Genetics . 2005 Jan 16; {Epub ahead of print} A novel recombination pathway initiated by the MRN complex eliminates palindromes during meiosis in Schizosaccharomyces pombe; Farah JA et al.; DNA palindromes are rare in humans but are associated with meiosis-specific transloca-tions . The conserved Mre11*Rad50*Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair . Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160 bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion . Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separate from the Rec12 (ortholog of Spo11) pathway . We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication . Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication . Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes. J Cell Sci, 2005 Jan 15, 118(Pt 2), 447 - 59 Mcp6, a meiosis-specific coiled-coil protein of Schizosaccharomyces pombe, localizes to the spindle pole body and is required for horsetail movement and recombination; Saito TT et al.; We report here that a meiosis-specific gene of Schizosaccharomyces pombe denoted mcp6(+) (meiotic coiled-coil protein) encodes a protein that is required for the horsetail movement of chromosomes at meiosis I . The mcp6(+) gene is specifically transcribed during the horsetail phase . Green fluorescent protein (GFP)-tagged Mcp6 appears at the start of karyogamy, localizes to the spindle-pole body (SPB) and then disappears before chromosome segregation at meiosis I . In the mcp6Delta strain, the horsetail movement was either hampered (zygotic meiosis) or abolished (azygotic meiosis) and the pairing of homologous chromosomes was impaired . Accordingly, the allelic recombination rates of the mcp6Delta strain were only 10-40% of the wild-type rates . By contrast, the ectopic recombination rate of the mcp6Delta strain was twice the wild-type rate . This is probably caused by abnormal homologous pairing in mcp6Delta cells because of aberrant horsetail movement . Fluorescent microscopy indicates that SPB components such as Sad1, Kms1 and Spo15 localize normally in mcp6Delta cells . Because Taz1 and Swi6 also localized with Sad1 in mcp6Delta cells, Mcp6 is not required for telomere clustering . In a taz1Delta strain, which does not display telomere clustering, and the dhc1-d3 mutant, which lacks horsetail movement, Mcp6 localized with Sad1 normally . However, we observed abnormal astral microtubule organization in mcp6Delta cells . From these results, we conclude that Mcp6 is necessary for neither SPB organization nor telomere clustering, but is required for proper astral microtubule positioning to maintain horsetail movement. J Microbiol, 2004 Dec, 42(4), 353 - 6 Transcriptional Regulation of the Schizosaccharomyces pombe Gene Encoding Glutathione S-Transferase I by a Transcription Factor Pap1; Kim HG et al.; In a previous study, a gst gene was isolated from the fission yeast Schizosaccharomyces pombe . This gene was dubbed gst I, and was characterized using the gstI-lacZ fusion plasmid pYSH2000 . In this work, four additional fusion plasmids, pYSHSD1, pYSHSD2, pYSHSD3 and pYSHSD4, were constructed, in order to carry (respectively) 770, 551, 358 and 151 bp upstream regions from the translational initiation point . The sequence responsible for induction by aluminum, mercury and hydrogen peroxide was located in the range between -1,088 and -770 bp upstream of the S . pombe gst I gene . The same region was identified to contain the nucleotide sequence responsible for regulation by Pap1, and has one putative Pap1 binding site, TTACGTAT, located in the range between -954 ~ -947 bp upstream of the gst I gene . Negatively acting sequences are located between -1,088 and -151 bp . These findings imply that the Pap1 protein is involved in basal and inducible transcription of the gst I gene in the fission yeast S . pombe. Mol Cells, 2004 Dec 31, 18(3), 332 - 9 Differential Regulation of Three Genes Encoding Glutathione S-Transferases in Schizosaccharomyces pombe; Kim HG et al.; Glutathione S-transferases (GSTs) are detoxifying enzymes that catalyze the conjugation of glutathione with a variety of reactive electrophilic compounds . Three GST genes were previously characterized in the fission yeast Schizosaccharomyces pombe . In this work, we examined the transcriptional regulation of these genes using individual GST-lacZ fusions and RT-PCR . Basal synthesis of b-galactosidase from the GSTII-lacZ fusion was higher than from the GSTI-lacZ and GSTIII-lacZ fusion . Diethylmaleate (0.2 mM) greatly enhanced the synthesis of b-galactosidase from the GSTII-lacZ fusion, but did not affect synthesis from the other two fusion genes . A switch to 0.3% glucose or 0.3% sucrose as sole carbon source enhanced expression from the GSTIII-lacZ fusion gene, while sodium nitroprusside (1.5 mM), tert-butylhydroquinone (0.2 mM), and L-buthionine-{S,R}-sulfoximine (0.01 mM) increased expression of the GSTII gene . The effects of these agents on GST mRNA levels were confirmed by measurements employing RT-PCR . Our results suggest that transcription of the three S . pombe GST genes is subjected to differential regulation under various stress conditions, and may be linked to their different physiological functions. Cell Mol Biol Lett, 2004, 9(4A), 675 - 83 The sensitivity of yeast and yeast-like cells to new lysosomotropic agents; Krasowska A et al.; The lysosomotropic action of the compounds DM-11 and DMAL-12s against Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans is species- and pH-dependent . At pH 6.0, DMAL-12s is less effective against S . cerevisiae and S . pombe but more effective against C . albicans than DM-11 . At pH 8.0, DMAL-12s strongly inhibits the growth of S . cerevisiae but has only a marginal effect on the resistant C . albicans . S . pombe did not grow at pH 8.0 . As shown by quinacrine accumulation, DM-11 causes a general intracellular acidification in all three species, while with DMAL-12s, the acidification is marginal . Morphological changes caused by DMAL-12s in S . cerevisiae affect the cell interior but not surface structures, while S . pombe cells exhibit a thickened and wrinkled cell wall, shrunken protoplast and "grainy" plasma membrane . A large number of blisters resembling lipid droplets were observed inside S . cerevisiae and S . pombe vacuoles . The high susceptibility of S . pombe cells to the action of DM-11 and DMAL-12s contrasts with the low sensitivity of S . pombe H(+)-ATPase to the agents . In our C . albicans isolate, DMAL 12s did not have an effect on cell morphology and appeared to be unable to penetrate the cells, especially at pH 8.0. J Biol Chem . 2005 Jan 12; {Epub ahead of print} The fission yeast protein, Ker1p, is an ortholog of RNA polymerase I subunit A14 in saccharomyces cerevisiae, and is required for stable association of Rrn3p and RPA21 in Pol I; Imazawa Y et al.; A heterodimer formed by the A14 and A43 subunits (A14/A43) of RNA polymerase I1 (pol I) in Saccharomyces cerevisiae is proposed to correspond to the Rpb4/Rpb7 and C17/C25 heterodimers in RNA polymerases II and III, respectively, and to play a role(s) in the recruitment of pol I to the promoter . However, the question of whether the A14/A43 heterodimer is conserved in eukaryotes other than S . cerevisiae remains unanswered, although both Rpb4/Rpb7 and C17/C25 are conserved from yeasts to human . To address this question, we have isolated a Schizosaccharomyces pombe gene named ker1+, using a yeast two-hybrid system including rpa21+, which encodes an ortholog of A43 as bait . Although no homolog of A14 has previously been found in the S . pombe genome, functional characterization of Ker1p and an alignment between Ker1p and A14 show that Ker1p is an ortholog of A14 . Disruption of ker1+ results in temperature-sensitive (ts) growth and the ts-deficit of ker1D is suppressed by either overexpression of rpa21+ or rrn3+, which encodes the rDNA transcription factor Rrn3p, suggesting that Ker1p is involved in stabilizing the association of RPA21 and Rrn3p in pol I . We also found that Ker1p dissociates from pol I in post-log-phase cells, suggesting that Ker1p is involved in growth-dependent regulation of rDNA transcription. Yeast . 2005 Jan 11;22(2):91-97 {Epub ahead of print} Pro-oxidant action of phloxine B on fission yeast Schizosaccharomyces pombe; Mutoh N et al.; A Schizosaccharomyces pombe mutant deficient in Cu,Zn-superoxide dismutase (sod1 mutant) was hypersensitive to phloxine B, which is used as a food-colouring agent and also to distinguish diploid strains of Sz . pombe from haploid strains, under illumination with light . The pro-oxidant nature of phloxine B was confirmed biochemically . The carbonyl content of proteins (which represents protein oxidation) increased, and the reduced form of glutathione was transiently decreased by phloxine B treatment under illumination with light . When cells were treated with phloxine B under light, carbonyl content of proteins in the sod1 mutant was greater than that in the wild-type and amount of glutathione was much decreased in the sod1 mutant compared with the wild-type . Genes induced by oxidative stress were induced by phloxine B under illumination with light and some were induced by phloxine B without light . Copyright (c) 2005 John Wiley & Sons, Ltd. Methods Enzymol, 2005, 392, 297 - 307 Labeling and Characterization of Small RNAs Associated with the RNA Interference Effector Complex RITS; Verdel A et al.; RNA interference (RNAi) is a gene silencing mechanism that acts at both the posttranscriptional and transcriptional levels . We have recently identified an RNA-containing complex, named RNA-induced transcriptional silencing (RITS), that directly links RNAi to transcriptional gene silencing in Schizosaccharomyces pombe . Here we review the affinity purification methods we use to isolate RITS and describe how to purify, detect, and analyze RNAs associated with this complex. Eukaryot Cell, 2005 Jan, 4(1), 55 - 62 Loss of Meiotic Rereplication Block in Saccharomyces cerevisiae Cells Defective in Cdc28p Regulation; Rice LM et al.; Cdc28p is the major cyclin-dependent kinase in Saccharomyces cerevisiae . Its activity is required for blocking the reinitiation of DNA replication during mitosis . Here, we show that under conditions where Cdc28p activity is improperly regulated-either through the loss of function of the Schizosaccharomyces pombe wee1 ortholog Swe1p or through the expression of a dominant CDC28 allele, CDC28AF-diploid yeast cells are able to complete several rounds of premeiotic DNA replication within a single meiotic cell cycle . Moreover, a percentage of mutant cells exhibit a "multispore" phenotype, possessing the ability to package more than four spores within a single ascus . These multispored asci contain both even and odd numbers of viable spores . In order for meiotic rereplication and multispore formation to occur, cells must initiate homologous recombination and maintain proper chromosome cohesion during meiosis I . Rad9p- or Rad17p-dependent checkpoint mechanisms are not required for multispore formation and neither are the B-type cyclin Clb6p and the cyclin-dependent kinase inhibitor Sic1p . Finally, we present evidence of a possible role for a Cdc55p-dependent protein phosphatase 2A in initiating meiotic replication. FEBS Lett, 2005 Jan 17, 579(2), 331 - 6 Constitutive activity of Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe implicates residues in addition to conserved cysteines in alpha-keto acid activation; Crichton PG et al.; Activity of the plant mitochondrial alternative oxidase (AOX) can be regulated by organic acids, notably pyruvate . To date, only two well-conserved cysteine residues have been implicated in this process . We report the functional expression of two AOX isozymes (Sauromatum guttatum Sg-AOX and Arabidopsis thaliana At-AOX1a) in Schizosaccharomyces pombe . Comparison of the response of these two isozymes to pyruvate in isolated yeast mitochondria and disrupted mitochondrial membranes reveals that in contrast to At-AOX1a, Sg-AOX activity is insensitive to pyruvate and appears to be in a constitutively active state . As both of these isozymes conserve the two cysteines, we propose that such contrasting behaviour must be a direct result of differences in their amino acid sequence and have subsequently identified novel candidate residues. Genome Biol . 