido, 1998) . To dissect the mechanisms of MDR development and to elucidate the physiological functions of ABC proteins, many efforts have been made during the past decade . Importantly, yeast orthologues of mammalian disease genes made this unicellular eukaryote an invaluable model system for studies on the molecular mechanisms of ABC proteins, in order to better understand and perhaps improve treatment of ABC gene-related disease . In this review, we provide an overview of ABC proteins and pleiotropic drug resistance in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe . Furthermore, we discuss the role of ABC proteins in clinical drug resistance development of certain fungal pathogens.

J Biol Chem, 2001 Aug 24, 276(34), 32247 - 56 Epub 2001 Jun 19.
Cloning of human PRP4 reveals interaction with Clk1; Kojima T et al.; Prp4 is a protein kinase of Schizosaccharomyces pombe identified through its role in pre-mRNA splicing, and belongs to a kinase family including mammalian serine/arginine-rich protein-specific kinases and Clks, whose substrates are serine/arginine-rich proteins . We cloned human PRP4 (hPRP4) full-length cDNA and the antiserum raised against a partial peptide of hPRP4 recognized 170-kDa polypeptide in HeLa S3 cell extracts . Northern blot analysis revealed that hPRP4 mRNA was ubiquitously expressed in multiple tissues . The extended NH(2)-terminal region of hPRP4 contains an arginine/serine-rich domain and putative nuclear localization signals . hPRP4 phosphorylated and interacted with SF2/ASF, one of the essential splicing factors . Indirect immunofluorescence analysis revealed that endogenous hPRP4 was distributed in a nuclear speckled pattern and colocalized with SF2/ASF in HeLa S3 cells . Furthermore, hPRP4 interacted directly with Clk1 on its COOH terminus, and the arginine/serine-rich domain of hPRP4 was phosphorylated by Clk1 in vitro . Overexpression of Clk1 caused redistribution of hPRP4, from the speckled to the diffuse pattern in nucleoplasm, whereas inactive mutant of Clk1 caused no change of hPRP4 localization . These findings suggest that the NH(2)-terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1.

Mol Cell Biol, 2001 Jul, 21(14), 4495 - 504
Schizosaccharomyces pombe cells lacking the amino-terminal catalytic domains of DNA polymerase epsilon are viable but require the DNA damage checkpoint control; Feng W et al.; In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol epsilon) is encoded by cdc20(+) and is essential for chromosomal DNA replication . Here we demonstrate that the N-terminal half of Pol epsilon that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae . However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol epsilon (cdc20(DeltaN-term)) are hypersensitive to DNA-damaging agents and have a cell cycle delay . Moreover, the viability of cdc20(DeltaN-term) cells is dependent on expression of rad3(+), hus1(+), and chk1(+), three genes essential for the DNA damage checkpoint control . These data suggest that in the absence of the N terminus of Pol epsilon, cells accumulate DNA damage that must be repaired prior to mitosis . Our observation that S phase occurs more slowly for cdc20(DeltaN-term) cells suggests that DNA damage might result from defects in DNA synthesis . We hypothesize that the C-terminal half of Pol epsilon is required for assembly of the replicative complex at the onset of S phase . This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.

Curr Genet, 2001 May, 39(3), 166 - 74
Fission yeast tor1 functions in response to various stresses including nitrogen starvation, high osmolarity, and high temperature; Kawai M et al.; A target of rapamycin (TOR) protein is a protein kinase that exerts cellular signal transduction to regulate cell growth in response to extracellular nutrient conditions . In the Schizosaccharomyces pombe genome database, there are two genes encoding TOR-related proteins, but their functions have not been analyzed . Here we report that one of the genes, referred to as tor1+, is required for sexual development induced by nitrogen starvation . Ste11 is a key transcription factor for the initiation of sexual development . The expression of ste11+ is normally regulated in tor1- cells; and overexpression of ste11+ hardly rescues the defect in fertility in tor1- . Upon nitrogen starvation, tor1+ cells promote two rounds of the cell cycle to become arrested at the G1 phase before initiation of sexual development . The tor1- cells do not promote such a cell cycle, suggesting that Tor1 is necessary for the response to nitrogen starvation . The tor1- cells show no growth or very slow growth under various stress conditions, including external high pH, high concentrations of salts or sorbitol, and high temperature . These results suggest that Tor1 is necessary for any response to a wide range of stresses . The vegetative growth of tor1- cells is inhibited by rapamycin, although tor1+ cells are resistant to the drug . The tor1- cells are hypersensitive to fluphenazine and cyclosporin A, which specifically inhibit calmodulin and calcineurin, respectively.

