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