2005;6(1):R1 . Epub 2004 Dec 15. Global expression changes resulting from loss of telomeric DNA in fission yeast; Mandell JG et al.; BACKGROUND: Schizosaccharomyces pombe cells lacking the catalytic subunit of telomerase (encoded by trt1+) lose telomeric DNA and enter crisis, but rare survivors arise with either circular or linear chromosomes . Survivors with linear chromosomes have normal growth rates and morphology, but those with circular chromosomes have growth defects and are enlarged . We report the global gene-expression response of S . pombe to loss of trt1+ . RESULTS: Survivors with linear chromosomes had expression profiles similar to cells with native telomeres, whereas survivors with circular chromosomes showed continued upregulation of core environmental stress response (CESR) genes . In addition, survivors with circular chromosomes had altered expression of 51 genes compared to survivors with linear chromosomes, providing an expression signature . S . pombe progressing through crisis displayed two waves of altered gene expression . One coincided with crisis and consisted of around 110 genes, 44% of which overlapped with the CESR . The second was synchronized with the emergence of survivors and consisted of a single class of open reading frames (ORFs) with homology both to RecQ helicases and to dh repeats at centromeres targeted for heterochromatin formation via an RNA interference (RNAi) mechanism . Accumulation of transcript from the ORF was found not only in trt1- cells, but also in dcr1- and ago1- RNAi mutants, suggesting that RNAi may control its expression . CONCLUSIONS: These results demonstrate a correlation between a state of cellular stress, short telomeres and growth defects in cells with circular chromosomes . A putative new RecQ helicase was expressed as survivors emerged and appears to be transcriptionally regulated by RNAi, suggesting that this mechanism operates at telomeres. Oncogene, 2005 Jan 10, 24(2), 299 - 305 The Plk3-Cdc25 circuit; Myer DL et al.; Polo-like kinases (Plks) are key regulators of the cell cycle, especially in the G2 phase and mitosis . They are incorporated into signaling networks that regulate many aspects of the cell cycle, including but not limited to centrosome maturation and separation, mitotic entry, chromosome segregation, mitotic exit, and cytokinesis . The Plks have well conserved 30-amino-acid elements, designated polo boxes (PBs), located in their carboxyl-termini, which with their flanking regions constitute a functional Polo-box domain (PBD) . Members of the Plk family exist in a variety of organisms including Polo in Drosophila melanogaster; Cdc5 in Saccharomyces cerevisiae; Plo1 in Schizosaccharomyces pombe; Plx1 in Xenopus laevis; and Plk1, Snk/Plk2, Fnk/Prk/Plk3, and Sak in mammals . Polo, Cdc5, and Plo1 are essential for viability . The Plks can be separated into two groups according to their functions . The first group (Polo, Cdc5, plo1, Plx1, and Plk1) primarily performs mitotic functions, whereas the second group (Plk2 and Plk3) appears to have additional functions during the G1, S, and G2 phases of the cell cycle . Several contributions to this issue will discuss different aspects of Plk involvement in cell-cycle regulation . This review, therefore, will focus on the role of Plk3 in regulating Cdc25 phosphatase function and its effect on the cell cycle. Mol Cell Biol, 2005 Jan, 25(2), 808 - 18 Human Protection of Telomeres 1 (POT1) Is a Negative Regulator of Telomerase Activity In Vitro; Kelleher C et al.; The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans . Human POT1 appears to interact with telomeres both through direct binding to the 3' overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex . The influence of POT1 on telomerase activity has not been studied at the molecular level . We show here that POT1 negatively effects telomerase activity in vitro . We find that the DNA binding activity of POT1 is required for telomerase inhibition . Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase. Mol Cell Biol, 2005 Jan, 25(2), 716 - 27 Functional comparison of the tup11 and tup12 transcriptional corepressors in fission yeast; Fagerstrom-Billai F et al.; Gene duplication is considered an important evolutionary mechanism . Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11(+) and tup12(+), that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein . Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles . Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast . However, tup11(-) and tup12(-) mutants have different phenotypes on media containing KCl and CaCl(2) . Consistent with the functional difference between tup11(-) and tup12(-) mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11(+) and tup12(+) genes . Many of these genes are differentially derepressed in tup11(-) mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions . Genes specifically derepressed in tup12(-) mutants require the Ssn6 protein for their repression . As for Tup12, Ssn6 is also required for efficient adaptation to KCl- and CaCl(2)-mediated stress . We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response. Mol Cell Biol, 2005 Jan, 25(2), 590 - 601 Global effects on gene expression in fission yeast by silencing and RNA interference machineries; Hansen KR et al.; Histone modifications influence gene expression in complex ways . The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs) . We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast) . The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation . Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres . Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions . We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi . Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing. Genes Dev, 2005 Jan 15, 19(2), 242 - 54 Epub 2004 Dec 29. Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin; Webb CJ et al.; Discrete sequence elements known as exonic splicing enhancers (ESEs) have been shown to influence both the efficiency of splicing and the profile of mature mRNAs in multicellular eukaryotes . While the existence of ESEs has not been demonstrated previously in unicellular eukaryotes, the factors known to recognize these elements and mediate their communication with the core splicing machinery are conserved and essential in the fission yeast Schizosaccharomyces pombe . Here, we provide evidence that ESE function is conserved through evolution by demonstrating that three exonic splicing enhancers derived from vertebrates (chicken ASLV, mouse IgM, and human cTNT) promote splicing of two distinct S . pombe pre-messenger RNAs (pre-mRNAs) . Second, as in extracts from mammalian cells, ESE function in S . pombe is compromised by mutations and increased distance from the 3'-splice site . Third, three-hybrid analyses indicate that the essential SR (serine/arginine-rich) protein Srp2p, but not the dispensable Srp1p, binds specifically to both native and heterologous purine-rich elements; thus, Srp2p is the likely mediator of ESE function in fission yeast . Finally, we have identified five natural purine-rich elements from S . pombe that promote splicing of our reporter pre-mRNAs . Taken together, these results provide strong evidence that the genesis of ESE-mediated splicing occurred early in eukaryotic evolution. Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 337 - 42 Epub 2004 Dec 28. DNA replication origins in the Schizosaccharomyces pombe genome; Dai J et al.; Origins of DNA replication in Schizosaccharomyces pombe lack a specific consensus sequence analogous to the Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, raising the question of how they are recognized by the replication machinery . Because all well characterized S . pombe origins are located in intergenic regions, we analyzed the sequence properties and biological activity of such regions . The AT content of intergenes is very high ( approximately 70%), and runs of A's or T's occur with a significantly greater frequency than expected . Additionally, the two DNA strands in intergenes display compositional asymmetry that strongly correlates with the direction of transcription of flanking genes . Importantly, the sequence properties of known S . pombe origins of DNA replication are similar to those of intergenes in general . In functional studies, we assayed the in vivo origin activity of 26 intergenes in a 68-kb region of S . pombe chromosome 2 . We also assayed the origin activity of sets of randomly chosen intergenes with the same length or AT content . Our data demonstrate that at least half of intergenes have potential origin activity and that the relative ability of an intergene to function as an origin is governed primarily by AT content and length . We propose a stochastic model for initiation of DNA replication in the fission yeast . In this model, the number of AT tracts in a given sequence is the major determinant of its probability of binding SpORC and serving as a replication origin . A similar model may explain some features of origins of DNA replication in metazoans. Anal Biochem, 2005 Jan 15, 336(2), 202 - 12 A glucanase-driven fractionation allows redefinition of Schizosaccharomyces pombe cell wall composition and structure: assignment of diglucan; Magnelli PE et al.; Purified endoglucanases have been used to determine the composition of Schizosaccharomyces pombe cell wall . This structure has been traditionally studied after isolating its components (mannoproteins, alpha1,3-glucan, beta1,3-glucan, and a branched beta-glucan) with hot alkali . Instead, we sequentially removed the polysaccharides by digesting with endo-beta1,3-glucanase and with a novel endo-alpha1,3-glucanase (mutanase) . After this gentle isolation we observed that a branched beta1,3-beta1,6-glucan is much more abundant than previously described . By scaling-up the new protocol we prepared large amounts of the highly branched glucan and determined its structural features . We have named this highly branched beta-glucan diglucan, reflecting its two types of beta linkages . We have also identified an insoluble endoglucanase-resistant type of 1,3-linked glucan present in S . pombe cell walls . We redefined the wall composition of S . pombe vegetative cells by this new method . Finally, to demonstrate its application, we determined the cell wall composition of known mutant strains. FEBS Lett, 2005 Jan 3, 579(1), 48 - 52 Glyceraldehyde-3-phosphate dehydrogenase and actin associate with RNA polymerase II and interact with its Rpb7 subunit; Mitsuzawa H et al.; RNA polymerase II (pol II) purified from the fission yeast Schizosaccharomyces pombe was previously reported to be associated with the general transcription factor TFIIF and the C-terminal domain phosphatase Fcp1, as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has recently been implicated in transcriptional activation in human cells . Here, we provide evidence that the Rpb7 subunit of pol II interacts with GAPDH . Two-hybrid screen identified GAPDH as an Rpb7-binding protein . In addition, GAPDH was affinity-purified from S . pombe extract by using an Rpb4/Rpb7-coupled column . We also identified actin as a pol II-associated protein and revealed the interaction between actin and Rpb7. Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2588 - 97 A recQ Gene Homolog from the Basidiomycetous Mushroom Lentinula edodes: Sequence Analysis and Expression; Katsukawa S et al.; We cloned and sequenced a recQ gene homolog from the basidiomycetous mushroom Lentinula edodes (Le.) . This gene, named Le.recQ, was found to have a coding capacity of 945 amino acids (aa) and be interrupted by 11 small introns . The deduced Le.RECQ protein was clearly smaller than other fungal RecQ proteins such as Neurospora crassa QDE3 (1955 aa), Schizosaccharomyces pombe Rqh1 (1328 aa), and Saccharomyces cerevisiae SGS1 (1447 aa) . It exhibited the highest homology to the Arabidopsis thaliana RecQl4A protein (1182 aa) in its size and aa sequence . Northern-blot analysis showed that the Le.recQ gene is transcribed at similar levels during mycelial development in L . edodes fruiting-body formation on a sawdust-corn bran medium . The L . edodes dikaryotic mycelial cells, which had been vegetatively grown in SMY liquid medium, were found to contain a clearly larger amount of Le.recQ transcript than the L . edodes two compatible monokaryotic mycelial cells . Expression of Le.recQ cDNA in S . cerevisiae might partially complement defects associated with the loss of its homolog S . cerevisiae SGS1 gene. Mol Biol Cell . 