Cancer Lett, 2001 Aug 10, 169(1), 51 - 8
Role of cAMP-dependent protein kinase in the regulation of DNA repair; Lee CH et al.; Enhanced DNA repair is an important factor in drug resistance in cancer . Using cell-free extracts derived from the fission yeast, Schizosaccharomyces pombe, we demonstrate in an in vitro system DNA repair system that increased cAMP levels, which activates cAMP-dependent protein kinase (PKA), inhibits repair of ultraviolet (UV)-damaged DNA . Supplementing the cell-free system with the catalytic kinase subunit of PKA also inhibits DNA repair . In contrast, addition of the PKA inhibitor H-89 enhances repair activity . These results show that PKA regulates DNA repair synthesis, thus implicating the cAMP signaling pathway in DNA damage response and repair of UV-damaged DNA lesions.

EMBO J, 2001 Jun 15, 20(12), 3114 - 23
Direct inhibition of caspase 3 is dispensable for the anti-apoptotic activity of XIAP; Silke J et al.; XIAP is a mammalian inhibitor of apoptosis protein (IAP) . To determine residues within the second baculoviral IAP repeat (BIR2) required for inhibition of caspase 3, we screened a library of BIR2 mutants for loss of the ability to inhibit caspase 3 toxicity in the yeast Schizosaccharomyces pombe . Four of the mutations, not predicted to affect the structure of the BIR fold, clustered together on the N-terminal region that flanks BIR2, suggesting that this is a site of interaction with caspase 3 . Introduction of these mutations into full-length XIAP reduced caspase 3 inhibitory activity up to 500-fold, but did not affect its ability to inhibit caspase 9 or interact with the IAP antagonist DIABLO . Furthermore, these mutants retained full ability to inhibit apoptosis in transfected cells, demonstrating that although XIAP is able to inhibit caspase 3, this activity is dispensable for inhibition of apoptosis by XIAP in vivo.

Biochim Biophys Acta, 2001 May 28, 1519(1-2), 111 - 6
Isolation and characterization of the Schizosaccharomyces pombe cDNA encoding the mitochondrial endonuclease(1); Ikeda S et al.; We isolated the cDNA of the fission yeast mitochondrial endonuclease SpNUC1, which consists of 322 amino acids and has a significant homology with the budding yeast NUC1 and mammalian endonuclease G . Comparison of the cDNA sequence with the genomic sequence showed that the gene consists of three exons and two introns and spans 1.31 kb . The enzyme localization in mitochondria was demonstrated by expressing the SpNUC1-green fluorescent protein fusion in the yeast . The endonuclease was activated by truncation of the amino-terminal region of the protein, indicating that the enzyme is encoded as an inactive precursor . The active enzyme degraded single-stranded DNA and RNA, the activity being dependent on Mg(2+) (Mn(2+)).

Mol Genet Genomics, 2001 May, 265(3), 424 - 35
A novel Cdc20-related WD-repeat protein, Fzr1, is required for spore formation in Schizosaccharomyces pombe; Asakawa H et al.; Ste9/Srw1 which shows sequence homology to Hctl from budding yeast, is an activator of the anaphase-promoting complex (APC) in the fission yeast Schizosaccharomyces pombe . By homology search of the S . pombe genome, we identified the gene fr1+, which encodes the protein with the highest homology to Ste9 among five Cdc20-like proteins . Like Ste9, Fzr1 contains seven WD-repeats in its C-terminal region . In spite of this structural similarity, however, overproduction of either of these proteins cannot complement mutants lacking the other . fzr1+ is transcribed exclusively during meiosis and sporulation, suggesting that it plays a role in these processes . In fact, the fzr1 disruptant formed aberrant asci, which contained only one or two mature spores, though meiotic nuclear divisions proceeded with kinetics similar to wild type, and meiotic segregation of chromosomes was normal . Structural alteration of spindle pole bodies, which is a prerequisite for the formation of the forespore membrane, occurred normally in fzr1delta during the second meiotic division . Localization of spore rim marker proteins fused to green fluorescent protein showed that nascent prespores were irregularly shaped, small in size and few in number in fzr1delta cells compared to wild-type cells . Furthermore, electron microscopy revealed that the outer layer of the spore walls was often missing in fzr1delta spores . These results show that Fzr1 is specifically involved in the assembly of the spore envelope and also in spore maturation . Fzr1, a structural homolog of the APC regulator, therefore plays an important role in spore morphogenesis.

