J Biol Chem, 2000 Sep 8, 275(36), 27909 - 16 HDAC1, a histone deacetylase, forms a complex with Hus1 and Rad9, two G2/M checkpoint Rad proteins; Cai RL et al.; HDAC1 is a member of the histone deacetylase family, which plays an important role in modulating the eukaryotic chromatin structure . Numerous studies have demonstrated its involvement in transcription and in tumorigenesis . To better understand the functions and regulation of HDAC1, a yeast two-hybrid screening approach was chosen to identify novel interactions involving HDAC1 . Human HDAC1 was found to interact specifically in yeast, mammalian cells, and in vitro with the human Hus1 gene product, whose Schizosaccharomyces pombe homolog has been implicated in G(2)/M checkpoint control . Both HDAC1 and Hus1 proteins localize to the nuclei . Furthermore, HDAC1 and Hus1 were found to exist in a complex with Rad9, a known Hus1-interacting factor . In addition, bioinformatics analysis of the protein sequences of Hus1, Rad1, and Rad9, three checkpoint Rad proteins that form a complex, revealed that they all contain a putative proliferating cell nuclear antigen (PCNA) fold, raising the possibility that these factors may bind to DNA in a PCNA-like ring structure . The results reported in this study strongly suggest a novel pathway involving HDAC1 in G(2)/M checkpoint control through the interaction with a functional Rad complex that may utilize a PCNA-like structure . Therefore, physically and functionally similar apparatus may function during G(2)/M checkpoint and DNA replication. Am J Respir Cell Mol Biol, 2000 Jun, 22(6), 722 - 31 Pneumocystis carinii uses a functional cdc13 B-type cyclin complex during its life cycle; Kottom TJ et al.; Pneumocystis carinii causes severe pneumonia in immunocompromised patients . Recent studies indicate that P . carinii uses a Cdc2 cyclin-dependent kinase to control its proliferation . To further study the regulation of the life cycle of P . carinii, we characterized the P . carinii B-type cyclin termed Cdc13, whose binding to Cdc2 is necessary for kinase activity . Antibodies to B-type cyclins (Cdc13) specifically immunoprecipitated Cdc2/ Cdc13 complexes with associated kinase activity from P . carinii extracts . To clone P . carinii cdc13, degenerate polymerase chain reaction was undertaken using primers generated from amino-acid motifs conserved in fungal Cdc13 proteins . This amplicon was used to obtain full-length genomic and complementary DNA (cDNA) clones . A specific synthetic peptide antibody generated to P . carinii Cdc13 further demonstrated differential Cdc2/Cdc13 activity over the life cycle of P . carinii, with greater activity in cysts compared with trophic forms of the organism . Finally, P . carinii cdc13 cDNA was used to rescue mutant Schizosaccharomyces pombe strains containing temperature-sensitive deficiencies of endogenous Cdc13 activity, thus verifying function of the P . carinii Cdc13 protein . Therefore, P . carinii contains a Cdc13 cyclin, which is variably active over its life cycle and which promotes fungal proliferation. Curr Biol, 2000 May 18, 10(10), 619 - 22 Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast; Hou MC et al.; A great deal is now known about how cells regulate entry into mitosis, but only recently have the mechanisms controlling exit from mitosis and cytokinesis begun to be revealed . In the budding yeast Saccharomyces cerevisiae, Mob1p interacts with the Dbf2p kinase and cells containing mutations in these genes arrest in late anaphase {1} {2} . Proteins related to Mob1p are present in both plants and animals, but information about Mob1p function has been obtained only from budding yeast . Here, we describe the identification and characterization of Mob1p from Schizosaccharomyces pombe . Mob1p associates with the Sid2p kinase and like Sid2p, Mob1p is required for the initiation of cytokinesis, but not for mitotic exit . Mob1p localizes to the spindle pole body (SPB) and to the cell-division site during cell division, suggesting that it might be involved in transducing the signal to initiate cell division from the SPB to the division site . Mob1p is required for Sid2p localization, and Mob1p localization requires the function of the cdc7, cdc11, cdc14, spg1, sid1, sid2, and sid4 genes, suggesting that together with Sid2p, Mob1p functions at the end of the signaling cascade required to regulate the onset of cytokinesis at the end of mitosis. RNA, 2000 May, 6(5), 653 - 8 Functional equivalence of hairpins in the RNA subunits of RNase MRP and RNase P in Saccharomyces cerevisiae; Lindahl L et al.; RNase MRP and RNase P are both ribonucleoprotein enzymes performing endonucleolytic cleavage of RNA . RNase MRP cleaves at a specific site in the precursor-rRNA transcript to initiate processing of the 5.8S rRNA . RNase P cleaves precursor tRNAs to create the 5' end of the mature tRNAs . In spite of their different specificities, the two RNases have significant structural similarities . For example, the two enzymes in Saccharomyces cerevisiae share eight protein subunits; only one protein is unique to each enzyme . The RNA components of the two nucleases also show striking secondary-structure similarity . To begin to characterize the role of the RNA subunits in enzyme function and substrate specificity, we swapped two hairpin structures (MRP3 and P3) between RNase MRP RNA and RNase P RNA of S . cerevisiae . The hairpins in the two enzymes could be exchanged without loss of function or specificity . On the other hand, when the MRP3 hairpin in RNase MRP of S . cerevisiae was replaced with the corresponding hairpin from the RNA of Schizosaccharomyces pombe or human RNase MRP, no functional enzyme was assembled . We propose that the MRP3 and P3 hairpins in S . cerevisiae perform similar functions and have coevolved to maintain common features that are different from those of MRP3 and P3 hairpins in other species. Genetics, 2000 Jun, 155(2), 623 - 31 Slm9, a novel nuclear protein involved in mitotic control in fission yeast; Kanoh J et al.; In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis . Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1 . Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2 . To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25 degrees ) . One of the genes, slm9, encodes a novel protein of 807 amino acids . Slm9 is most similar to Hir2, the histone gene regulator in budding yeast . Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle . The slm9 disruptant is delayed at the G(2)-M transition as indicated by cell elongation and analysis of DNA content . Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation . The slm9 mutant is defective in recovery from G(1) arrest after nitrogen starvation . The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation. Genetics, 2000 Jun, 155(2), 551 - 68 Four chromo-domain proteins of Schizosaccharomyces pombe differentially repress transcription at various chromosomal locations; Thon G et al.; Transcription is repressed in regions of the fission yeast genome close to centromeres, telomeres, or the silent mating-type cassettes mat2-P and mat3-M . The repression involves the chromo-domain proteins Swi6 and Clr4 . We report that two other chromo-domain proteins, Chp1 and Chp2, are also important for these position effects . Chp1 showed a specificity for centromeric regions . Its essentiality for the transcriptional repression of centromeric markers correlates with its importance for chromosome stability . Chp2 appeared more pleiotropic . Its effects on centromeric silencing were less pronounced than those of Chp1, and it participated in telomeric position effects and transcriptional silencing in the mating-type region . We also found that PolII-transcribed genes were repressed when placed in one of the Schizosaccharomyces pombe rDNA clusters, a situation analogous to that in the budding yeast Saccharomyces cerevisiae . Chp2, Swi6, Clr4, and, to a lesser extent, Chp1 participated in that repression. Genetics, 2000 Jun, 155(2), 539 - 49 Schizosaccharomyces pombe Ste7p is required for both promotion and withholding of the entry to meiosis; Matsuyama A et al.; The fission yeast ste7 mutant cannot mate and undergo meiosis, but shows no defect in vegetative growth . We cloned and characterized the ste7 gene . The deduced ste7 gene product (Ste7p) was a protein of 569 amino acids with no significant similarity to other proteins . Transcription of ste7 was induced by nutrient starvation via the function of the transcription factor Ste11p . Disruption of the ste7 gene blocked both conjugation and meiosis, showing that Ste7p plays a positive role in these two processes, probably activating the pheromone signal pathway . Unexpectedly, overexpression of ste7(+) promoted conjugation but inhibited meiosis in wild-type cells . The temperature-sensitive pat1-114 mutant underwent ectopic conjugation at the semirestrictive temperature when its genetic background was ste7(+), whereas the same mutant initiated haploid meiosis when its genetic background was ste7Delta . Two-hybrid analysis suggested that Ste7p interacts physically with both Pat1p and Mei2p, which together constitute the major switch to initiate meiosis . Ste7p tagged with green fluorescent protein accumulated in haploid cells under nutrient starvation until they completed conjugation, but this protein disappeared when they were to enter meiosis . These observations suggest that Ste7p may have a function to suppress the onset of meiosis until the conjugation process has been duly completed. EMBO J, 2000 Jun 1, 19(11), 2719 - 27 Promiscuous targeting of Bacillus subtilis cell division protein DivIVA to division sites in Escherichia coli and fission yeast; Edwards DH et al.; The Bacillus subtilis divIVA gene encodes a coiled-coil protein that shows weak similarity to eukaryotic tropomyosins . The protein is targeted to the sites of cell division and mature cell poles where, in B.subtilis, it controls the site specificity of cell division . Although clear homologues of DivIVA are present only in Gram-positive bacteria, and its role in division site selection is not conserved in the Gram-negative bacterium, Escherichia coli, a DivIVA-green fluorescent protein (GFP) fusion was targeted accurately to division sites and retained at the cell pole in this organism . Remarkably, the same fusion protein was also targeted to nascent division sites and growth zones in the fission yeast Schizosaccharomyces pombe, mimicking the localization of the endogenous tropomyosin-like cell division protein Cdc8p, and F-actin . The results show that a targeting signal for division sites is conserved across the eukaryote-prokaryote divide. EMBO J, 2000 Jun 1, 19(11), 2475 - 82 Human dolichol-phosphate-mannose synthase consists of three subunits, DPM1, DPM2 and DPM3; Maeda Y et al.; Dolichol-phosphate-mannose (DPM) synthase generates mannosyl donors for glycosylphosphatidylinositols, N-glycan and protein O- and C-mannosylation . In Saccharomyces cerevisiae, this enzyme is encoded by DPM1 . We reported previously that mammalian DPM synthase contains catalytic DPM1 and regulatory DPM2 subunits, and that DPM1 requires DPM2 for its stable expression in the endoplasmic reticulum . Here we report that human DPM synthase consists of three subunits . The third subunit, DPM3, comprises 92 amino acids associated with DPM1 via its C-terminal domain and with DPM2 via its N-terminal portion . The stability of DPM3 was dependent upon DPM2 . However, overexpression of DPM3 in Lec15 cells, a null mutant of DPM2, restored the biosynthesis of DPM with an increase in DPM1, indicating that DPM3 directly stabilized DPM1 . Therefore, DPM2 stabilizes DPM3 and DPM3 stabilizes DPM1 . DPM synthase activity was 10 times higher in the presence of DPM2, indicating that DPM2 also plays a role in the enzymatic reaction . Schizosaccharomyces pombe has proteins that resemble three human subunits; S.pombe DPM3 restored biosynthesis of DPM in Lec15 cells, indicating its orthologous relationship to human DPM3. Mol Plant Microbe Interact, 2000 Jun, 13(6), 629 - 36 High level activation of vitamin B1 biosynthesis genes in haustoria of the rust fungus Uromyces fabae; Sohn J et al.; In the rust fungus Uromyces fabae, the transition from the early stages of host plant invasion toward parasitic growth is accompanied by the activation of many genes (PIGs = in planta induced genes) . Two of them, PIG1 (= THI1) and PIG4 (= THI2), were found to be highly transcribed in haustoria, and are homologous to genes involved in thiamine (vitamin B1) biosynthesis in yeast . Their functional identity was confirmed by complementation of Schizosaccharomyces pombe thiamine auxotrophic thi3 (nmt1) and thi2 (nmt2) mutants, respectively . In contrast to thiamine biosynthesis genes of other fungi that are completely suppressed by thiamine, THI1 and THI2 expression was not affected by the addition of thiamine to rust hyphae grown either in vitro or in planta . Immunoblot analysis revealed decreasing amounts of THI1p in extracts from spores, germlings, and in vitro-grown infection structures with increasing time after inoculation . Immunofluorescence microscopy of rust-infected leaves detected high concentrations of THI1p in haustoria, and only low amounts in intercellular hyphae . In the sporulating mycelium, THI1p was found in the basal hyphae of the uredia, but not in the pedicels and only at very low levels in uredospores . These data indicate that the haustorium is an essential structure of the biotrophic rust mycelium not only for nutrient uptake but also for the biosynthesis of metabolites such as thiamine. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6367 - 72 Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomycespombe; Haering CH et al.; The chromosome end-replicating enzyme telomerase is composed of a template-containing RNA subunit, a reverse transcriptase (TERT), and additional proteins . The importance of conserved amino acid residues in Trt1p, the TERT of Schizosaccharomyces pombe, was tested . Mutation to alanine of the proposed catalytic aspartates in reverse transcriptase motifs A and C and of conserved amino acids in motifs 1 and B' resulted in defective growth, progressive loss of telomeric DNA, and loss of detectable telomerase enzymatic activity in vitro . Mutation of the phenylalanine (F) in the conserved FYxTE of telomerase-specific motif T had no phenotype in vivo or in vitro whereas mutation of a conserved amino acid in RT motif 2 had an intermediate effect . In addition to identifying single amino acids of TERT required for telomere maintenance in the fission yeast, this work provides useful tools for S . pombe telomerase research: a functional epitope-tagged version of Trt1p that allows detection of the protein even in crude cellular extracts, and a convenient and robust in vitro enzymatic activity assay based on immunopurification of telomerase. Mol Cell Biol, 2000 Jun, 20(12), 4288 - 94 Mechanism of caffeine-induced checkpoint override in fission yeast; Moser BA et al.; Mitotic checkpoints restrain the onset of mitosis (M) when DNA is incompletely replicated or damaged . These checkpoints are conserved between the fission yeast Schizosaccharomyces pombe and mammals . In both types of organisms, the methylxanthine caffeine overrides the synthesis (S)-M checkpoint that couples mitosis to completion of DNA S phase . The molecular target of caffeine was sought in fission yeast . Caffeine prevented activation of Cds1 and phosphorylation of Chk1, two protein kinases that enforce the S-M checkpoint triggered by hydroxyurea . Caffeine did not inhibit these kinases in vitro but did inhibit Rad3, a kinase that regulates Cds1 and Chk1 . In accordance with this finding, caffeine also overrode the G(2)-M DNA damage checkpoint that requires Rad3 function . Rad3 coprecipitated with Cds1 expressed at endogenous amounts, a finding that supports the hypothesis that Rad3 is involved in direct activation of Cds1. Biochemistry, 2000 May 9, 39(18), 5586 - 92 Novel substrates of Escherichia coli nth protein and its kinetics for excision of modified bases from DNA damaged by free radicals; Dizdaroglu M et al.; Escherichia coli Nth protein (endonuclease III) is a DNA glycosylase with a broad substrate specificity for pyrimidine derivatives . We discovered novel substrates of E . coli Nth protein using gas chromatography/isotope-dilution mass spectrometry and DNA samples, which were damaged by gamma-irradiation or by H(2)O(2)/Fe(III)-EDTA/ascorbic acid . These were 4, 6-diamino-5-formamidopyrimidine, 5,6-dihydroxyuracil, and 5, 6-dihydroxycytosine . The first compound was recognized for the first time as a purine-derived substrate of the enzyme . We also investigated kinetics of excision of a multitude of modified bases from three damaged DNA substrates . Excision of modified bases was determined as a function of enzyme concentration, incubation time, and substrate concentration . Excision followed Michaelis-Menten kinetics . Kinetic parameters were determined for the following modified bases: 4,6-diamino-5-formamidopyrimidine, cis- and trans-thymine glycols, 5-hydroxycytosine, cis- and trans-uracil glycols, 5-hydroxyuracil, 5-hydroxy-5-methylhydantoin, alloxan, 5, 6-dihydroxycytosine, 5,6-dihydroxyuracil, 5-hydroxy-6-hydrothymine, and 5-hydroxy-6-hydrouracil . The results show that three newly discovered substrates were excised by the enzyme with a preference similar to excision of its known major substrates such as thymine glycol and 5-hydroxycytosine . Excision kinetics significantly depended on the nature of the damaged DNA substrates in agreement with previous results on other DNA glycosylases . Specificity constants (k(cat)/K(M)) of E . coli Nth protein were compared to those of its previously investigated functional homologues such as human and Schizosaccharomyces pombe Nth proteins and Saccharomyces cerevisiae Ntg1 and Ntg2 proteins . This comparison shows that significant differences exist with respect to substrate specificity and kinetic parameters despite extensive structural conservation among the Nth homologues. Microsc Res Tech, 2000 Apr 15, 49(2), 161 - 7 Microtubule and actin-dependent movement of the formin cdc12p in fission yeast; Chang F; Although a number of gene products involved in cytokinesis have been identified, still little is known about how these proteins are localized to the proper site and assembled into a ring structure . How is the plane of cell division is positioned in the cell? Schizosaccharomyces pombe are simple rod-shaped eukaryotic cells that divide by medial fission using a medial contractile ring . S . pombe cdc12p encodes a member of the formin gene family, proteins with conserved roles in cytokinesis and actin organization . cdc12p is required specifically for the formation of the medial ring and is located in this ring during mitosis . Time-lapse microscopy of cells expressing GFP-cdc12p protein fusions reveals that during interphase, S . pombe cdc12p is present in a discrete, motile cytoplasmic particle that moves using both actin and microtubules . At the onset of mitosis, the spot moves to the future site of cell division and spreads out into a ring . These studies demonstrate that a cytokinesis factor may travel on both microtubule and actin networks to the site of contractile ring assembly . These findings suggest a potential mechanism for how the mitotic spindle positions the cell division plane in animal cells . Microsc Res Tech, 2000 Apr 15, 49(2), 152 - 60 Cytokinesis in fission yeast: a myosin pas de deux; Mulvihill DP et al.; Cytokinesis in the fission yeast, Schizosaccharomyces pombe consists of two distinct but overlapping events: the assembly and constriction of a cytokinetic actomyosin ring (CAR) and the formation of a cross wall or septum . These two processes must be spatially and temporally coordinated both with each other and with other cell cycle events, most notably spindle formation and anaphase chromosome segregation . In fission yeast, the CAR contains two unusual type II myosins, Myo2, encoded by the gene myo2(+), and Myp2, encoded by myp2(+) . The relationship of these two proteins to each other and their relative contribution to CAR assembly and contraction is largely unknown . Here we review what is known about the role of each myosin in cytokinesis and present some new information concerning their regulation and possible physical interaction . Biochem Pharmacol, 2000 Jul 1, 60(1), 91 - 4 Additive inhibitory effect of calcipotriol and anthralin on ribonuclease P activity; Papadimou E et al.; The effects of two antipsoriatic compounds, calcipotriol and anthralin, separately or in combination on ribonuclease P (RNase P), were investigated using a cell-free system from the slime mold Dictyostelium discoideum . RNase P is an ubiquitous and essential enzyme which endonucleolytically cleaves all tRNA precursors to produce the mature 5' end . The substrate for RNase P assays was an in vitro (32)P-labeled transcript of the Schizosaccharomyces pombe tRNA(Ser) gene supS1 . Enzyme assays were carried out at 37 degrees in 20 microL 50 mM Tris-HCL 7.6 buffer, containing 10 mM NH(4)Cl, 5 mM MgCl(2), and 10% isopropanol . Calcipotriol or anthralin alone exerted a dose-dependent inhibitory effect on RNase P activity, with the former being more active than the latter in this respect . Simultaneous exposure of the enzyme to both drugs resulted in an enhancement of RNase P inhibition, which was additive . Considering the lack of structural similarities between the substrate (precursor tRNA) of RNase P and the tested drugs, it seems reasonable to suggest that their effects may be due to binding to allosteric inhibition sites of the enzyme . Although our in vitro findings cannot be directly extrapolated to the in vivo human condition, they do suggest that the inhibitory effects of calcipotriol and anthralin on tRNA biogenesis may be implicated in the mechanisms of their antipsoriatic action . Moreover, the additive inhibitory effect of these compounds on RNase P activity provides an experimental basis for their possible combined therapeutic application in the management of psoriasis. Yeast, 2000 May, 16(7), 597 - 609 Multiple pathways regulating fission yeast mitosis upon environmental stresses; Kishimoto N et al.; Environmental signals, such as nutrient availability and physiological stresses, modulate the cell cycle and cell size of the fission yeast Schizosaccharomyces pombe . However, little is known about how these signals are transmitted to the central cell cycle regulator, Cdc2, the cyclin-dependent kinase that induces mitosis . We show here genetic evidence that medium alkalization stimulates mitosis and consequently reduces cell size, either through the Nim1-Wee1 cascade, which regulates the inhibitory phosphorylation of Cdc2 at Tyr(15), or through the Cdc2-activating phosphatase, Cdc25 . Alkaline stress stimulates phosphorylation of Nim1, accumulation of Cdc25 and dephosphorylation of Cdc2 at Tyr(15) . We also show that osmostress stimulates mitosis through two independent pathways: one stimulates accumulation of Cdc25, and another dephosphorylation of Cdc2 at Tyr(15) . However, our analysis demonstrates that these environmental stresses can stimulate mitosis independently of dephosphorylation of Cdc2 at Tyr(15) . The S . pombe MAP kinase, Spc1, was required for the steady-state level of Cdc25 in the normal cell cycle and for its accumulation in response to alkaline stress and nutritional starvation . Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5249 - 54 Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast; Chang L et al.; A mutation in the Schizosaccharomyces pombe sid4(+) (septation initiation defective) gene was isolated in a screen for mutants defective in cytokinesis . We have cloned sid4(+) and have found that sid4(+) encodes a previously unknown 76.4-kDa protein that localizes to the spindle pole body (SPB) throughout the cell cycle . Sid4p is required for SPB localization of key regulators of septation initiation, including the GTPase Spg1p, the protein kinase Cdc7p, and the GTPase-activating protein Byr4p . An N-terminally truncated Sid4p mutant does not localize to SPBs and when overproduced acts as a dominant-negative mutant by titrating endogenous Sid4p and Spg1p from the SPB . Conversely, the Sid4p N-terminal 153 amino acids are sufficient for SPB localization . Biochemical studies demonstrate that Sid4p interacts with itself, and yeast two-hybrid analysis shows that its self-interaction domain lies within the C-terminal half of the protein . Our data indicate that Sid4p SPB localization is a prerequisite for the execution of the Spg1p signaling cascade. Mol Cell Biol, 2000 Jun, 20(11), 4049 - 61 Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae; Longtine MS et al.; Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds . This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p . Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay . The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent . In contrast, mutations affecting the other two Nim1p-related kinases in S . cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization . However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p . As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation . Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G(2)/M phase . Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck . This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G(2) delay observed in such mutants . In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism . The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed). Mol Cell Biol, 2000 Jun, 20(11), 4016 - 27 Cpc2, a fission yeast homologue of mammalian RACK1 protein, interacts with Ran1 (Pat1) kinase To regulate cell cycle progression and meiotic development; McLeod M et al.; The Schizosaccharomyces pombe ran1/pat1 gene regulates the transition between mitosis and meiosis . Inactivation of Ran1 (Pat1) kinase is necessary and sufficient for cells to exit the cell cycle and undergo meiosis . The yeast two-hybrid interaction trap was used to identify protein partners for Ran1/Pat1 . Here we report the identification of one of these, Cpc2 . Cpc2 encodes a homologue of RACK1, a WD protein with homology to the beta subunit of heterotrimeric G proteins . RACK1 is a highly conserved protein, although its function remains undefined . In mammalian cells, RACK1 physically associates with some signal transduction proteins, including Src and protein kinase C . Fission yeast cells containing a cpc2 null allele are viable but cell cycle delayed . cpc2Delta cells fail to accumulate in G(1) when starved of nitrogen . This leads to defects in conjugation and meiosis . Copurification studies show that although Cpc2 and Ran1 (Pat1) physically associate, Cpc2 does not alter Ran1 (Pat1) kinase activity in vitro . Using a Ran1 (Pat1) fusion to green fluorescent protein, we show that localization of the kinase is impaired in cpc2Delta cells . Thus, in parallel with the proposed role of RACK1 in mammalian cells, fission yeast cpc2 may function as an anchoring protein for Ran1 (Pat1) kinase . All defects associated with loss of cpc2 are reversed in cells expressing mammalian RACK1, demonstrating that the fission yeast and mammalian gene products are indeed functional homologues. Mol Cell Biol, 2000 Jun, 20(11), 3807 - 16 Three yeast proteins related to the human candidate tumor suppressor p33(ING1) are associated with histone acetyltransferase activities; Loewith R et al.; Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombe proteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33(ING1) in their C-terminal regions . The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation . We show that GFP-Yng2, like human Ing1, is localized in the nucleus . Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine . These phenotypes are suppressed by expression of either human Ing1 or S . pombe Png1, suggesting that the yeast and human proteins are functionally conserved . Yng1- and Pho23-deficient cells also share some of these phenotypes . We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes . We further demonstrated by coimmunoprecipitation that HA-Yng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast . Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex . These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling . They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor. Biochemistry, 2000 May 16, 39(19), 5788 - 96 Ultraviolet damage endonuclease (Uve1p): a structure and strand-specific DNA endonuclease; Kaur B et al.; Schizosaccharomyces pombe ultraviolet damage endonuclease (UVDE or Uve1p) performs the initial step in an alternative excision repair pathway for UV-induced DNA damage . This DNA repair pathway was originally thought to be specific for UV damage . However, the broad substrate specificity of Uve1p suggests a more general role for this enzyme . Uve1p recognizes UV-induced bipyrimidine photoadducts and other non-UV-induced DNA adducts . Biochemical and genetic analysis also suggests that Uve1p may be involved in orchestrating mismatch repair in vivo . This study demonstrates that Uve1p recognizes and cleaves heteroduplex DNA with small unpaired loops but does not recognize loops six to eight nucleotides in length . In addition, the enzyme does not recognize DNA with palindromic insertions that could form base-paired hairpin structures . The cleavage efficiency of Uve1p depends on the distance of a mismatch from the DNA terminus, suggesting that the 3' terminus may contribute to the strand discrimination signal for Uve1p . These biochemical activities are discussed in the context of the role of Uve1p in DNA repair. J Biol Chem, 2000 May 12, 275(19), 14381 - 7 Byr4 localizes to spindle-pole bodies in a cell cycle-regulated manner to control Cdc7 localization and septation in fission yeast; Li C et al.; Cytokinesis and septation in the fission yeast Schizosaccharomyces pombe are studied as a model for mammalian cell division . In fission yeast, septation is positively regulated by Spg1, a Ras family GTPase that localizes to spindle-pole bodies (SPBs) throughout the cell cycle . As cells enter mitosis, Spg1 accumulates in an active, GTP-bound form and binds the Cdc7 protein kinase to cause Cdc7 translocation to SPBs . Cdc7 disappears from one SPB in mid-anaphase and from the second SPB in late mitosis . Byr4 plus Cdc16 negatively regulate septation by forming a two-component GTPase-activating protein for Spg1 . These results led us to hypothesize that Byr4 localization to SPBs regulated the nucleotide state of Spg1, due to its ability to form Spg1GAP activity with Cdc16 and thus the binding of Cdc7 to Spg1 at SPBs . To test this hypothesis, Byr4 localization was determined using indirect immunofluorescence . This analysis revealed that Byr4 was localized to SPBs that did not contain Cdc7 . In byr4(-) mutants, Cdc7 localized to interphase SPBs and only symmetrically localized to mitotic SPBs . In contrast, Byr4 overexpression prevented Spg1 and Cdc7 localization to SPBs . These results suggest that Byr4 localization to SPBs maintains Spg1 in an inactive form, presumably by stimulating Spg1 GTPase activity with Cdc16, and that loss of Byr4 from mitotic SPBs increases the active fraction of Spg1 and thereby increases Spg1-Cdc7 binding . Byr4 localization to SPBs was decreased in spg1, cdc16, sid4, and cdc11 mutants as well as in several mutants that affect medial F-actin structures, suggesting that multiple pathways regulate Byr4 localization to SPBs. J Biol Chem, 2000 May 12, 275(19), 14217 - 22 Complementation of a glucose transporter mutant of Schizosaccharomyces pombe by a novel Trypanosoma brucei gene; Bayele HK et al.; The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host . This is underscored by biochemical switches in its nutritional requirements . In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose . Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis . Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a lambdaEMBL4 genomic library of T . brucei EATRO 164 using a rat liver glucose transporter cDNA clone . Genomic analysis shows that the gene is present as a single copy within the parasite genome . The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily . Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence . It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains . However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants . The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides . The kinetics of glucose uptake reflected those in T . brucei . In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells. J Biol Chem, 2000 May 12, 275(19), 14095 - 101 cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme; Nuccio ML et al.; The N-methylation of phosphoethanolamine is the committing step in choline biogenesis in plants and is catalyzed by S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT, EC ) . A spinach PEAMT cDNA was isolated by functional complementation of a Schizosaccharomyces pombe cho2(-) mutant and was shown to encode a protein with PEAMT activity and without ethanolamine- or phosphatidylethanolamine N-methyltransferase activity . The PEAMT cDNA specifies a 494-residue polypeptide comprising two similar, tandem methyltransferase domains, implying that PEAMT arose by gene duplication and fusion . Data base searches suggested that PEAMTs with the same tandem structure are widespread among flowering plants . Size exclusion chromatography of the recombinant enzyme indicates that it exists as a monomer . PEAMT catalyzes not only the first N-methylation of phosphoethanolamine but also the two subsequent N-methylations, yielding phosphocholine . Monomethyl- and dimethylphosphoethanolamine are detected as reaction intermediates . A truncated PEAMT lacking the C-terminal methyltransferase domain catalyzes only the first methylation . Phosphocholine inhibits both the wild type and the truncated enzyme, although the latter is less sensitive . Salinization of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about 10-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis. Curr Genet, 2000 Mar, 37(3), 159 - 67 The Schizosaccharomyces pombe rfc3+ gene encodes a homologue of the human hRFC36 and Saccharomyces cerevisiae Rfc3 subunits of replication factor C; Gray FC et al.; In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe replication factor C (RF-C) plays key roles both in chromosomal DNA replication and in DNA replication checkpoint function . At the replication fork, the five-subunit RF-C complex functions to load the trimeric polymerase accessory factor PCNA onto DNA . PCNA then acts as a sliding clamp, tethering Pol delta to the DNA to maximise its processivity . Here we describe the cloning of the S . pombe rfc3+ gene, encoding a homologue of the S . cerevisiae Rfc3 and human hRFC36 proteins . The 1026 bp rfc3+ ORF is interrupted by five introns, ranging in size from 49 to 165 bp . The spliced ORF is predicted to encode a 342 amino-acid protein that is approximately 50% identical at the amino acid sequence level to the S . cerevisiae Rfc3 and human hRFC36 proteins . As expected, S . pombe rfc3+ is an essential gene, with rfc3delta cells being defective for DNA replication . Loss of rfc3+ function can be rescued by heterologous expression of either the S . cerevisiae Rfc3 or human hRFC36 proteins in S . pombe. Mol Microbiol, 2000 Apr, 36(2), 377 - 90 Sxa2 is a serine carboxypeptidase that degrades extracellular P-factor in the fission yeast Schizosaccharomyces pombe; Ladds G et al.; Stimulating the fission yeast Schizosaccharomyces pombe with mating pheromones brings about responses that lead to cell conjugation . Persistent stimulation does not, however, induce a continuous response as the cells become desensitized to the presence of the pheromone . One mechanism that contributes to desensitization in M-cells is the release of a carboxypeptidase that inactivates the extracellular P-factor pheromone . Production of the carboxypeptidase requires a functional sxa2 gene . In this study, we report the first molecular characterization of the Sxa2 protein and provide direct evidence that it is the carboxypeptidase that degrades P-factor . Sxa2 is synthesized as a precursor that undergoes an internal cleavage event catalysed by a protease with specificity for basic residues . This generates a series of catalytically active N-terminal fragments and an inactive C-terminal fragment . Cleavage is essential for activation of the carboxypeptidase and, although the C-terminal fragment is inactive, it is required for the N-terminal fragment to attain activity. Yeast, 2000 Apr, 16(6), 531 - 8 Molecular cloning of the CRM1 gene from Candida albicans; Raymond M et al.; In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated . The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04 . Crm1p from C . albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity) . Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S . cerevisiae Crm1p . CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S . cerevisiae, showing that CaCrm1p and S . cerevisiae Crm1p are functionally conserved . Southern blot analysis suggests that CaCRM1 is present at a single locus within the C . albicans genome . The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No . AF178855 . Yeast, 2000 Apr, 16(6), 523 - 9 Cyclic AMP regulates cell size of Schizosaccharomyces pombe through Cdc25 mitotic inducer; Kishimoto N et al.; Nutritional state modulates the cell size of the fission yeast Schizosaccharomyces pombe, such that cells grown in rich medium are larger in size than those in poor medium . This signal is transduced partly through the cyclic AMP-dependent protein kinase cascade . However, little is known about how cyclic AMP interacts with the central cell cycle machinery, Cdc2, the cyclin-dependent kinase that induces mitosis . We show here that cyclic AMP regulates mitosis and cell size, in part, through regulation of protein stability of the Cdc2-activating phosphatase, Cdc25 . However, our analysis demonstrates that cyclic AMP can negatively regulate mitosis independently of dephosphorylation of Cdc2 at Tyr(15) . FEBS Lett, 2000 Apr 28, 472(2-3), 254 - 8 Rad24 is essential for proliferation of diploid cells in fission yeast; Tanaka Y et al.; The rad24(+) gene of Schizosaccharomyces pombe encodes a ubiquitously expressed 14-3-3 protein . We report here that Deltarad24 cells displayed a defect in diploid colony formation, although they conjugated efficiently . We found that a cumulative deletion of mei2(+) gene almost completely suppressed this defect, and demonstrated using two-hybrid analysis that Rad24 protein directly associates with Mei2 protein by recognizing Ser-438 which is a phosphorylation target of Pat1 kinase . We conclude that constitutive progression to meiosis, caused by lack of Mei2 inhibition due to the absence of Rad24 protein, is the primary cause of the proliferative deficiency observed in Deltarad24 cells. RNA, 2000 Apr, 6(4), 638 - 50 REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export; Stutz F et al.; Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus . TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE) . Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions . Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein . This interaction is evolutionarily conserved as Yra1p also interacts with TAP . Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs . Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants . The murine members of the family directly interact with TAP . Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins . Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs. Mol Cell Biol, 2000 May, 20(10), 3728 - 41 Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression; Melcher M et al.; SUV39H1, a human homologue of the Drosophila position effect variegation modifier Su(var)3-9 and of the Schizosaccharomyces pombe silencing factor clr4, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis . Using a detailed structure-function analysis of SUV39H1 mutant proteins in transfected cells, we now show that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organization . First, forced expression of full-length SUV39H1 (412 amino acids) redistributes endogenous M31 (HP1beta) and induces abundant associations with inter- and metaphase chromatin . These properties depend on the C-terminal SET domain, although the major portion of the SUV39H1 protein (amino acids 89 to 412) does not display affinity for nuclear chromatin . By contrast, the M31 interaction surface, which was mapped to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin . Second, cells overexpressing full-length SUV39H1 display severe defects in mitotic progression and chromosome segregation . Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G(2)-specific distribution of phosphorylated histone H3 at serine 10 (phosH3) is dispersed in these cells . This phosH3 shift is not observed with C-terminally truncated mutant SUV39H1 proteins or with deregulated M31 . Together, our data reveal a dominant role(s) for the SET domain of SUV39H1 in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal SU(VAR) protein and histone H3. Genomics, 2000 Apr 1, 65(1), 24 - 33 Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the regulation of cell cycle progression; Hang H et al.; Schizosaccharomyces pombe hus1 promotes radioresistance and hydroxyurea resistance, as well as S and G2 phase checkpoint control . We isolated a human cDNA homologous to hus1, called HUS1 . The major focus of this report is on a detailed analysis of the physical interactions of the HUS1-encoded protein and two other checkpoint control proteins, RAD1p and RAD9p, implicated in the cellular response to DNA damage . We found that HUS1p interacts with itself and the N-terminal region of RAD1p . In contrast, the C-terminal portion of the checkpoint protein RAD9p is essential for interacting with HUS1p and the C-terminal region of RAD1p . Since the N-terminal portion of RAD9p was previously demonstrated to participate in apoptosis, this protein likely has at least two functional domains, one that regulates programmed cell death and another that regulates cell cycle checkpoint control . Truncated versions of HUS1p are unable to bind RAD1p, RAD9p, or another HUS1p molecule . RAD1p-RAD1p and RAD9p-RAD9p interactions can be demonstrated by coimmunoprecipitation, but not by two-hybrid analysis, suggesting that the proteins associate as part of a complex but do not interact directly . Northern blot analysis indicates that HUS1 is expressed in different tissues, but the mRNA is most predominant in testis where high levels of RAD1 and RAD9 message have been detected . These studies suggest that HUS1p, RAD9p, and RAD1p form a complex in human cells and may function in a meiotic checkpoint in addition to the cell cycle delays induced by incomplete DNA replication or DNA damage . EMBO J, 2000 Apr 17, 19(8), 1907 - 17 Conservation of polyamine regulation by translational frameshifting from yeast to mammals; Ivanov IP et al.; Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme . Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe . The expression of mammalian antizyme genes requires a specific +1 translational frameshift . The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop . As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression . Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts . The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals. EMBO J, 2000 Apr 17, 19(8), 1803 - 15 The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast; Guertin DA et al.; Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability . In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis . Here we describe the characterization of a novel protein kinase, Sid1p . Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase . Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation . Additionally, we found that Cdc14p is in a complex with Sid1p . Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p . Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit. J Biol Chem, 2000 Jun 23, 275(25), 18871 - 8 Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe; Lee JK et al.; Minichromosome maintenance (Mcm) proteins 2-7 are highly conserved in eukaryotes and play an essential role in DNA replication . Here, we describe the reconstitution of the various complexes of the Mcm proteins of Schizosaccharomyces pombe using the baculovirus expression system . The simultaneous expression of all six of the Mcm proteins, as well as different combinations of these proteins, yielded several stable complexes that included the heterohexamer of Mcm2/3/4/5/6/7, the Mcm2/4/6/7 heterotetramer, the dimer of the Mcm4/6/7 heterotrimer, and the Mcm3/5 heterodimer . The purification and characterization of the biochemical properties of these complexes showed that only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities . Consistent with these results, the interaction of either Mcm2 or Mcm3/5 with the Mcm4/6/7 complex resulted in the disassembly of the dimeric complex of Mcm4/6/7 and the loss of DNA helicase activity . These results suggest that the Mcm4/6/7 complex is a catalytic core of the Mcm complex and that Mcm2 and Mcm3/5 may be involved in the regulation of the activity of this complex. J Cell Sci, 2000 May, 113 ( Pt 10), 1813 - 25 Molecular mechanism of myosin-II assembly at the division site in Schizosaccharomyces pombe; Motegi F et al.; Schizosaccharomyces pombe cells divide by virtue of the F-actin-based contractile ring (F-actin ring) . Two myosin-II heavy chains, Myo2 and Myp2/Myo3, have been localized to the F-actin ring . Here, we investigated the mechanism of myosin-II assembly at the division site in S . pombe cells . First, we showed that Cdc4, an EF-hand protein, appears to be a common myosin light chain associated with both Myo2 and Myo3 . Loss of function of both Myo2 and Myo3 caused a defect in F-actin assembly at the division site, like the phenotype of cdc4 null cells . It is suggested that Myo2, Myo3 and Cdc4 function in a cooperative manner in the formation of the F-actin ring during mitosis . Next, we investigated the dynamics of myosin-II during mitosis in S . pombe cells . In early mitosis when accumulation of F-actin cables in the medial region was not yet observed, Myo2 was detected primarily as dots widely located in the medial cortex . Myo2 fibers also became visible following the appearance of the dots . The Myo2 dots and fibers then fused with each other to form a medial cortical network . Some Myo2 dots appeared to be localized with F-actin cables which are also accumulated in the medial region . Finally these structures were packed into a thin contractile ring . In mutant cells that cannot form the F-actin ring such as cdc3(ts), cdc8(ts) and cdc12(ts), Myo2 was able to accumulate as dots in the medial cortex, whereas no accumulation of Myo2 dots was detected in cdc4(ts) cells . Moreover, disruption of F-actin in the cell by applying latrunculin-A did not affect the accumulation of Myo2 dots, suggesting that F-actin is not required for their accumulation . A truncated Myo2 which lacks putative Cdc4-binding sites (Myo2dIQs) was able to rescue myo2 null cells, myo3 null cells, cdc4(ts) mutant cells and cdc4 null cells . The Myo2dIQs could assemble into a normal-shaped ring in these cells . Therefore, its assembly at the division site does not require the function of either Cdc4 or Myo3. J Cell Sci, 2000 May, 113 ( Pt 10), 1727 - 36 The G(2) DNA damage checkpoint targets both Wee1 and Cdc25; Raleigh JM et al.; The onset of mitosis is controlled by the cyclin dependent kinase Cdc2p . Cdc2p activity is controlled through the balance of phosphorylation and dephosphorylation of tyrosine-15 (Y15) by the Wee1p kinase and Cdc25p phosphatase . In the fission yeast Schizosaccharomyces pombe, detection of DNA damage in G(2) activates a checkpoint that prevents entry into mitosis through the maintenance of Y15 phosphorylation of Cdc2p, thus ensuring DNA repair precedes chromosome segregation . The protein kinase Chk1p is the endpoint of this checkpoint pathway . We have previously reported that overexpression of Chk1p causes a wee1(+)-dependent G(2) arrest, and this or irradiation leads to hyperphosphorylation of Wee1p . Moreover, Chk1p directly phosphorylates Wee1p in vitro . These data suggested that Wee1p is a key target of Chk1p action in checkpoint control . However, cells lacking wee1(+) are checkpoint proficient and sustained Chk1p overexpression arrests cell cycle progression independently of Wee1p . Therefore, up-regulation of Wee1p alone cannot enforce a checkpoint arrest . Chk1p can also phosphorylate Cdc25p in vitro . These phosphorylation events are thought to promote the interaction with 14-3-3 proteins the cytoplasmic retention of the 14-3-3/Cdc25p complexes . However, we show here that the G(2) DNA damage checkpoint is intact in cells that regulate mitotic entry independently of Cdc25p . Further, these cells are still sensitive to Chk1p-mediated arrest, and so down-regulation of Cdc25p is also insufficient to regulate checkpoint arrest . Conversely, inactivation of both wee1(+) and cdc25(+ )abolishes checkpoint control . We also show that activation of the G(2) DNA damage checkpoint induces a transient increase in Wee1p levels . We conclude that the G(2) DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p and down-regulation of Cdc25p, thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability. J Cell Sci, 2000 May, 113 ( Pt 10), 1695 - 704 The S . pombe orthologue of the S . cerevisiae mob1 gene is essential and functions in signalling the onset of septum formation; Salimova E et al.; We have isolated the Schizosaccharomyces pombe orthologue of the Saccharomyces cerevisiae MOB1 