2004 Dec 22; {Epub ahead of print} Identification of Cell Cycle-regulated Genes in Fission Yeast; Peng X et al.; Monitoring Editor: John Pringle Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes . To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray . Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle . Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression . Peaks of gene expression were found to be distributed throughout the entire cell cycle . Furthermore, we found that four promoter motifs exhibited strong association with cell-cycle-phase-specific expression . Examination of the regulation of MCB motif-containing genes through the perturbation of DSC/MBF-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements . Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found approximately 140 genes that are cell cycle-regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Nucleic Acids Res, 2004 Dec 22, 32(22), 6706 - 15 Print 2004. Tfg3, a subunit of the general transcription factor TFIIF in Schizosaccharomyces pombe, functions under stress conditions; Kimura M et al.; TFIIF is a general transcription factor (GTF) that binds to RNA polymerase II (pol II) for subsequent recruitment of pol II to a promoter . TFIIF of Saccharomyces cerevisiae contains a small subunit, designated Tfg3, in addition to two conserved subunits, TFIIFalpha (Tfg1) and TFIIFbeta (Tfg2) . In this study, we characterized Tfg3 of Schizosaccharomyces pombe . Using Tfg3 fused to green fluorescent protein (GFP), we found that Tfg3 is located in nuclei, and it is assembled into the C-terminal domain phosphatase (Fcp1)/TFIIF/pol II complex via interactions with TFIIFalpha and TFIIFbeta . As in the case of S.cerevisiae, Tfg3 in S.pombe forms part of another GTF, namely TFIID . The TFIID complex isolated from S.pombe that had been cultured at elevated temperatures included increased levels of Tfg3 . The interaction of recombinant Tfg3 with TATA-binding protein (TBP), the central subunit of TFIID, was temperature-dependent . Moreover, a mutant of S.pombe that lacked the gene for Tfg3 was sensitive to a battery of stresses including temperature up-shift . Starting from a mutant with tfg3- mutation, we isolated five species of multicopy suppressors . Expression levels of the suppressor genes were lower in the mutant cell than in wild-type cell at an elevated temperature . Taken together, we propose that Tfg3 is involved in transcriptional regulation under stress conditions, in particular, at high temperatures. J Cell Sci, 2005 Jan 1, 118(Pt 1), 199 - 210 Nak1 interacts with Hob1 and Wsp1 to regulate cell growth and polarity in Schizosaccharomyces pombe; Huang TY et al.; We have previously reported that Nak1, a group-II germinal center (GC) kinase, is essential for polarized growth in Schizosaccharomyces pombe . Here, we provide evidence that Nak1 regulates cell growth and polarity, in part, through its interactions with Hob1 (an Rvs167/amphiphysin homolog) and Wsp1 (Wiskott-Aldrich-syndrome-protein homolog) . We found that Nak1, Hob1 and Wsp1 interact physically, and that both Hob1/green-fluorescent-protein (Hob1-GFP) and Wsp1-GFP fusion proteins localized to F-actin patches at growing cell ends and medial division sites . Hob1-GFP was dissociated from patches in cells lacking Wsp1 . Also, Hob1 overexpression dissociated Wsp1-GFP from foci, inhibited Wsp1-directed F-actin formation in vitro and partially restored polarity defects associated with Wsp1 overexpression or nak1 repression . Furthermore, loss of both Wsp1 and Hob1 resulted in rounded cells, slow growth and multiple septae . Together, these observations suggest that Hob1 and Wsp1 cooperate to mediate cell polarity, growth and division . Repression of nak1 resulted in a random redistribution of Hob1-GFP and Wsp1-GFP foci, and inhibition of Wsp1-directed F-actin formation in vitro . Furthermore, hob1Delta and wsp1Delta mutants exhibited synthetic growth defects in combination with nak1 repression, suggesting that Nak1 has redundant functions with Hob1 and Wsp1 . Collectively, our results suggest that Nak1 both regulates and cooperates with Hob1 and Wsp1 to promote F-actin formation and polarized cell growth. J Cell Sci, 2005 Jan 1, 118(Pt 1), 157 - 174 The novel fission yeast (1,3){beta}-D-glucan synthase catalytic subunit Bgs4p is essential during both cytokinesis and polarized growth; Cortes JC et al.; Schizosaccharomyces pombe contains four putative (1,3)beta-D-glucan synthase (GS) catalytic subunits, Bgs1p-4p . In this work, we cloned bgs4(+) and show that Bgs4p is the only subunit found to be a part of the GS enzyme and essential for maintaining cell integrity during cytokinesis and polarized growth . Here we show that bgs4(+), cwg1(+) (cwg1-1 shows reduced cell-wall beta-glucan and GS catalytic activity) and orb11(+) (orb11-59 is defective in cell morphogenesis) are the same gene . bgs4(+) is essential for spore germination and bgs4(+) shut-off produces cell lysis at growing poles and mainly at the septum prior to cytokinesis, suggesting that Bgs4p is essential for cell wall growth and to compensate for an excess of cell wall degradation during cytokinesis . Shut-off and overexpression analysis suggest that Bgs4p forms part of a GS catalytic multiprotein complex and that Bgs4p-promoted cell-wall beta-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)alpha-D-glucan synthase . Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and at each stage of wall synthesis or remodeling that occurs during sexual differentiation: mating, zygote and spore formation, and spore germination . Bgs4p timing and requirements for proper positioning during cytokinesis and its localization pattern during spore maturation differ from those of Bgs1p . Bgs4p localizes overlapping the contractile ring once Bgs1p is present and a Calcofluor white-stained septum material is detected, suggesting that Bgs4p is involved in a late process of secondary or general septum synthesis . Unlike Bgs1p, Bgs4p needs the medial ring but not the septation initiation network proteins to localize with the other septation components . Furthermore, Bgs4p localization depends on the polarity establishment proteins . Finally, F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, but it is not needed for the stable maintenance of Bgs4p at the growing sites, poles and septum . All these data show for the first time an essential role for a Bgs subunit in the synthesis of a (1,3)beta-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed. Mol Microbiol, 2005 Jan, 55(1), 104 - 14 Rpc25, a conserved RNA polymerase III subunit, is critical for transcription initiation; Zaros C et al.; Summary Rpc25 is a strongly conserved subunit of RNA polymerase III with homology to Rpa43 in RNA polymerase I, Rpb7 in RNA polymerase II and the archaeal RpoE subunit . A central domain of Rpc25 can replaced the corresponding region of Rpb7 with little or no growth defect, underscoring the functional relatedness of these proteins . Rpc25 forms a heterodimer with Rpc17, another conserved component of RNA polymerase III . A conditional mutant (rpc25-S100P) impairs this interaction . rpc25-S100P and another conditional mutant obtained by complementation with the Schizosaccharomyces pombe subunit (rpc25-Sp) were investigated for the properties of their purified RNA polymerase III . The mutant enzymes were defective in the specific synthesis of pre-tRNA transcripts but acted at a wild-type level on poly{d(A-T)} templates . They were also indistinguishable from wild type in transcript elongation, cleavage and termination . These data indicate that Rpc25 is needed for transcription initiation but is not critical for the elongating properties of RNA polymerase III. Genetics, 2004 Dec, 168(4), 1867 - 75 Differential Activation of eIF2 Kinases in Response to Cellular Stresses in Schizosaccharomyces pombe; Zhan K et al.; Phosphorylation of eukaryotic initiation factor-2 (eIF2) is an important mechanism mitigating cellular injury in response to diverse environmental stresses . While all eukaryotic organisms characterized to date contain an eIF2 kinase stress response pathway, the composition of eIF2 kinases differs, with mammals containing four distinct family members and the well-studied lower eukaryote Saccharomyces cerevisiae expressing only a single eIF2 kinase . We are interested in the mechanisms by which multiple eIF2 kinases interface with complex stress signals and elicit response pathways . In this report we find that in addition to two previously described eIF2 kinases related to mammalian HRI, designated Hri1p and Hri2p, the yeast Schizosaccharomyces pombe expresses a third eIF2 kinase, a Gcn2p ortholog . To delineate the roles of each eIF2 kinase, we constructed S . pombe strains expressing only a single eIF2 kinase gene or deleted for the entire eIF2 kinase family . We find that Hri2p is the primary activated eIF2 kinase in response to exposure to heat shock, arsenite, or cadmium . Gcn2p serves as the primary eIF2 kinase induced during a nutrient downshift, treatment with the amino acid biosynthetic inhibitor 3-aminotriazole, or upon exposure to high concentrations of sodium chloride . In one stress example, exposure to H(2)O(2), there is early tandem activation of both Hri2p and Gcn2p . Interestingly, with extended stress conditions there is activation of alternative secondary eIF2 kinases, suggesting that eukaryotes have mechanisms of coordinate activation of eIF2 kinase in their stress remediation responses . Deletion of these eIF2 kinases renders S . pombe more sensitive to many of these stress conditions. Genetics, 2004 Dec, 168(4), 1843 - 53 In Vivo Activation of Protein Kinase A in Schizosaccharomyces pombe Requires Threonine Phosphorylation at Its Activation Loop and Is Dependent on PDK1; Tang Y et al.; Phosphoinositide-dependent protein kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases . This family includes protein kinase C (PKC), protein kinase B (PKB), p70/p90 ribosomal S6 kinases (RSK and S6K), and the catalytic subunit of cAMP-dependent protein kinase (PKA) . Although PDK1 phosphorylates and activates PKC, PKB, and RSK in vivo, PDK1 regulation of PKA remains controversial . We isolated ksg1, the fission yeast ortholog of mammalian PDK1, as a suppressor of growth defects caused by loss of the stress-activated MAP kinase, Spc1 . Here, we demonstrate that Ksg1 is required for activation of PKA . Cells containing the ksg1.12 thermolabile allele exhibit pleiotropic phenotypes, including the failure to arrest in G(1) and an inability to conjugate . The ksg1.12 allele strongly suppresses defects associated with unregulated PKA . Pka1, the catalytic subunit of cAMP-dependent protein kinase, is phosphorylated in vivo at Thr-356, which is located in the activation loop of the kinase and corresponds to Thr-197 in mammalian PKA . Phosphorylation of Thr-356 is required for in vivo activation of Pka1 and is dependent upon Ksg1 . These data provide experimental evidence that PKA is a physiological substrate for PDK1. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D476 - 80 Inparanoid: a comprehensive database of eukaryotic orthologs; O'Brien KP et al.; The Inparanoid eukaryotic ortholog database is a collection of pairwise ortholog groups between 17 whole genomes; Anopheles gambiae, Caenorhabditis briggsae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Takifugu rubripes, Gallus gallus, Homo sapiens, Mus musculus, Pan troglodytes, Rattus norvegicus, Oryza sativa, Plasmodium falciparum, Arabidopsis thaliana, Escherichia coli, Saccharomyces cerevisiae and Schizosaccharomyces pombe . Complete proteomes for these genomes were derived from Ensembl and UniProt and compared pairwise using Blast, followed by a clustering step using the Inparanoid program . An Inparanoid cluster is seeded by a reciprocally best-matching ortholog pair, around which inparalogs (should they exist) are gathered independently, while outparalogs are excluded . The ortholog clusters can be searched on the website using Ensembl gene/protein or UniProt identifiers, annotation text or by Blast alignment against our protein datasets . The entire dataset can be downloaded, as can the Inparanoid program itself. Mutat Res, 2005 Jan 6, 569(1-2), 13 - 27 Yeast signaling pathways in the oxidative stress response; Ikner A et al.; Oxidative stress that generates the reactive oxygen species (ROS) is one of the major causes of DNA damage and mutations . The "DNA damage checkpoint" that arrests cell cycle and repairs damaged DNA has been a focus of recent studies, and the genetically amenable model systems provided by yeasts have been playing a leading role in the eukaryotic checkpoint research . However, means to eliminate ROS are likely to be as important as the DNA repair mechanisms in order to suppress mutations in the chromosomal DNA, and yeasts also serve as excellent models to understand how eukaryotes combat oxidative stress . In this article, we present an overview of the signaling pathways that sense oxidative stress and induce expression of various anti-oxidant genes in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the pathogenic yeast Candida albicans . Three conserved signaling modules have been identified in the oxidative stress response of these diverse yeast species: the stress-responsive MAP kinase cascade, the multistep phosphorelay and the AP-1-like transcription factor . The structure and function of these signaling modules are discussed. Mol Cell Biol, 2005 Jan, 25(1), 303 - 11 Molecular and cellular dissection of mating-type switching steps in Schizosaccharomyces pombe; Holmes AM et al.; A strand-specific imprint (break) controls mating-type switching in fission yeast . By introducing a thiamine repressible promoter upstream of the mat1 locus, we can force transcription through the imprinted region, erasing the imprint and inhibiting further mating-type switching, in a reversible manner . Starting from a synchronized, virgin M-cell population, we show that the site- and strand-specific break is formed when DNA replication intermediates appear at mat1 during the first S phase . The formation of the break is concomitant with a replication fork pause and binding of the Swi1 protein at mat1 until early G(2) and then rapidly disappears . Upon its formation, the break remains stable throughout the cell cycle and triggers mating-type switching during the second S phase . Finally, we have recreated the mating-type switching pedigree at the molecular and single-cell levels, allowing for the first time separation between the establishment of imprinting and its developmental fate. Mol Cell Biol, 2005 Jan, 25(1), 185 - 96 Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage; Andrews EA et al.; The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M(r) complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62 . The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint . We have characterized Nse2 and show here that it is a SUMO ligase . Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein . Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation . nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal . Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain . Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex. Mol Cell Biol, 2005 Jan, 25(1), 172 - 84 Composition and architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) complex; Sergeant J et al.; The rad18 gene of Schizosaccharomyces pombe is an essential gene that is involved in several different DNA repair processes . Rad18 (Smc6) is a member of the structural maintenance of chromosomes (SMC) family and, together with its SMC partner Spr18 (Smc5), forms the core of a high-molecular-weight complex . We show here that both S . pombe and human Smc5 and -6 interact through their hinge domains and that four independent temperature-sensitive mutants of Rad18 (Smc6) are all mutated at the same glycine residue in the hinge region . This mutation abolishes the interactions between the hinge regions of Rad18 (Smc6) and Spr18 (Smc5), as does mutation of a conserved glycine in the hinge region of Spr18 (Smc5) . We purified the Smc5-6 complex from S . pombe and identified four non-SMC components, Nse1, Nse2, Nse3, and Rad62 . Nse3 is a novel protein which is related to the mammalian MAGE protein family, many members of which are specifically expressed in cancer tissue . In initial steps to understand the architecture of the complex, we identified two subcomplexes containing Rad18-Spr18-Nse2 and Nse1-Nse3-Rad62 . The subcomplexes are probably bridged by a weaker interaction between Nse2 and Nse3. Proc Natl Acad Sci U S A, 2004 Dec 28, 101(52), 17952 - 7 Epub 2004 Dec 14. Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA; Gaczynska M et al.; In eukaryotes, the initiation of DNA replication requires the interaction between origin sequences and the origin recognition complex (ORC), which is highly conserved . In this report, atomic force microscopy (AFM) was used to examine the binding of Schizosaccharomyces pombe (sp) ORC and the spOrc4 protein with the sp autonomously replicating sequence 1 (ars1) . AFM imaging revealed that spORC binding to ars1 occurred solely through spOrc4p and depended on the N-terminal AT-hook domains present in spOrc4p . At high molar ratios of spORC (or spOrc4p alone) to DNA (6:1), all of the input ars1 was bound in a one protein complex to one plasmid manner . Restriction digestion and AFM analysis of protein-DNA fragments revealed the presence of two binding sites in ars1 . One site mapped to a region centered at nucleotide 838 of ars1 previously detected by DNase I protection that was reported to be essential for the autonomously replicating sequence activity of ars1 . The second site mapped to a previously uncharacterized region centered at nucleotide 1148 . AFM showed that the length of the DNA fragment complexed with either spORC or spOrc4p was shortened by approximately 140 bp, suggesting the wrapping of two turns of the DNA around the spOrc4p alone as well as the spOrc4p in spORC . We also show that treatment of the spORC (spOrc4p)-ars1 complex with topoisomerase I induced a negative shift in the topoisomer distribution . These findings suggest that the binding of spORC to origin DNA alters the structure of the DNA . Thus, in the case of spORC, due to its unusual spOrc4p, at least two factors are likely to influence ars1 activation . These include the selective binding of the complex to A- and T-rich regions and the alteration of the DNA structure due to its wrapping around spOrc4p. J Cell Sci, 2005 Jan 1, 118(Pt 1), 39 - 50 Epub 2004 Dec 07. Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function; Dunaway S et al.; The protein kinase Chk1 is required for proper arrest of the cell cycle in response to DNA damage . We have previously shown in Schizosaccharomyces pombe, that upon DNA damage, phosphorylation of Chk1 correlates with checkpoint activation and that phosphorylated Chk1 is capable of interacting with the 14-3-3 proteins, Rad24 and Rad25 . The interaction between Rad24 and Chk1 is stimulated tenfold after exposure to DNA damaging agents and we postulate that it is an important event in the DNA damage checkpoint response pathway in fission yeast . We identified a stretch of leucine residues as the domain in Chk1 that mediates the interaction with 14-3-3 proteins . Substitution of leucine residues with alanine disrupts the interaction with Rad24 and also prevents Chk1 from becoming phosphorylated in response to DNA damaging agents . Cells expressing the mutants are sensitive to UV radiation . In this study, we also show that Chk1 accumulates in the nucleus in response to DNA damage and this behavior is dependent on Rad24 . Interestingly, the 14-3-3 binding domain mutants also fail to localize to the nucleus prompting a search for localization sequences within Chk1 . Our investigations have identified the presence of both functional nuclear import and nuclear export sequences encoded in S . pombe Chk1 that, in conjunction with 14-3-3 proteins, may play a prominent role in regulating Chk1 localization and function. Yeast, 2005 Jan 15, 22(1), 31 - 41 Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2; Goddard A et al.; To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted alpha-galactosidase encoded by the Sz . pombe ORF SPAC869.07c (CAB60017), which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae . The alpha-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-alpha-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of alpha-galactosidase developed the most intense blue colour . Unlike assays based on beta-galactosidase, the Sz . pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis . It is therefore suitable for screening large numbers of colonies . To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway . The sxa2 > mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library . There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection . The mel1 alpha-galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz . pombe . Copyright (c) 2004 John Wiley & Sons, Ltd. Methods Mol Biol, 2004, 296, 181 - 8 In situ assay for analyzing the chromatin binding of proteins in fission yeast; Kearsey SE et al.; An in situ technique for studying the chromatin binding of proteins in single fission yeast cells (Schizosaccharomyces pombe) is described . Cells are permeabilized by enzymatic digestion and extracted with a detergent-containing buffer . This procedure removes soluble proteins, but proteins that are bound to insoluble cell structures such as chromatin are retained, and overall cell morphology is maintained . Extraction of proteins is monitored by fluorescence microscopy, either using fluorescently tagged proteins or by indirect immunofluorescence . This method allows the chromatin association of proteins to be correlated with other cell cycle events without the need for cell synchronization. Methods Mol Biol, 2004, 296, 3 - 30 Cell cycle molecules and mechanisms of the budding and fission yeasts; Humphrey T et al.; The cell cycles of the budding yeast Saccharomyces cerevisiae and the fission yeast, Schizosaccharomyces pombe are currently the best understood of all eukaryotes . Studies in these two evolutionarily divergent organisms have identified common control mechanisms, which have provided paradigms for our understanding of the eukaryotic cell cycle . This chapter provides an overview of our current knowledge of the molecules and mechanisms that regulate the mitotic cell cycle in these two yeasts. Nat Cell Biol, 2004 Dec, 6(12), 1245 - 6 Intra-nuclear microtubules and a mitotic spindle orientation checkpoint; Zimmerman S et al.; Cells of the fission yeast Schizosaccharomyces pombe have a checkpoint mechanism that reportedly monitors the orientation of the mitotic spindle . Astral microtubules in pre-anaphase spindles are thought to contact the contractile actin ring at the plasma membrane in order to rotate the spindle and to sense spindle orientation . Here, we show that these microtubules are actually inside the nuclear envelope. Mol Cell Biol, 2004 Dec, 24(24), 10621 - 35 C-terminal anchoring of mid1p to membranes stabilizes cytokinetic ring position in early mitosis in fission yeast; Celton-Morizur S et al.; mid1p is a key factor for the central positioning of the cytokinetic ring in Schizosaccharomyces pombe . In interphase and early mitosis, mid1p forms a medial cortical band overlying the nucleus, which may represent a landmark for cytokinetic ring assembly . It compacts before anaphase into a tight ring with other cytokinetic ring components . We show here that mid1p binds to the medial cortex by at least two independent means . First, mid1p C-terminus association with the cortex requires a putative amphipathic helix adjacent to mid1p nuclear localization sequence (NLS), which is predicted