Curr Genet, 2001 Apr, 39(2), 83 - 91
L1-like non-LTR retrotransposons in the yeast Candida albicans; Goodwin TJ et al.; Non-LTR retrotransposons (also known as LINEs) have had a major influence on the structure of many eukaryote genomes . They are abundant in many multicellular eukaryotes, including mammals, but appear to be absent from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . This absence has, to date, precluded the development of a yeast model system for the study of non-LTR retrotransposons . In this report we describe several families of non-LTR retrotransposons from the yeast Candida albicans . These elements, which we call Zorro elements, are all members of the L1 clade of non-LTR retrotransposons . Some are intact, transcriptionally active and have transposed recently . This finding should allow the development of a yeast model system.

Mol Biol Cell, 2001 Jun, 12(6), 1583 - 94
Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex; Taylor EM et al.; The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control . It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex . We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5 . Both proteins are about 1100 amino acids in length and are 27-28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains . The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer . In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton . In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters . Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle . Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

J Biol Chem, 2001 Aug 17, 276(33), 30766 - 72 Epub 2001 Jun 14.
Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA; Hohl M et al.; Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents . Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages . Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1 . Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli . Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance . In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides . Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA . Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair . Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.

J Biol Chem, 2001 Aug 17, 276(33), 31376 - 87 Epub 2001 Jun 11.
Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase; Ogino K et al.; Dfp1/Him1 protein of fission yeast, Schizosaccharomyces pombe, encodes the regulatory subunit for Hsk1 kinase, a homologue of budding yeast Cdc7 kinase essential for initiation and progression of the S phase of the cell cycle . This protein binds and activates Hsk1 kinase, which phosphorylates the MCM2 protein . Comparison of the amino acid sequences of the Cdc7 regulatory subunits from various eukaryotes revealed the presence of three small stretches of conserved amino acid sequences, namely Dbf4 motifs N, M, and C . We report here that the Dbf4 motif M, a unique proline-rich motif, and the Dbf4 motif C, a C(2)H(2)-type zinc finger motif, are essential for mitotic functions of Dfp1/Him1 protein as well as for full-level activation of Hsk1 kinase . In vitro, a small segment containing the Dbf4 motif M or C alone binds to and partially activates Hsk1 . Co-expression of these two segments augments the extent of activation . Furthermore, a fused polypeptide containing only Dbf4 motifs M and C without any spacer can activate Hsk1 and is capable of rescuing the growth defect of him1 null cells . Insertion of a long stretch of amino acids between the motif M and motif C can be tolerated for mitotic functions . On the other hand, internal deletion of Dbf4 motif N, which has some similarity with the BRCA C-terminal domain motif, results in a defect in hydroxyurea-induced checkpoint responses and sensitivity to methyl methane sulfonate, yet mitotic functions and kinase activation are intact . In one-hybrid assays with budding yeast Dbf4, motif N mutants exhibit reduced interaction with a replication origin . Our observations suggest the molecular architecture of Cdc7.Dbf4-related kinase complexes at the origins, in which they are tethered to replication machinery through Dbf4 motif N and the catalytic subunits are activated through bipartite binding of Dbf4 motifs M and C of the regulatory subunits.

J Mol Biol, 2001 Jun 22, 309(5), 1101 - 15
Biochemical characterization of the structure-specific DNA-binding protein Cmb1 from Schizosaccharomyces pombe; Sassoon J et al.; Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation . It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution . Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested.As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor . Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch . Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding . Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region . The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA . The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling . The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA . Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent . In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA .

J Biol Chem, 2001 Aug 10, 276(32), 30399 - 406 Epub 2001 Jun 06.
BRCT domain-containing protein TopBP1 functions in DNA replication and damage response; Makiniemi M et al.; Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101 . We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon . In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication . Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks . DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase . We also show that TopBP1 interacts with the checkpoint protein hRad9 . Thus, these results implicate TopBP1 in replication and checkpoint functions.

Mol Cell, 2001 May, 7(5), 1095 - 101
Genetic and molecular characterization of Skb15, a highly conserved inhibitor of the fission yeast PAK, Shk1; Kim HW et al.; The p21-activated kinase, Shk1, is essential for viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe . Here we describe the characterization of a highly conserved, WD repeat protein, Skb15, which negatively regulates Shk1 in fission yeast . A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle . Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions . A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution.