gene in a screen designed to enrich for septation mutants . The gene is essential, and cells lacking it display a phenotype typical of septation signalling network mutants . mob1p is located on both spindle pole bodies throughout mitosis . In addition it is also co-localised with the medial ring later in mitosis, and flanks the septum as the medial ring contracts . We also demonstrate that mob1p can be precipitated from cells in a complex with the septation regulating kinase sid2p. Gene, 2000 Apr 4, 246(1-2), 285 - 93 Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors; Durand B et al.; The RFX family of transcription factors is characterized by a unique DNA binding domain . Five genes have been isolated in mammals, one gene in Caenorhabditis elegans and in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae . Whereas the roles of the RFX genes are beginning to be understood in yeasts, no clear function has been reported in multicellular organisms, except for RFX5, the most divergent member of the family . To study the physiological role of RFX transcription factors using an alternative multicellular model, we report the isolation and characterization of the Drosophila RFX gene (dRFX) . The fruit fly protein shares highly conserved domains with the mammalian factors RFX1 to 3 and is more closely related to this subgroup . It binds DNA with the same target specificity as mammalian factors RFX1 to 3 . dRFX is located on chromosome III and we characterized the entire locus . dRFX expression was analyzed during embryogenesis . dRFX mRNAs are detected only in the peripheral nervous system and in the brain of the embryo. J Biol Chem, 2000 Apr 21, 275(16), 11824 - 8 Alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe operates both in nucleus and in mitochondria; Yasuhira S et al.; The fission yeast, Schizosaccharomyces pombe, possesses a UV-damaged DNA endonuclease-dependent excision repair (UVER) pathway in addition to nucleotide excision repair pathway for UV-induced DNA damage . We examined cyclobutane pyrimidine dimer removal from the myo2 locus on the nuclear genome and the coI locus on the mitochondrial genome by the two repair pathways . While nucleotide excision repair repairs damage only on the nuclear genome, UVER efficiently removes cyclobutane pyrimidine dimers on both nuclear and mitochondrial genomes . The ectopically expressed wild type UV-damaged DNA endonuclease was localized to both nucleus and mitochondria, while modifications of N-terminal methionine codons restricted its localization to either of two organelles, suggesting an alternative usage of multiple translation initiation sites for targeting the protein to different organelles . By introducing the same mutations into the chromosomal copy of the uvde(+) gene, we selectively inactivated UVER in either the nucleus or the mitochondria . The results of UV survival experiments indicate that although UVER efficiently removes damage on the mitochondrial genome, UVER in the mitochondria hardly contributes to UV resistance of S . pombe cells . We suggest a possible UVER function in mitochondria as a backup system for other UV damage tolerance mechanisms. Genes Dev, 2000 Apr 1, 14(7), 783 - 91 Distinct protein interaction domains and protein spreading in a complex centromere; Partridge JF et al.; Fission yeast (Schizosaccharomyces pombe) centromeres are composed of large (40-100 kb) inverted repeats that display heterochromatic features, thus providing a good model for higher eukaryotic centromeres . The association of three proteins that mediate region-specific silencing across centromere 1 has been mapped by quantitative chromatin immunoprecipitation . Swi6 and Chp1 are confined to the flanking outer repeats and Swi6 can spread across at least 3 kb of extraneous chromatin in cen1 . In contrast, Mis6 coats the inner repeats and central core . tRNA genes demarcate this transition zone . These analyses clearly define two distinct domains within this complex centromere which interact with different proteins. Plant J, 2000 Mar, 21(6), 507 - 18 AtRAD1, a plant homologue of human and yeast nucleotide excision repair endonucleases, is involved in dark repair of UV damages and recombination; Gallego F et al.; Plants are unique in the obligatory nature of their exposure to sunlight and consequently to ultraviolet (UV) irradiation . However, our understanding of plant DNA repair processes lags far behind the current knowledge of repair mechanisms in microbes, yeast and mammals, especially concerning the universally conserved and versatile dark repair pathway called nucleotide excision repair (NER) . Here we report the isolation and functional characterization of Arabidopsis thaliana AtRAD1, which encodes the plant homologue of Saccharomyces cerevisiae RAD1, Schizosaccharomyces pombe RAD16 and human XPF, endonucleolytic enzymes involved in DNA repair and recombination processes . Our results indicate that AtRAD1 is involved in the excision of UV-induced damages, and allow us to assign, for the first time in plants, the dark repair of such DNA lesions to NER . The low efficiency of this repair mechanism, coupled to the fact that AtRAD1 is ubiquitously expressed including tissues that are not accessible to UV light, suggests that plant NER has other roles . Possible 'UV-independent' functions of NER are discussed with respect to features that are particular to plants. Genetics, 2000 Mar, 154(3), 1025 - 38 Isolation and characterization of par1(+) and par2(+): two Schizosaccharomyces pombe genes encoding B' subunits of protein phosphatase 2A; Jiang W et al.; Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases found in eukaryotic cells . We cloned two genes, par1(+) and par2(+), encoding distinct B' subunits of PP2A in fission yeast . They share 52% identity at the amino acid sequence level . Neither gene is essential but together they are required for normal septum positioning and cytokinesis, for growth at both high and low temperature, and for growth under a number of stressful conditions . Immunofluorescence microscopy revealed that Par2p has a cell-cycle-related localization pattern, being localized at cell ends during interphase and forming a medial ring in cells that are undergoing septation and cytokinesis . Our analyses also indicate that Par1p is more abundant than Par2p in the cell . Cross-organism studies showed that both par1(+) and par2(+) could complement the rts1Delta allele in Saccharomyces cerevisiae, albeit to different extents, in spite of the fact that neither contains a serine/threonine-rich N-terminal domain like that found in the S . cerevisiae homolog Rts1p . Thus, while Schizosaccharomyces pombe is more similar to higher eukaryotes with respect to its complement of B'-encoding genes, the function of those proteins is conserved relative to that of Rts1p. Rev Med Chir Soc Med Nat Iasi, 1997 Jan-Jun, 101(1-2), 87 - 91 {Inositol is necessary to sexual differentiation in Schizosaccharomyces pombe}; Poitelea M et al.; Sexual differentiation in fission yeast Schizosaccharomyces pombe was found to be dependent on the glucose and inositol contents of culture media . Certain combinations in the amounts of these two compounds suppressed mating and sporulation, while maintaining normal viability . Inositol was found to be required for mating and sporulation in both nitrogen-rich and nitrogen-poor media (the lack of nitrogen is most well-known factor that induces the sexual differentiation) . The experiments were performed using the wild-type strains and standard media . Mutants carrying the pat1-114 temperature-sensitive allele circumvented the need for inositol at the restrictive temperature. Nucleic Acids Res, 2000 May 1, 28(9), 2004 - 11 Fission yeast hrp1, a chromodomain ATPase, is required for proper chromosome segregation and its overexpression interferes with chromatin condensation; Yoo EJ et al.; Hrp1 of Schizosaccharomyces pombe is a member of the CHD protein family, characterized by a chromodomain, a Myb-like telobox-related DNA-binding domain and a SNF2-related helicase/ATPase domain . CHD proteins are thought to be required for modification of the chromatin structure in transcription, but the exact roles of CHD proteins are not known . Here we examine the sub-cellular localization and biochemical activity of Hrp1 and the phenotypes of hrp1 Delta and Hrp1-overexpressing strains . Fluorescence microscopy revealed that Hrp1 protein is targeted to the nucleus . We found that Hrp1 exhibited DNA-dependent ATPase activity, stimulated by both single- and double-stranded DNA . Overexpression of Hrp1 caused slow cell growth accompanied by defective chromosome condensation in anaphase resulting in a 'cut' (celluntimelytorn) phenotype and chromosome loss . The hrp1 Delta mutation also caused abnormal anaphase and mini-chromosome loss phenotypes . Electron micrographs demonstrated that aberrantly shaped nucleoli appeared in Hrp1-overexpressing cells . Therefore, these results suggest that Hrp1 may play a role in mitotic chromosome segregation and maintenance of chromatin structure by utilizing the energy from ATP hydrolysis. J Biol Chem, 2000 Jun 23, 275(25), 18739 - 44 Identification of a fourth subunit of mammalian DNA polymerase delta; Liu L et al.; A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase delta by conventional chromatography . A 16-mer peptide sequence was obtained from the 12-kDa polypeptide which matched a new open reading frame from a human EST () encoding a hypothetical protein of unknown function . The protein was designated as p12 . Human EST was identified as the putative human homologue of Schizosaccharomyces pombe Cdm1 by a tBlastn search of the EST data base using S . pombe Cdm1 . The open reading frame of human EST encoded a polypeptide of 107 amino acids with a predicted molecular mass of 12.4 kDa, consistent with the experimental findings . p12 is 25% identical to S pombe Cdm1 . Both of the 25-kDa polypeptide sequences matched the hypothetical KIAA0039 protein sequence, recently identified as the third subunit of pol delta . Western blotting of immunoaffinity purified calf thymus pol delta revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12 . With the identification of p12 mammalian pol delta can now be shown to consist of four subunits . These studies pave the way for more detailed analysis of the possible functions of the mammalian subunits of pol delta. J Cell Sci, 2000 May, 113 ( Pt 9), 1503 - 13 Tying the knot: linking cytokinesis to the nuclear cycle; Balasubramanian MK et al.; For the survival of both the parent and the progeny, it is imperative that the process of their physical division (cytokinesis) be precisely coordinated with progression through the mitotic cell cycle . Recent studies in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are beginning to unravel the nature of the links between cytokinesis and the nuclear division cycle . The cyclin-dependent kinases and a novel surveillance mechanism that monitors cytokinesis and/or morphogenesis appear to play important regulatory roles in forging these links . It is becoming increasingly clear that the inactivation of the mitosis-promoting cyclin-dependent kinase, which marks the completion of the nuclear division cycle, is essential for actomyosin ring constriction and division septum assembly in both yeasts . Additionally, the spindle pole bodies are emerging as important transient locale for proteins that might play a key role in coupling the completion of mitosis to the onset of cytokinesis. Hum Mol Genet, 2000 Mar 22, 9(5), 675 - 84 Characterization of the Schizosaccharomyces pombe orthologue of the human survival motor neuron (SMN) protein; Owen N et al.; Childhood onset spinal muscular atrophy (SMA) is a common autosomal recessive disorder primarily characterized by the loss of lower alpha motor neurons . The underlying chromosomal defects causing SMA have been found in the survival motor neuron (SMN) gene . SMN has been shown previously to play a role in both snRNP biogenesis and mRNA processing, although direct evidence for the relationship between SMN and disease pathology has not been elucidated . SMN orthologues have been isolated in many species including Caenorhabditis elegans and Danio rerio . To study the function of SMN, we have identified and characterized the Schizosaccharomyces pombe orthologue of human SMN, smn1 (+) . We have demonstrated that smn1 (+) is essential for viability in S.pombe and yeast expressing missense mutations in Smn1p, which