to insert directly into the lipid bilayer . This association is stabilized by the polybasic NLS . mid1p mutated within the helix and the NLS forms abnormal filaments in early mitosis that are not properly anchored to the medial cortex . Misplaced rings assemble in late mitosis, indicating that mid1p C-terminus binding to membranes stabilizes cytokinetic ring position . Second, the N terminus of mid1p has the ability to associate faintly with the medial cortex and is sufficient to form tight rings . In addition, we show that mid1p oligomerizes . We propose that membrane-bound oligomers of mid1p assemble recruitment "platforms" for cytokinetic ring components at the medial cortex and stabilize the ring position during its compaction. Genes Cells, 2004 Dec, 9(12), 1189 - 97 Hub1 is an essential ubiquitin-like protein without functioning as a typical modifier in fission yeast; Yashiroda H et al.; Hub1 exhibits 23% sequence identity to ubiquitin . However, Hub1 lacks the C-terminal Gly, which is essential for covalent attachment to target protein(s) of ubiquitin and other ubiquitin-like (UBL) modifiers . Instead, Hub1 proteins in all eukaryotes retain the di-Tyr just before a single variable residue at the C-terminus, so one intriguing question is whether Hub1 could be linked to substrate through the conserved Tyr or not . Here we studied Hub1 in Schizosaccharomyces pombe . Gene disruption experiment revealed that hub1+ is essential . Remarkably, the mutant cells harbouring Hub1 lacking the di-Tyr could grow similar to wild-type cells, indicating that the di-Tyr is dispensable for the essential function of Hub1 . Moreover, we could not observe cleavage of Flag-tag fused with C-terminus of Hub1 . It suggests that the processing for conjugation via conserved Tyr is not likely to occur in Hub1, and Hub1 is a novel class of the UBL protein family . Finally, we isolated a temperature-sensitive allele, hub1-1 . This temperature sensitivity could be suppressed by overproduction of Rpb10 or Snu66, the former of which is one of the common subunits of the RNA polymerases and the other is the component of the spliceosome . We also observed that pre-mRNA splicing was impaired in hub1-1. Mol Biol Cell . 2004 Nov 24; {Epub ahead of print} The Role of the Kinesin Motor KipA in Microtubule Organization and Polarized Growth of Aspergillus nidulans; Konzack S et al.; Monitoring Editor: John Pringle Polarized growth in filamentous fungi requires the integrity of the microtubule (MT) cytoskeleton . We found that growing MTs in Aspergillus nidulans merge at the center of fast growing tips and discovered that a kinesin motor protein, KipA, related to Tea2p of Schizosaccharomyces pombe, is required for this process . In a DeltakipA strain, MT plus ends reach the tip but show continuous lateral movement . Hyphae lose directionality and grow in curves, apparently due to mislocalization of the vesicle supply center (Spitzenkorper) in the apex . GFP-KipA accumulates at MT plus ends, whereas a KipA rigor mutant protein, GFP-KipA(G223E), coated MTs evenly . These findings suggest that KipA requires its intrinsic motor activity to reach the MT plus end . Using KipA as a MT plus-end marker, we found bidirectional organization of MTs and determined the locations of MTOCs at nuclei, in the cytoplasm, and at septa. Nat Struct Mol Biol, 2004 Dec, 11(12), 1223 - 9 Epub 2004 Dec. Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection; Lei M et al.; The POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryotes . It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells . Here, we report the crystal structure at a resolution of 1.73 A of the N-terminal half of human POT1 (hPOT1) protein bound to a telomeric single-stranded DNA (ssDNA) decamer, TTAGGGTTAG, the minimum tight-binding sequence indicated by in vitro binding assays . The structure reveals that hPOT1 contains two oligonucleotide/ oligosaccharide-binding (OB) folds; the N-terminal OB fold binds the first six nucleotides, resembling the structure of the S . pombe Pot1pN-ssDNA complex, whereas the second OB fold binds and protects the 3' end of the ssDNA . These results provide an atomic-resolution model for chromosome end-capping. Cell, 2004 Nov 24, 119(5), 603 - 14 Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage; Sanders SL et al.; Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities . Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe . Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function . Rather, we find the modification has a role in DNA damage response . Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest . Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage . These results argue that H4-K20 methylation functions as a "histone mark" required for the recruitment of the checkpoint protein Crb2. Cell, 2004 Nov 24, 119(5), 583 - 6 Making the right choice--long-range chromosomal interactions in development; Broach JR; Schizosaccharomyces pombe has the remarkable potential to switch mating type as often as every generation, through selective interaction of an expressor locus with either of two transcriptionally silent donor loci . Recent results demonstrate that selection of the appropriate donor locus likely occurs through mating-type and heterochromatin-dependent spreading of a protein complex that marks the correct donor locus. J Cell Sci, 2004 Dec 1, 117(Pt 25), 6163 - 6174 Epub 2004 Nov 16. Schizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that regulates cell wall {beta}-glucan biosynthesis through the GTPase Rho1p; Tajadura V et al.; Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell-wall synthesis . Here, we describe the cloning and characterization of rgf3(+), a member of the Rho family of guanine nucleotide exchange factors (Rho GEFs) . The rgf3(+) gene was cloned by complementation of a mutant (ehs2-1) hypersensitive to drugs that interfere with cell-wall biosynthesis . The rgf3(+) gene was found to be essential for cell viability and depletion of Rgf3p afforded phenotypes similar to those obtained following depletion of Rho1p . However, the cell death caused by Rgf3p depletion could be rescued by the presence of 1.2 M sorbitol, whereas depletion of Rho1 was lethal under the same conditions . We show that Rgf3p is a specific Rho1-GEF . The hypersensitivity to drugs affecting the cell wall of the ehs2-1 mutant was suppressed by overexpression of rho1(+) but not by any of the other GTPases of the Rho family . Rgf3p interacted with the GDP-bound form of Rho1p and promoted the GDP-GTP exchange . In addition, we show that overexpression of Rgf3p produces multiseptated cells and increases beta-1,3-glucan synthase activity and the amount of cell wall beta-1,3-glucan . Rgf3p localized to the septum and the mRNA level was regulated in a cell-cycle-dependent manner peaking during septation . Our results suggest that Rgf3p acts as a positive activator of Rho1p, probably activating the Rho functions that coordinate cell-wall biosynthesis to maintain cell integrity during septation. Yeast, 2004 Nov, 21(15), 1289 - 305 pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast; Matsuyama A et al.; A novel series of plasmid vectors named pDUAL have been developed . These vectors enable one to introduce not only multicopies of genes with episomal maintenance but also a single copy with chromosomal integration into the fission yeast, Schizosaccharomyces pombe . The multicopy plasmids can be easily converted to fragments for chromosomal integration by digestion of the plasmids with a certain restriction endonuclease before transformation of the yeast cells . The resultant fragments, lacking the autonomously replicating sequence, are designed for targeting into the chromosomal leu1 locus by homologous recombination . Whether the transformants are the results of episomal maintenance of the plasmid or homologous gene targeting can be readily checked by their requirement for uracil or leucine, or by the PCR diagnostic analysis . Furthermore, we propose the use of pDUAL derivatives for PCR-based chromosomal tagging of a gene to introduce several tags into 5'-terminus of a gene, employing a set of primers . Using these all-in-one vectors, a suitable mode of expression of a cloned gene can be selected for individual analysis without any complicated subcloning processes. Yeast, 2004 Nov, 21(15), 1279 - 88 Posttranslational activation, site-directed mutation and phylogenetic analyses of the lysine biosynthesis enzymes alpha-aminoadipate reductase Lys1p (AAR) and the phosphopantetheinyl transferase Lys7p (PPTase) from Schizosaccharomyces pombe; Guo S et al.; Alpha-aminoadipate reductase (AAR), the signature enzyme for lysine biosynthesis in fungi, catalyses the conversion of alpha-aminoadipate to alpha-aminoadipate-semiadehyde in the presence of ATP and NADPH . In Saccharomyces cerevisiae and Candida albicans, the LYS2-encoded AAR is posttranslationally activated by CoA and the LYS5-encoded PPTase . The fission yeast Schizosaccharomyces pombe is evolutionarily highly diverged from S . cerevisiae and C . albicans . We report here several unusual activation characteristics of Sz . pombe Lys1p and Lys7p, isofunctional to Lys2p (AAR) and Lys5p (PPTase), respectively . Unlike the Lys2p from S . cerevisiae and C . albicans, the Sz . pombe Lys1p was active when expressed in E . coli and exhibited significant AAR activity without the addition of CoA or the Sz . pombe Lys7p intron free PPTase . Somewhat higher AAR activity was obtained with the addition of CoA and the Sz . pombe Lys7p PPTase . Substitution of G910A, S913T or S913A in the Sz . pombe Lys1p activation domain (IGGHSI) resulted in no AAR activity . Similarly, substitutions of several amino acid residues in the Sz . pombe Lys7p PPTase domain (G79A, R80K and P81A in Core 1; F93W, D94E, F95W and N96D in Core 1a; G124A, V125I and D126E in Core 2; K172R, E173D and K177R in Core 3) also resulted in no activation of Lys1p and no AAR activity . The Sz . pombe Lys1p amino acid sequence showed a high degree of similarity to other fungal Lys2p proteins; however, the Lys7p amino acid sequence showed much less similarity to other bacterial, fungal and animal PPTases representing several phylogenetic groups. Genetics . 2004 Nov 15; {Epub ahead of print} Conserved locus-specific silencing functions of S . pombe sir2+ Freeman-Cook L, Gomez EB, Spedale EJ, Marlett J, Forsburg SL, Pillus L, Laurenson P. In Schizosaccharomyces pombe, three genes, sir2(+), hst2(+) and hst4(+), encode members of the Sir2 family of conserved NAD(+) dependent protein deacetylases . The S . pombe sir2(+) gene encodes a nuclear protein that is neither essential for viability nor for resistance to treatment with UV nor a microtubule- destabilizing agent . However, sir2(+) is essential for full transcriptional silencing of centromeres, telomeres and the cryptic mating-type loci . Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions . Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p . The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci . However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci . Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S . pombe sir2(+), suggesting overlapping deacetylation substrates in both species . These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin. Genetics . 2004 Nov 15; {Epub ahead of print} RNA silencing in Aspergillus nidulans is independent of RNA dependent RNA polymerases; Hammond TM et al.; The versatility of RNA dependent RNA polymerases (RDRPs) in eukaryotic gene silencing is perhaps best illustrated in the Kingdom Fungi . Biochemical and genetic studies of Schizosaccharomyces pombe and Neurospora crassa show that these types of enzymes are involved in a number of fundamental gene silencing processes, including heterochromatin regulation and RNA silencing in S . pombe and meiotic silencing and RNA silencing in N . crassa . Here we show that Aspergillus nidulans, another model fungus, does not require an RDRP for inverted repeat transgene (IRT) induced RNA silencing . However, RDRP requirements may vary within the Aspergillus genus as genomic analysis indicates that A . nidulans, but not Aspergillus fumigatus or Aspergillus oryzae, has lost a QDE-1 ortholog, an RDRP associated with RNA silencing in N . crassa . We also provide evidence suggesting that 5'-->3' transitive RNA silencing is not a significant aspect of A . nidulans IRT-RNA silencing . These results indicate a lack of conserved kingdom wide requirements for RDRPs in fungal RNA silencing. Genetics . 