J Biol Chem, 2001 Jul 27, 276(30), 28075 - 82 Epub 2001 May 31.
The length, phosphorylation state, and primary structure of the RNA polymerase II carboxyl-terminal domain dictate interactions with mRNA capping enzymes; Pei Y et al.; The carboxyl-terminal domain (CTD) of elongating RNA polymerase II serves as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing . The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation . The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD . In vivo interactions of Pct1 and Pce1 with the CTD in a two-hybrid assay require 12 and 14 tandem repeats of the CTD heptapeptide, respectively . Pct1 and Pce1 bind in vitro to synthetic CTD peptides containing phosphoserine uniquely at position 5 or doubly at positions 2 and 5 of each of four tandem YSPTSPS repeats, but they bind weakly (Pce1) or not at all (Pct1) to a peptide containing phosphoserine at position 2 . These results illustrate how remodeling of the CTD phosphorylation array might influence the recruitment and dissociation of the capping enzymes during elongation . But how does the CTD structure itself dictate interactions with the RNA processing enzymes independent of the phosphorylation state? Using CTD-Ser5 phosphopeptides containing alanine substitutions at other positions of the heptad, we define essential roles for Tyr-1 and Pro-3 (but not Thr-4 or Pro-6) in the binding of Schizosaccharomyces pombe guanylyltransferase . Tyr-1 is also essential for binding and allosteric activation of mammalian guanylyltransferase by CTD Ser5-PO4, whereas alanine mutations of Pro-3 and Pro-6 reduce the affinity for the allosteric CTD-binding site . These are the first structure-activity relationships deduced for an effector function of the phosphorylated CTD.

EMBO J, 2001 Jun 1, 20(11), 2857 - 66
A role for DNA polymerase alpha in epigenetic control of transcriptional silencing in fission yeast; Nakayama Ji et al.; In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures . Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division . Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint . Here, we show that a mutation in DNA polymerase alpha (pol(alpha)) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state . We also demonstrate that pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres . Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway . Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro . Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein . This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures.

Bioessays, 2001 Jun, 23(6), 526 - 33
How do meiotic chromosomes meet their homologous partners?: lessons from fission yeast; Yamamoto A et al.; Homologous chromosome pairing is required for proper chromosome segregation and recombination during meiosis . The mechanism by which a pair of homologous chromosomes contact each other to establish pairing is not fully understood . When pairing occurs during meiotic prophase in the fission yeast, Schizosaccharomyces pombe, the nucleus oscillates between the cell poles and telomeres remain clustered at the leading edge of the moving nucleus . These meiosis-specific activities produce movements of telomere-bundled chromosomes . Several lines of evidence suggest that these movements facilitate homologous chromosome pairing by aligning homologous chromosomes and promoting contact between homologous regions . Since telomere clustering and nuclear or chromosome movements in meiotic prophase have been observed in a wide range of eukaryotic organisms, it is suggested that telomere-mediated chromosome movements are general activities that facilitate homologous chromosome pairing .

J Biol Chem, 2001 Aug 10, 276(32), 29740 - 7 Epub 2001 May 29.
A screening for high copy suppressors of the sit4 hal3 synthetically lethal phenotype reveals a role for the yeast Nha1 antiporter in cell cycle regulation; Simon E et al.; A screening for multicopy suppressors of the G(1)/S blockage of a conditional sit4 hal3 mutant yielded the NHA1 gene, encoding a Na(+),K(+)/H(+) antiporter, composed of a transmembrane domain and a large carboxyl-terminal tail, which has been related to cation detoxification processes . Expression of either the powerful Saccharomyces cerevisiae Ena1 Na(+)/H(+)-ATPase or the Schizosaccharomyces pombe Sod2 Na(+)/H(+) antiporter, although increasing tolerance to sodium, was unable to mimic the Nha1 function in the cell cycle . Mutation of the conserved Asp residues Asp(266)-Asp(267) selectively abolished Na(+) efflux without modifying K(+) efflux and did not affect the capacity of Nha1 to relieve the G(1) blockage . Mutagenesis analysis revealed that the region near the carboxyl-terminal end of Nha1 comprising residues 800-948 is dispensable for sodium detoxification but necessary for transport of K(+) cations . Therefore, this portion of the protein contains structural elements that selectively modulate Nha1 antiporter functions . This region is also required for Nha1 to function in the cell cycle . However, expression of the closely related Cnh1 antiporter from Candida albicans, which also contains a long carboxyl-terminal extension, although allowing efficient K(+) transport does not relieve cell cycle blockage . This indicates that although the determinants for Nha1-mediated regulation of potassium transport and the cell cycle map very closely in the protein, most probably the function of Nha1 on cell cycle is independent of its ability to extrude potassium cations.

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