mimic mutations in patients with Type I SMA, show significant mislocalization of the protein and a decrease in cell viability . Wild-type Smn1p is localized predominantly in the nucleus whereas yeast expressing Smn1p with missense mutations or deletions of specific domains of the protein accumulate cytoplasmic aggregates . Overexpression of Smn1p results in an increase in the growth rate of cells . Furthermore, mutations within two highly conserved protein interaction domains have a dominant-negative effect on growth, indicating that each domain is of functional significance in S.pombe . These dominant phenotypes can be suppressed by overexpression of murine Smn in the same cell . Given the structural and functional similarities between the protein in fission yeast and higher eukaryotes, S.pombe will be an ideal organism to study the role of SMN in RNA processing. Hum Mol Genet, 2000 Mar 22, 9(5), 663 - 74 The Schizosaccharomyces pombe protein Yab8p and a novel factor, Yip1p, share structural and functional similarity with the spinal muscular atrophy-associated proteins SMN and SIP1; Hannus S et al.; The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of functional survival of motor neurons (SMN) protein . Previous studies have shown that SMN binds to the SMN-interacting protein SIP1 and mediates the assembly of spliceosomal U snRNPs in the cytoplasm . In addition, a nuclear function for SMN in pre-mRNA splicing has recently been proposed . Here, we describe the analysis of the Schizo-saccharomyces pombe protein Yab8p and provide evidence that it is structurally and functionally related to SMN found in higher eukaryotes . We show that Yab8p interacts via its N-terminus with a novel protein termed Yip1p . Importantly, Yip1p exhibits homology to SIP1, and the mode of binding to Yab8p is remarkably similar to the SMN-SIP1 interaction . Hence, Yip1p is likely to be the homologue of SIP1 in S.pombe . Yab8p and Yip1p localize predominantly in the nucleus . Genetic studies demonstrate that Yab8p is essential for viability . Strikingly, suppression of YAB8 expression in a conditional knock-out strain causes nuclear accumulation of poly(A) mRNA and inhibition of splicing . These data identify Yab8p as a novel factor involved in splicing and suggest that Yab8p exerts a function similar or identical to the nuclear pool of SMN . Our studies provide a model system to study the cellular function of SMN in yeast, and should help in understanding the molecular events leading to SMA. Mol Biol Cell, 2000 Apr, 11(4), 1169 - 81 Multistep phosphorelay proteins transmit oxidative stress signals to the fission yeast stress-activated protein kinase; Nguyen AN et al.; In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants . Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeast Schizosaccharomyces pombe . The fission yeast mpr1(+) gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1 . Spc1 activation upon oxidative stress is severely impaired in the Deltampr1 mutant as well as in the mpr1HQ strain, in which the putative phosphorylation site Mpr1-His221 is substituted with glutamine . In response to oxidative stress, Mpr1 binds to the Mcs4 response regulator that functions upstream of the Spc1 cascade, suggesting that Mcs4 is a cognate response regulator for Mpr1 . Unexpectedly, when exposed to hydrogen peroxide, Deltampr1 cells can induce the catalase gene ctt1(+), one of the transcriptional targets of the Spc1 pathway, and survive oxidative stress in the absence of significant Spc1 activation . We have found that Pap1, a bZIP transcription factor homologous to human c-Jun, can mediate induction of ctt1(+) expression upon oxidative stress independently of the Spc1 stress-activated protein kinase . These studies show that oxidative stress stimuli are transmitted by multiple pathways to induce specific gene expression. J Biol Chem, 2000 Jun 9, 275(23), 17677 - 82 Fidelity of eucaryotic DNA polymerase delta holoenzyme from Schizosaccharomyces pombe; Chen X et al.; The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template . Synthesis by pol delta alone was distributive . Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP . "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB . The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs . The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged . The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E . coli pol III HE at the same template position . This relatively low fidelity was caused by inefficient proofreading by the S . pombe polymerase-associated proofreading exonuclease . The S . pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA . A comparison of pol delta HE with E . coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair. J Biol Chem, 2000 Jun 16, 275(24), 18029 - 33 Nic1p, a relative of bacterial transition metal permeases in Schizosaccharomyces pombe, provides nickel ion for urease biosynthesis; Eitinger T et al.; The Schizosaccharomyces pombe genome sequencing project identified an open reading frame (O74869 and O74912, named Nic1p in the present study) with significant similarity to members of a family of bacterial transition metal permeases . These uptake systems transport Ni(2+) ion with extremely high affinity across the bacterial cytoplasmic membrane, but they differ in selectivity toward divalent transition metal cations . An S . pombe mutant harboring an interrupted nic1 allele (nic1-1) was strongly impaired in (63)Ni(2+) uptake in the presence of a high molar ratio of Mg(2+) relative to Ni(2+), conditions that reflect the natural situation . Under these conditions, the nic1-1 mutant contained only background activities of the nickel-dependent cytoplasmic enzyme urease and could not catabolize urea . Among a series of divalent transition metal cations tested (Cd(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+)), only Co(2+) caused considerable inhibition of Nic1p-mediated Ni(2+) uptake . On the other hand, experiments with (57)Co(2+) (at nm concentrations) did not show significant differences in Co(2+) uptake between the nic1-1 mutant and the parental strain . Our data suggest that Nic1p acts as a plasma-membrane nickel transporter in fission yeast, a finding that invites searches for isologous counterparts in higher eukaryotes. Genetics, 2000 Apr, 154(4), 1497 - 508 Autoregulated expression of Schizosaccharomyces pombe meiosis-specific transcription factor Mei4 and a genome-wide search for its target genes; Abe H et al.; The Schizosaccharomyces pombe mei4(+) gene encoding a forkhead transcription factor is necessary for the progression of meiosis and sporulation . We searched for novel meiotic genes, the expression of which is dependent on Mei4p, since only the spo6(+) gene has been assigned to its targets . Six known genes responsible for meiotic recombination were examined by Northern blotting, but none were Mei4 dependent for transcription . We determined the important cis-acting element, designated FLEX, to which Mei4p can bind . The S . pombe genome sequence database (The Sanger Centre, UK) was scanned for the central core heptamer and its flanking 3' sequence of FLEX composed of 17 nucleotides, and 10 candidate targets of Mei4 were selected . These contained a FLEX-like sequence in the 5' upstream nontranslatable region within 1 kb of the initiation codon . Northern blotting confirmed that 9 of them, named mde1(+) to mde9(+), were transcriptionally induced during meiosis and were dependent on mei4(+) . Most mde genes have not been genetically defined yet, except for mde9(+), which is identical to spn5(+), which encodes one of the septin family of proteins . mde3(+) and a related gene pit1(+) encode proteins related to Saccharomyces cerevisiae Ime2 . The double disruptant frequently produced asci having an abnormal number and size of spores, although it completed meiosis . We also found that the forkhead DNA-binding domain of Mei4p binds to the FLEX-like element in the putative promoter region of mei4 and that the maximum induction level of mei4 mRNA required functional mei4 activity . Furthermore, expression of a reporter gene driven by the authentic mei4 promoter was induced in vegetative cells by ectopic overproduction of Mei4p . These results suggest that mei4 transcription is positively autoregulated. Genetics, 2000 Apr, 154(4), 1451 - 61 A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene; Tsutsui Y et al.; To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation . One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype . The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae . An rhp57 (RAD57 homolog of S . pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination . The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene . An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57 . These characteristics of the rhp57 mutants are very similar to those of S . cerevisiae rad57 mutants . Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins. EMBO J, 2000 Apr 3, 19(7), 1691 - 702 A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein; Fousteri MI et al.; In Schizosaccharomyces pombe, rad18 is an essential gene involved in the repair of DNA damage produced by ionizing radiation and in tolerance of UV-induced DNA damage . The Rad18 protein is a member of the SMC (structural maintenance of chromosomes) superfamily, and we show that, like the other SMC proteins in condensin and cohesin, Rad18 is a component of a high-molecular-weight complex . This complex contains at least six other proteins, the largest of which is Spr18, a novel SMC family member closely related to Rad18, and likely to be its heterodimeric partner . SMC proteins have ATP-binding domains at the N- and C-termini, and two extended coiled-coil domains separated by a hinge in the middle . We show that the N-terminal ATP-binding domain of Rad18 is essential for all functions, and overexpression of an N-terminal mutant has a dominant-negative effect . We have identified an important mutation (S1045A) near the C-terminus of Rad18 that separates its repair and essential roles . Potential models for the role of the Rad18-Spr18 complex during DNA repair are discussed. EMBO J, 2000 Apr 3, 19(7), 1681 - 90 Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18; Kearsey SE et al.; We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe . After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent . Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence . Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures . We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast . Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast) . Release of mcm4 from chromatin occurs during S phase and requires DNA replication . Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication. Curr Genet, 2000 Feb, 37(2), 94 - 103 A gene associated with filamentous growth in Ophiostoma novo-ulmi has RNA-binding motifs and is similar to a yeast gene involved in mRNA splicing; Pereira V et al.; The COL1 gene was isolated from Ophiostoma novo-ulmi using the techniques of insertional mutagenesis and plasmid rescue . Sequence analyses suggest that the COL1 gene encodes a unique protein of 826 amino acids with consensus-type RNA-binding domains, most similar to a putative protein from Schizosaccharomyces pombe which resembles the C-terminus of the Saccharomyces cerevisiae U4/U6 splicing factor PRP24 . Disruption of the COL1 gene produced the yeast-like col1 mutant . The inability of the mutant to synthesize the COL1 gene product was confirmed by transcript analysis . Transformation of the col1 mutant with the COL1 gene restored the wild phenotype and production of the 4.0-kb mRNA . The results from this study demonstrate that the COL1 RNA-binding protein is associated with filamentous growth in O . novo-ulmi. Curr Genet, 2000 Feb, 37(2), 75 - 8 Importance of the Sgs1 helicase activity in DNA repair of Saccharomyces cerevisiae; Saffi J et al.; The Saccharomyces cerevisiae Sgs1 protein, together with Schizosaccharomyces pombe Rqh1 and the human Bloom and Werner proteins, is a DNA helicase of the Escherichia coli RecQ family . Mutation of SGS1 causes premature aging in yeast cells, including the accumulation of extrachromosomal rDNA circles . We have recently shown that Sgs1p interacts with the DNA repair Rad16p protein and is epistatic to Rad16p for UVC, 4-NQO and H2O2 lesions . Therefore we tested sgs1 strains containing mutations in the helicase and C-terminal domains . We demonstrate here that the helicase activity of the Sgs1 is important for most elements of the sgs1 mutation phenotype, including sensitivity to UVC, 4-NQO, H2O2, MMS and hydroxyurea. Biosci Biotechnol Biochem, 2000 Feb, 64(2), 438 - 42 Basidiomycete fungal gene encoding a regulatory subunit A homologue of protein phosphatase 2A; Ishizaki T et al.; A gene, Le.paa, encoding a regulatory subunit A (PR65) homologue of protein phosphatase 2A was isolated from the basidiomycete mushroom Lentinus edodes . The deduced Le.paa gene product (Le.PR65) had the highest sequence similarity to the Schizosaccharomyces pombe PR65 protein (54.1% similarity) . The Le.paa gene was shown to be transcribed more actively during the late stages of fruiting development of the fungus . Gill tissue in which basidiospores are formed contained abundant Le.paa transcript as compared with gill-depleted pileus and stipe. J Bacteriol, 2000 Apr, 182(8), 2104 - 12 Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe; Memisoglu A et al.; DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability . To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene . We report that S . pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism . We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance . Mutations in genes involved in nucleotide excision repair (rad13) and recombinational repair (rhp51) are much more alkylation sensitive than mag1 mutants . In addition, S . pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants . Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction . A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S . pombe is discussed. J Biol Chem, 2000 Mar 31, 275(13), 9557 - 62 Interaction of the somatostatin receptor subtype 1 with the human homolog of the Shk1 kinase-binding protein from yeast; Schwarzler A et al.; Interaction between the C terminus of a G-protein-coupled receptor and intracellular constituents may represent a crucial step in regulating signal transduction . To identify potential interacting candidates the C terminus of the somatostatin receptor subtype 1 was used as bait in a yeast two hybrid screen of a human brain cDNA library . We identified the human Skb1 sequence (Skb1Hs) as interacting protein, which is homologous to the yeast protein known Skb1 to down-regulate mitosis in Schizosaccharomyces pombe via binding to the Shk1 protein kinase; the latter is a homolog to the mammalian p21(cdc42/Rac)-activated protein kinases . Interaction required almost the entire C terminus of the somatostatin receptor subtype 1 including the conserved NPXXY motif of transmembrane region seven; in the case of the Skb1Hs most of the N terminus and an S-adenosylmethionine binding domain were mandatory, whereas the C terminus was not essential . Interaction was verified by coexpression experiments in human embryonic kidney cells . As revealed by immunocytochemical analysis Skb1Hs expressed alone aggregates in large cytosolic clusters . When coexpressed, receptor subtype 1 and Skb1Hs were colocalized at the cell surface; these cells showed a strong increase in somatostatin binding compared with cells expressing the receptor only . This may suggest that Skb1Hs acts like a chaperone by correctly targeting the receptor to the cell surface. Mol Cell Biol, 2000 Apr, 20(8), 2852 - 64 Fission yeast homologs of human CENP-B have redundant functions affecting cell growth and chromosome segregation; Baum M et al.; Two functionally important DNA sequence elements in centromeres of the fission yeast Schizosaccharomyces pombe are the centromeric central core and the K-type repeat . Both of these DNA elements show internal functional redundancy that is not correlated with a conserved DNA sequence . Specific, but degenerate, sequences in these elements are bound in vitro by the S . pombe DNA-binding proteins Abp1p (also called Cbp1p) and Cbhp, which are related to the mammalian centromere DNA-binding protein CENP-B . In this study, we determined that Abp1p binds to at least one of its target sequences within S . pombe centromere II central core (cc2) DNA with an affinity (K(s) = 7 x 10(9) M(-1)) higher than those of other known centromere DNA-binding proteins for their cognate targets . In vivo, epitope-tagged Cbhp associated with centromeric K repeat chromatin, as well as with noncentromeric regions . Like abp1(+)/cbp1(+), we found that cbh(+) is not essential in fission yeast, but a strain carrying deletions of both genes (Deltaabp1 Deltacbh) is extremely compromised in growth rate and morphology and missegregates chromosomes at very high frequency . The synergism between the two null mutations suggests that these proteins perform redundant functions in S . pombe chromosome segregation . In vitro assays with cell extracts with these proteins depleted allowed the specific assignments of several binding sites for them within cc2 and the K-type repeat . Redundancy observed at the centromere DNA level appears to be reflected at the protein level, as no single member of the CENP-B-related protein family is essential for proper chromosome segregation in fission yeast . The relevance of these findings to mammalian centromeres is discussed. Mol Cell Biol, 2000 Apr, 20(8), 2774 - 82 Conservation of glutamine-rich transactivation function between yeast and humans; Escher D et al.; Several eukaryotic transcription factors such as Sp1 or Oct1 contain glutamine-rich domains that mediate transcriptional activation . In human cells, promoter-proximally bound glutamine-rich activation domains activate transcription poorly in the absence of acidic type activators bound at distal enhancers, but synergistically stimulate transcription with these remote activators . Glutamine-rich activation domains were previously reported to also function in the fission yeast Schizosaccharomyces pombe but not in the budding yeast Saccharomyces cerevisiae, suggesting that budding yeast lacks this pathway of transcriptional activation . The strong interaction of an Sp1 glutamine-rich domain with the general transcription factor TAF(II)110 (TAF(II)130), and the absence of any obvious TAF(II)110 homologue in the budding yeast genome, seemed to confirm this notion . We reinvestigated the phenomenon by reconstituting in the budding yeast an enhancer-promoter architecture that is prevalent in higher eukaryotes but less common in yeast . Under these conditions, we observed that glutamine-rich activation domains derived from both mammalian and yeast transcription factors activated only poorly on their own but strongly synergized with acidic activators bound at the remote enhancer position . The level of activation by the glutamine-rich activation domains of Sp1 and Oct1 in combination with a remote enhancer was similar in yeast and human cells . We also found that mutations in a glutamine-rich domain had similar phenotypes in budding yeast and human cells . Our results show that glutamine-rich activation domains behave very similarly in yeast and mammals and that their activity in budding yeast does not depend on the presence of a TAF(II)110 homologue. J Biochem (Tokyo), 2000 Feb, 127(2), 233 - 8 Role of Atf1 and Pap1 in the induction of the catalase gene of fission yeast schizosaccharomyces pombe; Nakagawa CW et al.; We examined the induction of the catalase gene (ctt1(+)) of fission yeast Schizosaccharomyces pombe in response to several stresses by using mutants of transcription factors (Atf1 and Pap1) and a series of deletion mutants of the ctt1(+) promoter region . A transcription factor, Atf1, and its binding site are necessary for the induction of ctt1(+) by osmotic stress, UV irradiation, and heat shock . Induction by menadione treatment, which produces superoxide anion, required element A, the region from -111 to -90 (numbered with the transcription start site as +1) . The factor responsible for the induction of the gene by oxidative stress via element A was identified as the transcription factor Pap1 . We also found that Atf1 is activated by menadione treatment in pap1 mutant cells, although it is not activated by menadione treatment in pap1(+) cells . The activity of catalase is not increased in pap1 cells by several stresses, despite mRNA induction, suggesting that Pap1 plays some role in the expression of catalase activity. Methods Cell Sci, 1999, 21(2-3), 87 - 93 Synchronization of yeast cell populations; Walker GM; The study of synchronous populations of yeast cells has provided a wealth of information into regulatory aspects of the eukaryotic cell division cycle . Synchronized yeast cultures may also have potential benefit when exploiting yeasts in biotechnology . This paper provides an overview of the methods which have been used in the synchronization of cell division in budding and fission yeasts . The relative merits of these methods are outlined and protocols for preferred synchronization methods, based on size selection techniques, are described . In particular, centrifugal elutriation protocols for Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) are detailed as this method is regarded as one of the best ways of preparing 'unperturbed' synchronous yeast cultures for cell cycle studies. Antisense Nucleic Acid Drug Dev, 2000 Feb, 10(1), 29 - 34 The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe; Raponi M et al.; A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression . Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene . No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo . Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number. Biotechniques, 2000 Mar, 28(3), 532 - 6, 538, 540 Mutagenesis and gene cloning in Schizosaccharomyces pombe using nonhomologous plasmid integration and rescue; Hoffman CS et al.; Genes are commonly cloned in yeasts and bacteria by plasmid complementation, where the introduction of the gene of interest into a host strain carrying a recessive mutation in that gene suppresses the host's mutant phenotype . However, a lack of low copy cloning vectors in the fission yeast Schizosaccharomyces pombe can complicate this approach especially when overexpression of one gene may suppress a defect in another gene or when overexpression of the desired gene is detrimental, if not lethal, to the cell . We describe here a method of identifying mutations in S . pombe that allows for the rapid and direct cloning of the defective gene . This involves the nonhomologous integration of a marked plasmid into the yeast genome and its subsequent rescue into Escherichia coli, so that DNA at the site of insertion is incorporated into the recovered plasmid . As two of three insertions obtained in this study occurred outside of the affected gene's open reading frame, this method should be applicable to cloning both essential genes and nonessential genes. Gene, 2000 Jan 25, 242(1-2), 183 - 92 Fission yeast contains an rDNA binding activity that interacts specifically with regulatory sequences for ribosomal RNA synthesis; Guo A et al.; Basal level transcriptional initiation of fission yeast ribosomal RNA genes is dependent on the core ribosomal RNA gene promoter and is stimulated by an upstream rDNA promoter element and by regulatory sequences located in its approximately 3.5 kb intergenic rDNA spacer . A Schizosaccharomyces pombe sequence-specific rDNA binding activity was characterized that interacted with the upstream rDNA promoter region and that associated with required RNA polymerase I transcription components in initial fractionation steps . The rDNA binding activity was further purified and found to specifically associate with a region of the rDNA promoter between -80 and -56 . The promoter region required for stable binding correlates with that mediating activated levels of transcriptional initiation . This rDNA binding activity stimulates in vitro rRNA synthesis supported by templates bearing this upstream promoter domain but not by templates lacking it. DNA Res, 2000 Feb 28, 7(1), 27 - 30 mRNAs encoding zinc finger protein isoforms are expressed by alternative splicing of an in-frame intron in fission yeast; Okazaki K et al.; We report here that a gene encoding a protein with three zinc fingers is expressed predominantly to produce a protein containing only two zinc fingers in the fission yeast Schizosaccharomyces pombe . A third zinc finger resides within the in-frame intron that is normally spliced out . By RT-PCR analysis, we detected a minor transcript encoding a protein with three zinc fingers . Such alternative splicing for assortment of zinc finger domains have been