2004 Nov 15; {Epub ahead of print} A Natural Meiotic DNA Break Site in Schizosaccharomyces pombe is a Hotspot of Gene Conversion, Highly Associated with Crossing-over; Cromie G et al.; In Schizosaccharomyces pombe meiosis-specific DNA breaks that initiate recombination are observed at prominent but widely separated sites . We investigated the relationship between breakage and recombination at one of these sites, the mbs1 locus on chromosome I . A total of 10% breakage was mapped to four clusters spread over a 2.1 kb region . Gene conversion of markers within the clusters occurred in 3% of meiotic chromatids (11% of tetrads), making mbs1 a conversion hotspot compared to other fission yeast markers . Approximately 80% of these conversions were associated with crossing-over of flanking markers, suggesting strong bias in meiotic break repair towards the generation of crossovers . This bias was observed in conversion events at three other loci, ade6, ade7 and ura1 . 50-80% of all crossovers seen in a 90 kb region flanking mbs1 occurred in a 4.8 kb interval containing the break sites . Thus, mbs1 is also a hotspot of crossing-over, with breakage at mbs1 generating most of the crossovers in the 90 kb interval . Neither Rec12 (Spo11 ortholog) nor I-SceI-induced breakage at mbs1 was significantly associated with crossing-over in an apparently break-free interval >25 kb away . Possible mechanisms for generating crossovers in such break-free intervals are discussed. FEMS Microbiol Rev, 2004 Nov, 28(5), 581 - 601 Non-homologous end-joining factors of Saccharomyces cerevisiae; Dudasova Z et al.; DNA double-strand breaks (DSB) are considered to be a severe form of DNA damage, because if left unrepaired, they can cause a cell death and, if misrepaired, they can lead to genomic instability and, ultimately, the development of cancer in multicellular organisms . The budding yeast Saccharomyces cerevisiae repairs DSB primarily by homologous recombination (HR), despite the presence of the KU70, KU80, DNA ligase IV and XRCC4 homologues, essential factors of the mammalian non-homologous end-joining (NHEJ) machinery . S . cerevisiae, however, lacks clear DNA-PKcs and ARTEMIS homologues, two important additional components of mammalian NHEJ . On the other hand, S . cerevisiae is endowed with a regulatory NHEJ component, Nej1, which has not yet been found in other organisms . Furthermore, there is evidence in budding yeast for a requirement for the Mre11/Rad50/Xrs2 complex for NHEJ, which does not appear to be the case either in Schizosaccharomyces pombe or in mammals . Here, we comprehensively describe the functions of all the S . cerevisiae NHEJ components identified so far and present current knowledge about the NHEJ process in this organism . In addition, this review depicts S . cerevisiae as a powerful model system for investigating the utilization of either NHEJ or HR in DSB repair. Mol Biol Cell, 2005 Jan, 16(1), 358 - 71 Epub 2004 Nov 10. The Nuclear Kinase Lsk1p Positively Regulates the Septation Initiation Network and Promotes the Successful Completion of Cytokinesis in Response to Perturbation of the Actomyosin Ring in Schizosaccharomyces pombe; Karagiannis J et al.; Cytokinesis in fission yeast requires the function of an actomyosin-based contractile ring whose constriction is dependent on a signaling module termed the septation initiation network (SIN) . In response to minor perturbation of the ring, the duration of SIN signaling is extended concurrently with a delay in nuclear cycle progression . These mechanisms require the conserved phosphatase Clp1p/Flp1p and facilitate the successful completion of cytokinesis, thereby increasing cellular viability . To isolate novel components of this cytokinesis monitoring system, we screened a genome-wide bank of protein kinase deletion mutants and identified Lsk1p, a nuclear-localized protein kinase . Similar to clp1Delta mutants, and in contrast to wild type, lsk1Delta cells are unable to maintain the integrity of the actomyosin ring upon treatment with low doses (0.3 muM) of latrunculin A . However, unlike clp1Delta mutants, lsk1Delta cells are competent to delay nuclear cycle progression after cytokinetic failure . In addition, lsk1Delta mutants suppress the lethal, multiseptate phenotype conferred by hyperactivation of the SIN, demonstrating that Lsk1p is a positive regulator of this module . In this report, we demonstrate that Lsk1p acts in parallel to Clp1p to promote actomyosin ring stability upon checkpoint activation . Our studies also establish that actomyosin ring maintenance and nuclear cycle delay in response to cytokinetic perturbation can be genetically resolved into independent pathways. Cell, 2004 Nov 12, 119(4), 469 - 80 Heterochromatin regulates cell type-specific long-range chromatin interactions essential for directed recombination; Jia S et al.; Mating-type switching in Schizosaccharomyces pombe involves replacing genetic information at the expressed mat1 locus with sequences copied from one of two silent donor loci, mat2-P or mat3-M, located within a 20-kb heterochromatic domain . Donor selection is dictated by cell type: mat2 is the preferred donor in M cells, and mat3 is the preferred donor in P cells . Here we show that a recombination-promoting complex (RPC) containing Swi2 and Swi5 proteins exhibits cell type-specific localization pattern at the silent mating-type region and this differential localization modulates donor preference during mating-type switching . In P cells, RPC localization is restricted to a recombination enhancer located adjacent to mat3, but in M cells, RPC spreads in cis across the entire silent mating-type interval in a heterochromatin-dependent manner . Our analyses implicate heterochromatin in long-range regulatory interactions and suggest that heterochromatin imposes at the mating-type region structural organization that is important for the donor-choice mechanism. Biochem J . 2004 Nov 10; {Epub ahead of print} A microbial TRP-like polycystic kidney disease related ion channel gene; Palmer CP et al.; Ion channel genes have been discovered in many microbial organisms . We have investigated a microbial transient receptor potential (TRP) ion channel gene which has most homology to polycystic kidney disease related ion channel genes . We have shown that this gene (pkd2) is essential for cellular viability and involved in cell growth and cell wall synthesis . Expression of this gene increases following damage to the cell wall . This fission yeast pkd2 gene, homologs of which are found in all eukaryotic cells, appears to be a key signaling component in the regulation of cell shape and cell wall synthesis in yeast through an interaction with a Rho1-GTPase . A model for the mode of action of this Schizosaccharomyces pombe protein in a Ca 2+ signaling pathway is hypothesized. J Biol Chem, 2005 Jan 7, 280(1), 408 - 17 Epub 2004 Nov 08. Interaction of Checkpoint Proteins Hus1/Rad1/Rad9 with DNA Base Excision Repair Enzyme MutY Homolog in Fission Yeast, Schizosaccharomyces pombe; Chang DY et al.; The DNA glycosylase MutY homolog (MYH) is responsible for removing adenines misincorporated opposite DNA strands containing guanine or 7,8-dihydro-8-oxoguanine by base excision repair thereby preventing G:C to T:A mutations . MYH has been shown to interact with the proliferating cell nuclear antigen (PCNA) in both human and fission yeast Schizosaccharomyces pombe systems . Here we show that S . pombe (Sp) MYH physically interacts with all subunits of the PCNA-like checkpoint protein heterotrimer, SpRad9/SpRad1/SpHus1, in yeast extracts and when the individual subunits are expressed in bacteria . The SpHus1 and SpPCNA binding sites are located in discrete regions of SpMYH . Immunoprecipitation assays reveal that the interaction between SpHus1 and SpMYH increases dramatically after hydrogen peroxide treatment, and this increase in the SpHus1-SpMYH interaction correlates with the presence of SpHus1 phosphorylation . In contrast, the interaction between SpPCNA and SpMYH after hydrogen peroxide treatment remains nearly unchanged . SpMYH associates with SpHus1 in a complex of approximately 450 kDa, the reported native molecular mass of the SpRad9/SpRad1/SpHus1-MYC complex . A larger portion of SpMYH shifts to the 150-500-kDa regions after hydrogen peroxide treatment in comparison with untreated extracts . SpHus1 phosphorylation is substantially reduced in SpMYHDelta cells after hydrogen peroxide treatment . These data suggest that MYH may act as an adaptor to recruit checkpoint proteins to the DNA lesions. Biochem Biophys Res Commun, 2004 Dec 10, 325(2), 439 - 44 The Xenopus laevis morphogenetic factor, tumorhead, causes defects in polarized growth and cytokinesis in the fission yeast, Schizosaccharomyces pombe; Wu CF et al.; Tumorhead (TH) is a maternally expressed gene product that regulates neural tube morphogenesis in the amphibian, Xenopus laevis . Here we describe the effects of TH expression in the rod-shaped fission yeast, Schizosaccharomyces pombe . Expression of TH in S . pombe resulted in severe morphological defects, including ovoid, bottle-shaped, and enlarged cells . Multi-septated cells were also observed in TH expressing cultures, indicating that TH is inhibitory to a process required for the completion of cytokinesis . TH expression caused significant actin and microtubule cytoskeletal defects, including depolarization of the cortical F-actin cytoskeleton and increased microtubule formation . Immunostaining experiments showed that TH is localized to the cell cortex, cell tips, and septum in S . pombe cells . Localization of TH to the cell cortex was dependent on the S . pombe PAK homolog, Shk1 . Moreover, TH expression was inhibitory to the growth of a mutant defective in Shk1 function, suggesting that TH may interact with a component(s) of a PAK-mediated morphogenetic regulatory pathway in S . pombe . Taken together, our findings demonstrate that S . pombe may be a useful model organism for identifying potential TH interacting factors. Curr Biol, 2004 Nov 9, 14(21), 1924 - 8 A programmed strand-specific and modified nick in S . pombe constitutes a novel type of chromosomal imprint; Kaykov A et al.; The sexual locus mat1, in the fission yeast Schizosaccharomyces pombe, efficiently switches between the two mating types, P and M, by a process similar to gene conversion, using the silent mat2-P and mat3-M loci, respectively, as donors of the P and M genetic information . It has been proposed that an asymmetrically inherited, site- and strand-specific imprint at mat1 initiates the mating-type switching process . The molecular nature of the imprint is controversial; it was initially described as a double-strand break and then as a single-strand lesion or a strand-specific, alkali-labile modification . Here, we use E . coli DNA ligase in vitro to demonstrate that the imprint is a nick with no resection of nucleotides . By using ligation-mediated PCR, we show that the nick contains 3'OH and 5'OH unphosphorylated termini resistant to RNase treatments . This nonmutational mark on one of the DNA strands provides the first example of a novel type of imprint. Mol Cells, 2004 Oct 31, 18(2), 242 - 8 Transcriptional regulation of glutathione synthetase in the fission yeast Schizosaccharomyces pombe; Kim SJ et al.; Glutathione (GSH), an important antioxidant involved in the stress response, is synthesized in two sequential reactions involving glutamylcysteine synthetase (GCS), followed by glutathione synthetase (GS) . Expression of the unique GS gene in the fission yeast Schizosaccharomyces pombe was previously found to be regulated by nitric oxide and by L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GCS . In this work, expression of S . pombe GS gene is shown to be induced by menadione (MD), which generates superoxide . The responsible DNA sequence between -365 and -234 bp from the translation start site, was convinced using five GS-lacZ fusion plasmids . Expression of GS gene is also induced by low glucose, fructose and disaccharides, apparently dependent on Pap1 protein; GS mRNA increases in low concentrations of glucose in wild type S . pombe but not in Pap1-negative cells . Although nonfermentable carbon sources such as acetate and ethanol stimulate expression of GS gene, they also arrest the growth of the yeast cells . These results indicate that the biosynthesis of glutathione is regulated by superoxide radicals and carbon source limitation. Mol Biol Cell, 2005 Jan, 16(1), 385 - 95 Epub 2004 Nov 03. The A78V Mutation in the Mad3-like Domain of Schizosaccharomyces pombe Bub1p Perturbs Nuclear Accumulation and Kinetochore Targeting of Bub1p, Bub3p, and Mad3p and Spindle Assembly Checkpoint Function; Kadura S et al.; During mitosis, the spindle assembly checkpoint (SAC) responds to faulty attachments between kinetochores and the mitotic spindle by imposing a metaphase arrest until the defect is corrected, thereby preventing chromosome missegregation . A genetic screen to isolate SAC mutants in fission yeast yielded point mutations in three fission yeast SAC genes: mad1, bub3, and bub1 . The bub1-A78V mutant is of particular interest because it produces a wild-type amount of protein that is mutated in the conserved but uncharacterized Mad3-like region of Bub1p . Characterization of mutant cells demonstrates that the alanine at position 78 in the Mad3-like domain of Bub1p is required for: 1) cell cycle arrest induced by SAC activation; 2) kinetochore accumulation of Bub1p in checkpoint-activated cells; 3) recruitment of Bub3p and Mad3p, but not Mad1p, to kinetochores in checkpoint-activated cells; and 4) nuclear accumulation of Bub1p, Bub3p, and Mad3p, but not Mad1p, in cycling cells . Increased targeting of Bub1p-A78V to the nucleus by an exogenous nuclear localization signal does not significantly increase kinetochore localization or SAC function, but GFP fused to the isolated Bub1p Mad 3-like accumulates in the nucleus . These data indicate that Bub1p-A78V is defective in both nuclear accumulation and kinetochore targeting and that a threshold level of nuclear Bub1p is necessary for the nuclear accumulation of Bub3p and Mad3p. Nat Cell Biol, 2004 Nov, 6(11), 1142 - 4 Epub 2004 Nov 01. Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeast; Takeda T et al.; Many membrane processes occur in discrete membrane domains containing lipid rafts, but little is known about how these domains are organized and positioned . In the fission yeast Schizosaccharomyces pombe, a sterol-rich membrane domain forms at the cell-division site . Here, we show that formation of this membrane domain is independent of the contractile actin ring, septation, mid1p and the septins, and also requires cdc15p, an essential contractile ring protein that associates with lipid rafts . cdc15 mutants have membrane domains in the shape of spirals . Overexpression of cdc15p in interphase cells induces abnormal membrane domain formation in an actin-independent manner . We propose that cdc15p functions to organize lipid rafts at the cleavage site for cytokinesis. Mikrobiol Z, 2004 Jul-Aug, 66(4), 69 - 77 {Effect of radio-frequency of electromagnetic radiation on yeast sensitivity to fungicide antibiotics}; Genomic specification and epigenetic regulation of eukaryotic DNA replication origins; Instituto de Microbiologia Bioquimica, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, Salamanca, SpainIdentification of DNA replication origins (ORIs) at a genome-wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences . Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals . This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals . It also discusses the possible additional involvement of ORIs in processes other than DNA replication. J Cell Sci, 2004 Nov 1, 117(Pt 23), 5623 - 32 Fkh2p and Sep1p regulate mitotic gene transcription in fission yeast; Buck V et al.; In the fission yeast Schizosaccharomyces pombe, several genes including cdc15+, spo12+, fin1+, slp1+, ace2+ and plo1+ are periodically expressed during M phase . The products of these genes control various aspects of cell cycle progression including sister chromatid separation, septation and cytokinesis . We demonstrate that periodic expression of these genes is regulated by a common promoter sequence element, named a PCB . In a genetic screen for cell cycle regulators we have identified a novel forkhead transcription factor, Fkh2p, which is periodically phosphorylated in M phase . We show that Fhk2p and another forkhead transcription factor, Sep1p, are necessary for PCB-driven M-phase-specific transcription . In a previous report we identified a complex by electrophoretic mobility shift assay, which we termed PBF, that binds to a 150 bp region of the cdc15+ promoter that contains the PCB element . We have identified Mbx1p, a novel MADS box protein, as a component of PBF . However, although Mbx1p is periodically phosphorylated in M phase, Mbx1p is not required for periodic gene transcription in M phase . Moreover, although PBF is absent in strains bearing a C-terminal epitope tag on Fkh2p, simultaneous deletion of fkh2+ and sep1+ does not abolish PBF binding activity . This suggests that Mbx1p binds to gene promoters, but is not required for transcriptional activation . Together these results suggest that the activation of the Fkh2p and Sep1p forkhead transcription factors triggers mitotic gene transcription in fission yeast. J Cell Sci, 2004 Nov 1, 117(Pt 23), 5543 - 56 The p150-Glued Ssm4p regulates microtubular dynamics and nuclear movement in fission yeast; Niccoli T et al.; During vegetative growth of the fission yeast Schizosaccharomyces pombe, microtubules nucleate from multiple microtubule organising centres (MTOCs) close to the nucleus, polymerising until they reach the end of the cell and then shrinking back to the cell centre . In response to mating pheromone, S . pombe undergoes a morphological switch from a vegetative to a shmooing growth pattern . The switch in growth mode is paralleled by a switch in microtubular dynamics . Microtubules nucleate mostly from a single MTOC and pull on the ends of the cell to move the nucleus back and forth . This movement continues after cellular and nuclear fusion in the zygote and is important to ensure correct chromosome pairing, recombination and segregation during meiosis . Here we show that Ssm4p, a p150-Glued protein, is induced specifically in response to pheromone and is required for this nuclear movement . Ssm4p is associated with the cytoplasmic dynein complex and together with the CLIP-170 homologue Tip1p regulates dynein heavy chain localisation . We also show that Ssm4p collaborates with Tip1p in establishing the shmooing microtubular array. Mol Cell Biol, 2004 Nov, 24(22), 9813 - 22 Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast; Lee BS et al.; DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting . The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1 . swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site . With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences . First, we report the identification and DNA sequence of the swi3 gene . swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting . Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation . Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs . Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1) . These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions. Mol Cell Biol, 2004 Nov, 24(22), 9786 - 801 Kinetochore targeting of fission yeast Mad and Bub proteins is essential for spindle checkpoint function but not for all chromosome segregation roles of Bub1p; Vanoosthuyse V et al.; Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochore-microtubule attachment . Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores . We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions . Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization . We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response . Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation. Genes Cells, 2004 Nov, 9(11), 1069 - 82 An interactive gene network for securin-separase, condensin, cohesin, Dis1/Mtc1 and histones constructed by mass transformation; Yuasa T et al.; The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that approximately 850 are essential for viability . To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones . Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected . Each plasmid interacted positively or negatively with the average 14 strains . Identification of the mutant gene products by cloning revealed many hitherto unknown interactions . The interactive networks of segregation therefore may consist of genes with a variety of functions . For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase . Surprisingly, S . pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/cut1 mutants . This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype. Biochem Soc Trans, 2004 Dec, 32(Pt 6), 967 - 72 Cell cycle-regulated transcription in fission yeast; McInerny CJ; A fundamental process in biology is the mechanism by which cells duplicate and divide to produce two identical daughter cells . The fission yeast, Schizosaccharomyces pombe, has proved to be an excellent model organism to study the role that gene expression plays in this process . The basic paradigm emerging is that a number of groups of genes are expressed in successive waves at different cell cycle times . Transcription of a particular group is controlled by a common DNA motif present in each gene's promoter, bound by a transcription factor complex . Each motif and transcription factor complex is specific to the time in the cell cycle when the group of genes is expressed . Examples of this are the MBF (MCB-binding factor)/MCB (MluI cell cycle box) system controlling gene expression at the start of S-phase, and PBF (PCB-binding factor)/PCB (Pombe cell cycle box) regulation of transcription at the end of mitosis . In some cases, these transcription control systems also operate during the alternative form of cell division, meiosis. J Cell Biol, 2004 Oct 25, 167(2), 315 - 25 UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II; Lord M et al.; We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe . The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p . Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity . Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2 . The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2 . Thus, Rng3p contributes directly to the motility activity of native Myo2 . Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring . The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations . In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis. Curr Opin Drug Discov Devel, 2004 Sep, 7(5), 683 - 91 Analysis of human GPCRs in fission yeast; Ladds G et al.; G protein-coupled receptors (GPCRs) regulate diverse biological processes in all eukaryotes, including yeast, insects, plants and humans . This evolutionary conservation allows an almost unrestricted interchange of signaling components between different cell types . A large number of model systems have been developed for the study of GPCRs, and yeasts provide one of the more attractive hosts since they are amenable to both genetic and biochemical manipulation, while their robustness, low cost and lack of endogenous GPCRs are ideal starting points for the development of assays suitable for high-throughput screening . The purpose of this review is to introduce readers to the possibilities of using the fission yeast Schizosaccharomyces pombe for analysis of GPCRs . We describe the endogenous signaling pathways, the development of assays for heterologous GPCRs, and some of the technology available to elucidate GPCR structure and activity. Nat Cell Biol, 2004 Nov, 6(11), 1135 - 41 Epub 2004 Oct 24. A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1; Obuse C et al.; Defects in kinetochore proteins often lead to aneuploidy and cancer . Mis12-Mtw1 is a conserved, essential kinetochore protein family . Here, we show that a Mis12 core complex exists in Schizosaccharomyces pombe and human cells . Nine polypeptides bind to human hMis12; two of these, HEC1 and Zwint-1, are authentic kinetochore proteins . Four other human proteins of unknown function (c20orf172, DC8, PMF1 and KIAA1570) correspond to yeast Mis12-Mtw1 complex components and are shown to be required for chromosome segregation in HeLa cells using RNA interference (RNAi) . Surprisingly, hMis12 also forms a stable complex with the centromeric heterochromatin components HP1alpha and HP1gamma . Double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8 . Therefore, centromeric HP1 may be the base to anchor the hMis12 core complex that is enriched with coiled coils and extends to outer Zwint-1 during mitosis. BMC Cell Biol . 