reported in animals and implicated in switching of the target genes expressed specifically during development . This is the first report of the occurrence of such zinc finger assortment in lower eucaryotes. EMBO J, 2000 Mar 15, 19(6), 1389 - 96 Fission yeast switches mating type by a replication-recombination coupled process; Arcangioli B et al.; Fission yeast exhibits a homothallic life cycle, in which the mating type of the cell mitotically alternates in a highly regulated fashion . Pedigree analysis of dividing cells has shown that only one of the two sister cells switches mating type . It was shown recently that a site- and strand-specific DNA modification at the mat1 locus precedes mating-type switching . By tracking the fate of mat1 DNA throughout the cell cycle with a PCR assay, we identified a novel DNA intermediate of mating-type switching in S-phase . The time and rate of appearance and disappearance of this DNA intermediate are consistent with a model in which mating-type switching occurs through a replication-recombination coupled pathway . Such a process provides experimental evidence in support of a copy choice recombination model in Schizosaccharomyces pombe mating-type switching and is reminiscent of the sister chromatid recombination used to complete replication in the presence of certain types of DNA damage. J Biol Chem, 2000 Mar 17, 275(11), 7451 - 4 Human DNA damage checkpoint protein hRAD9 is a 3' to 5' exonuclease; Bessho T et al.; Human RAD9 protein (hRAD9) is a homolog of the fission yeast Rad9 protein, one of the six so-called checkpoint Rad proteins involved in the early steps of DNA damage checkpoint response in Schizosaccharomyces pombe . It has been shown previously that, in vivo, a highly modified form of hRAD9 makes a ternary complex with two other checkpoint Rad proteins, hRAD1 and hHUS1 (Volkmer, E., and Karnitz, L . M . (1999) J . Biol . Chem . 274, 567-570; St . Onge, R . P., Udell, C . M., Casselman, R., and Davey, S . (1999) Mol . Biol . Cell . 10, 1985-1995) . However, the function of this complex is not known at present . To help define the functions of checkpoint Rad proteins in humans, we expressed hRAD9 in Escherichia coli, purified the recombinant protein and characterized it . We found that hRAD9 is a 3' to 5' exonuclease and located the nuclease active site to the region between residues 51 and 91 of the 391-amino acid-long protein . Our results suggest that exonucleolytic processing of primary DNA lesion by hRAD9 may contribute to DNA damage checkpoint response in humans. Mol Microbiol, 2000 Mar, 35(5), 1135 - 45 The respiratory gene OXA1 has two fission yeast orthologues which together encode a function essential for cellular viability; Bonnefoy N et al.; The Saccharomyces cerevisiae nuclear gene OXA1, which is conserved from prokaryotes to human, was shown to be essential for cytochrome c oxidase and F1F0-ATP synthase biogenesis . We have searched for an orthologue of OXA1 in Schizosaccharomyces pombe, another yeast that is highly diverged from S . cerevisiae and which could more closely model higher eukaryotes . In particular, S . pombe exhibits a limited growth under anaerobic conditions and is petite negative, that is it does not tolerate large deletions of its mitochondrial DNA . Surprisingly, two S . pombe cDNAs able to complement an S . cerevisiae oxa1 mutation were isolated . The corresponding genes have different chromosomal locations and intron contents . They encode distinct proteins, both sharing a weak sequence identity one with the other and with Oxa1p . A phenotypic analysis of both single inactivations demonstrates that only one gene is essential for respiration in S . pombe . However, the double inactivation is lethal . This work gives new insight into the dependence of S . pombe viability upon oxa1 function, providing evidence of a connection between petite negativity, a functional respiratory chain and F1F0-ATP synthase complex in S . pombe. Mol Biol Cell, 2000 Mar, 11(3), 873 - 86 Mechanisms of sod2 gene amplification in Schizosaccharomyces pombe; Albrecht EB et al.; Gene amplification in eukaryotes plays an important role in drug resistance, tumorigenesis, and evolution . The Schizosaccharomyces pombe sod2 gene provides a useful model system to analyze this process . sod2 is near the telomere of chromosome I and encodes a plasma membrane Na(+)(Li(+))/H(+) antiporter . When sod2 is amplified, S . pombe survives otherwise lethal concentrations of LiCl, and >90% of the amplified sod2 genes are found in 180- and 225-kilobase (kb) linear amplicons . The sequence of the novel joint of the 180-kb amplicon indicates that it is formed by recombination between homologous regions near the telomeres of the long arm of chromosome I and the short arm of chromosome II . The 225-kb amplicon, isolated three times more frequently than the 180-kb amplicon, is a palindrome derived from a region near the telomere of chromosome I . The center of symmetry of this palindrome contains an inverted repeat consisting of two identical 134-base pair sequences separated by a 290-base pair spacer . LiCl-resistant mutants arise 200-600 times more frequently in strains deficient for topoisomerases or DNA ligase activity than in wild-type strains, but the mutant cells contain the same amplicons . These data suggest that amplicon formation may begin with DNA lesions such as breaks . In the case of the 225-kb amplicon, the breaks may lead to a hairpin structure, which is then replicated to form a double-stranded linear amplicon, or to a cruciform structure, which is then resolved to yield the same amplicon. Microbiol Immunol, 2000, 44(1), 51 - 6 Characterization of the cyclophilin of Trichophyton mentagrophytes; Kano R et al.; A genetic approach to cyclophilins in a dermatophyte, Trichophyton mentagrophytes, was carried out . The nucleotide and deduced amino acid sequences of the cyclophilin of T . mentagrophytes shared about 70% sequence similarity with those of Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans . However, the first 21 amino acid and the C-terminal amino acid regions of 188 to 226 of the T . mentagrophytes cyclophilin were distinct from those of the other fungal cyclophilins . The recombinant glutathione S-transferase (GST)-T . mentagrophytes cyclophilin fusion protein produced by Escherichia coli was purified . The protease digest of the fusion protein had a molecular weight of about 13 kDa and peptidyl-prolyl cis-trans isomerase (PPI) activity . This digest protein from T . mentagrophytes was confirmed to be cyclophilin by proving PPI activity. Biosci Biotechnol Biochem, 2000 Jan, 64(1), 149 - 59 Identification of a 14-3-3 protein from Lentinus edodes that interacts with CAP (adenylyl cyclase-associated protein), and conservation of this interaction in fission yeast; Zhou GL et al.; We previously identified a gene encoding a CAP (adenylyl cyclase-associated protein) homologue from the edible Basidiomycete Lentinus edodes . To further discover the cellular functions of the CAP protein, we searched for CAP-interacting proteins using a yeast two-hybrid system . Among the candidates thus obtained, many clones encoded the C-terminal half of an L . edodes 14-3-3 homologue (designated cip3) . Southern blot analysis indicated that L . edodes contains only one 14-3-3 gene . Overexpression of the L . edodes 14-3-3 protein in the fission yeast Schizosaccharomyces pombe rad24 null cells complemented the loss of endogenous 14-3-3 protein functions in cell morphology and UV sensitivity, suggesting functional conservation of 14-3-3 proteins between L . edodes and S . pombe . The interaction between L . edodes CAP and 14-3-3 protein was restricted to the N-terminal domain of CAP and was confirmed by in vitro co-precipitation . Results from both the two-hybrid system and in vivo co-precipitation experiments showed the conservation of this interaction in S . pombe . The observation that a 14-3-3 protein interacts with the N-terminal portion of CAP but not with full-length CAP in L . edodes and S . pombe suggests that the C-terminal region of CAP may have a negative effect on the interaction between CAP and 14-3-3 proteins, and 14-3-3 proteins may play a role in regulation of CAP function. J Cell Sci, 2000 Apr, 113 ( Pt 7), 1223 - 30 A checkpoint that monitors cytokinesis in Schizosaccharomyces pombe; Liu J et al.; Cell division in Schizosaccharomyces pombe is achieved through the use of a medially positioned actomyosin ring . A division septum is formed centripetally, concomitant with actomyosin ring constriction . Genetic screens have identified mutations in a number of genes that affect actomyosin ring or septum assembly . These cytokinesis-defective mutants, however, undergo multiple S and M phases and die as elongated cells with multiple nuclei . Recently, we have shown that a mutant allele of the S . pombe drc1(+)/cps1(+) gene, which encodes a 1,3-(beta)-glucan synthase subunit, is defective in cytokinesis but displays a novel phenotype . drc1-191/cps1-191 cells are capable of assembling actomyosin rings and completing mitosis, but are incapable of assembling the division septum, causing them to arrest as binucleate cells with a stable actomyosin ring . Each nucleus in arrested cps1-191 cells is able to undergo S phase but these G(2) nuclei are significantly delayed for entry into the M phase . In this study we have investigated the mechanism that causes cps1-191 to block with two G(2) nuclei . We show that the inability of cps1-191 mutants to proceed through multiple mitotic cycles is not related to a defect in cell growth . Rather, the failure to complete some aspect of cytokinesis may prevent the G(2)/M transition of the two interphase-G(2) nuclei . The G(2)/M transition defect of cps1-191 mutants is suppressed by a mutation in the wee1 gene and also by the dominant cdc2 allele cdc2-1w, but not the cdc2-3w allele . Transient depolymerization of all F-actin structures also allowed a significant proportion of the cps1-191 cells to undergo a second round of mitosis . We conclude that an F-actin and Wee1p dependent checkpoint blocks G(2)/M transition until previous cytokinesis is completed. Biochemistry, 2000 Mar 14, 39(10), 2659 - 66 In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway; Alleva JL et al.; Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions . AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion . Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER . This study represents the first report of the biochemical reconstitution of the AER pathway . A base mispair-containing substrate is repaired in a reaction requiring S . pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase . Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site. Genome, 2000 Feb, 43(1), 205 - 7 Identification of rpaP1-5 and rpaP2-6 genes encoding two additional variants of the 60S acidic ribosomal proteins of Schizosaccharomyces pombe; Bonnet C et al.; In the fission yeast, four genes (rpaP1-1, rpaP1-3, rpaP2-2, and rpaP2-4) encoding two variants of the RpaP1 and RpaP2 ribosomal proteins (rp) have been characterized . We have identified cDNA for additional variants called RpaP1.5 and RpaP2.6 . Sequence comparison suggests that RpaP1.5 diverged before RpaP1.1 and RpaP1.3 and that RpaP2.6 is closer to RpaP2.2 than to RpaP2.4 . The corresponding genes, rpaP1-5 and rpaP2-6, are transcribed coordinately with other rp genes. EMBO J, 2000 Mar 1, 19(5), 1108 - 18 Essential interaction between the fission yeast DNA polymerase delta subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21(Cip1)-like PCNA binding motif; Reynolds N et al.; Direct interaction between DNA polymerase delta and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear . We show that the 54 kDa subunit of DNA polymerase delta from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro . Binding is effected via a short sequence at the C-terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21(Cip1) protein . This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo . We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B-subunit of polymerase delta, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously . Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding. Biochemistry, 2000 Mar 7, 39(9), 2149 - 63 Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity; Arnold SM et al.; UDP-glucose:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that operates as a gatekeeper for quality control by preventing transport of improperly fol