2004 Oct 21;5(1):40. Germinating fission yeast spores delay in G1 in response to UV irradiation; Nilssen EA et al.; BACKGROUND: Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired . In fission yeast, checkpoint mechanisms are known to regulate entry into mitosis, but so far no checkpoint inhibiting S phase entry has been identified . RESULTS: We have studied the response of germinating Schizosaccharomyces pombe spores to UV irradiation in G1 . When germinating spores are irradiated in early G1 phase, entry into S phase is delayed . We argue that the observed delay is caused by two separate mechanisms . The first takes place before entry into S phase, does not depend on the checkpoint proteins Rad3, Cds1 and Chk1 and is independent of Cdc2 phosphorylation . Furthermore, it is not dependent upon inhibiting the Cdc10-dependent transcription required for S phase entry, unlike a G1/S checkpoint described in budding yeast . We show that expression of Cdt1, a protein essential for initiation of DNA replication, is delayed upon UV irradiation . The second part of the delay occurs after entry into S phase and depends on Rad3 and Cds1 and is probably due to the intra-S checkpoint . If the germinating spores are irradiated in late G1, they enter S phase without delay and arrest in S phase, suggesting that the delay we observe upon UV irradiation in early G1 is not caused by nonspecific effects of UV irradiation . CONCLUSIONS: We have studied the response of germinating S . pombe spores to UV irradiation in G1 and shown that S phase entry is delayed by a mechanism that is different from classical checkpoint responses . Our results point to a mechanism delaying expression of proteins required for S phase entry. Nucleic Acids Res, 2004 Oct 19, 32(18), 5649 - 57 Print 2004. Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid; Vermeersch JJ et al.; The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the epsilon-amino group of an active site lysine . PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K(i) = 40 microM) that blocks the cleavable complex formation . Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein . The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR) . Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505) . In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity . The spatial orientation of K532-PLP within the active site was approached by molecular modeling using available crystallographic data . The PLP moiety was found at close proximity of several active residues . PLP could be involved in the cellular control of topoisomerases IB . It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid. Genetics . 2004 Oct 16; {Epub ahead of print} The Meiotic Recombination Hot-Spot ura4A in Schizosaccharomyces pombe; Baur M et al.; A meiotic recombination hot spot ura4A (formerly ura4-aim) of Schizosaccharomyces pombe was observed at the insertion of the ura4+ gene 15 kb centromere-proximal to ade6 on chromosome III . Crosses heterozygous for the insertion showed frequent conversion at the heterology with preferential loss of the insertion . This report concerns the characterization of 12 spontaneous ura4A mutants . A gradient of conversion ranging from 18% at the 5' end to 6% at the 3' end was detected . A novel phenomenon was discovered: a mating-type related bias of conversion . The allele entering with the h+ parent acts preferentially as the acceptor for conversion (ratio of 3:2) . Tetrad analysis of two-factor crosses showed that heteroduplex DNA is predominantly asymmetrical, enters from the 5' end, and more often than not covers the entire gene . Restoration repair of markers at the 5' end was inferred . Random spore analyses of two-factor crosses and normalization of prototroph-recombinant frequencies to physical distance led to the demonstration of map expansion: Crosses involving distant markers yielded higher recombinant frequencies than the sum of the frequencies measured in the subintervals . Finally, marker effects on recombination were defined for two of the ura4A mutations. Biochim Biophys Acta, 2004 Oct 21, 1680(2), 93 - 102 A cDNA homologue of Schizosaccharomyces pombe cdc5(+) from the mushroom Lentinula edodes: characterization of the cDNA and its expressed product; Miyazaki Y et al.; A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA . The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S . pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs) . The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5'GCAATGT3' (complementary; 5'ACATTGC3') . Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box . The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus . There exists a possible leucine zipper between the two phosphorylation sites . The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase . Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper . Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L . edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation. Mol Cell Biol, 2004 Nov, 24(21), 9557 - 67 Fission yeast Dna2 is required for generation of the telomeric single-strand overhang; Tomita K et al.; It has been suggested that the Schizosaccharomyces pombe Rad50 (Rad50-Rad32-Nbs1) complex is required for the resection of the C-rich strand at telomere ends in taz1-d cells . However, the nuclease-deficient Rad32-D25A mutant can still resect the C-rich strand, suggesting the existence of a nuclease that resects the C-rich strand . Here, we demonstrate that a taz1-d dna2-2C double mutant lost the G-rich overhang at a semipermissive temperature . The amount of G-rich overhang in S phase in the dna2-C2 mutant was lower than that in wild-type cells at the semipermissive temperature . Dna2 bound to telomere DNA in a chromatin immunoprecipitation assay . Moreover, telomere length decreased with each generation after shift of the dna2-2C mutant to the semipermissive temperature . These results suggest that Dna2 is involved in the generation of G-rich overhangs in both wild-type cells and taz1-d cells . The dna2-C2 mutant was not gamma ray sensitive at the semipermissive temperature, suggesting that the ability to process double-strand break (DSB) ends was not affected in the dna2-C2 mutant . Our results reveal that DSB ends and telomere ends are processed by different mechanisms. Mol Cell Biol, 2004 Nov, 24(21), 9401 - 13 Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast; Morikawa H et al.; Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently . This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs) . This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants) . rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs . rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1 . Rad62 protein physically associates with the Smc5-6 complex . rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16 . These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination. J Biol Chem, 2004 Dec 17, 279(51), 53023 - 7 Epub 2004 Oct 06. DNA binding domain in the replication checkpoint protein Mrc1 of Schizosaccharomyces pombe; Zhao H et al.; The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides . This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis . In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1 . In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45 . Whether or not Mrc1 interacts directly with DNA is unknown . Here we define a approximately 150 amino acid DNA binding domain (DBD) in the N-terminal region of S . pombe Mrc1 . The DBD interacts preferentially with branched DNA structures in vitro . Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea . These mutations also impair the replication checkpoint arrest . The DBD has a helix-loop-helix motif that is predicted to bind DNA . This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins. Eukaryot Cell, 2004 Oct, 3(5), 1124 - 35 Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis; Cortes JC et al.; The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C . Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally . Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear . The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions . The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p . The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C) . The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins . Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium . The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells . These results indicate that S . pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally . Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth. Glycobiology . 2004 Oct 6; {Epub ahead of print} The structure of cell-wall {alpha}-glucan from fission yeast; Grun CH et al.; Morphology and structural integrity of fungal cells depend on cell-wall polysaccharides . The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan . Here, we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe . Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length . These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-D-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end . By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and with reduced alpha-glucan levels consisted of a single chain only . We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell-wall alpha-glucan . This mature form of cell-wall alpha-glucan is essential for fission-yeast morphogenesis. Microbiology, 2004 Oct, 150(Pt 10), 3163 - 73 Rot1p of Saccharomyces cerevisiae is a putative membrane protein required for normal levels of the cell wall 1,6-beta-glucan; Machi K et al.; Although ROT1 is essential for growth of Saccharomyces cerevisiae strain BY4741, the growth of a rot1Delta haploid was partially restored by the addition of 0.6 M sorbitol to the growth medium . Rot1p is predicted to contain 256 amino acids, to have a molecular mass of 29 kDa, and to possess a transmembrane domain near its C-terminus . Candida albicans and Schizosaccharomyces pombe have Rot1p homologues with high identity that also have predicted transmembrane domains . To explore the role of Rot1p, the phenotypes of the rot1Delta haploid were analysed . Deletion of ROT1 caused cell aggregation and an abnormal morphology . Analysis of the cell cycle showed that rot1Delta cells are delayed at the G2/M phase . The rot1Delta cells were resistant to K1 killer toxin and hypersensitive to SDS and hygromycin B, suggesting that they had cell wall defects . Indeed, greatly reduced levels of alkali-soluble and -insoluble 1,6-beta-glucan, and increased levels of chitin and 1,3-beta-glucan, were found in rot1Delta cells . Furthermore, the phenotypes of rot1Delta cells resemble those of disruption mutants of the KRE5 and BIG1 genes, which show greatly reduced levels of cell wall 1,6-beta-glucan . Incorporation of glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in big1Delta and rot1Delta cells was examined using a GFP-Flo1 fusion protein . GFP fluorescence was detected both on the cell surface and in the culture medium, suggesting that, in these mutants, mannoproteins may become only weakly bound to the cell wall and some of these proteins are released into the medium . Electron microscopic analyses of rot1Delta and big1Delta cells showed that the electron-dense mannoprotein rim staining was more diffuse and paler than that in the wild-type