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| 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, and that the outer boundary of the cell wall was irregular . A big1Deltarot1Delta double mutant had a growth rate similar to the corresponding single mutants, suggesting that Rot1p and Big1p have related functions in 1,6-beta-glucan synthesis. Genetics, 2004 Dec, 168(4), 1891 - 8 Epub 2004 Sep 30. Swi5 Acts in Meiotic DNA Joint Molecule Formation in Schizosaccharomyces pombe; Ellermeier C et al.; Previously isolated Schizosaccharomyces pombe swi5 mutants are defective in mitotic mating-type switching and in repair of meiotic recombination-related DNA double-strand breaks . Here, we identify the swi5 gene, which encodes an 85-amino-acid polypeptide, similar to Sae3 of Saccharomyces cerevisiae, with an N-terminal predicted coiled-coil domain . A swi5 complete deletion mutant had normal mitotic growth rate but was hypersensitive to DNA-damaging agents and defective in mating-type switching . In meiosis, recombinant frequencies were reduced by a factor of approximately 10 . The swi5 deletion strongly reduced the viable spore yields of mutants lacking Rhp55 or Rhp57, proteins thought to aid joint molecule formation . Furthermore, the swi5 deletion strongly suppressed the low viable spore yield of mutants lacking Mus81*Eme1, which resolves joint molecules such as Holliday junctions . These and previous results indicate that the small Swi5 polypeptide acts in a branched pathway of joint molecule formation to repair meiotic DNA breaks. Genetics . 2004 Sep 30; {Epub ahead of print} Regulation of leucine uptake by tor1+in Schizosaccharomyces pombe is sensitive to rapamycin; Weisman R et al.; TOR protein kinases are key regulators of cell growth in eukaryotes . TOR is also known as the target protein for the immunosuppressive and potentially anti-cancerous drug rapamycin . The fission yeast, Schizosaccharomyces pombe, has two TOR homologues . tor1+ is required under starvation and a variety of stresses, while tor2+ is an essential gene . Surprisingly, to date no rapamycin-sensitive TOR-dependent function has been identified in S . pombe . Herein, we show that S . pombe auxotrophs, in particular leucine auxotrophs, are sensitive to rapamycin . This sensitivity is suppressed by deletion of the S . pombe FKBP12 or by introducing a rapamycin-binding defective tor1 allele, suggesting that rapamycin inhibits a tor1p-dependent function . Sensitivity of leucine auxotrophs to rapamycin is observed when ammonia is used as the nitrogen source and can be suppressed by its replacement with proline . Consistently, using radioactive labeled leucine, we show that cells treated with rapamycin or disrupted for tor1+ are defective in leucine uptake when the nitrogen source is ammonia but not proline . Recently, it has been reported that tsc1+ and tsc2+, the S . pombe homologues for the mammalian TSC1 and TSC2, are also defective in leucine uptake . TSC1 and TSC2 may antagonize TOR signaling in mammalian cells and Drosophila . We show that reduction of leucine uptake in tor1 mutants is correlated with decreased expression of three putative amino acid permeases that are also down regulated in tsc1 or tsc2 . These findings suggest a possible mechanism for regulation of leucine uptake by tor1p and indicate that tor1p, as well as tsc1p and tsc2p, positively regulate leucine uptake in S . pombe. Plant Physiol, 2004 Oct, 136(2), 2971 - 81 Epub 2004 Oct 01. Evidence for serine/threonine and histidine kinase activity in the tobacco ethylene receptor protein NTHK2; Zhang ZG et al.; Ethylene plays important roles in plant growth, development, and stress responses . Two ethylene receptors, ETR1 from Arabidopsis and NTHK1 from tobacco (Nicotiana tabacum), have been found to have His kinase (HK) activity and Ser/Thr kinase activity, respectively, although both show similarity to bacterial two-component HK . Here, we report the characterization of another ethylene receptor homolog gene, NTHK2, from tobacco . This gene also encodes a HK-like protein and is induced by dehydration and CaCl(2) but not significantly affected by NaCl and abscisic acid treatments . The biochemical properties of the yeast (Schizosaccharomyces pombe)-expressed NTHK2 domains were further characterized . We found that NTHK2 possessed Ser/Thr kinase activity in the presence of Mn(2+) and had HK activity in the presence of Ca(2+) . Several lines of evidence supported this conclusion, including hydrolytic stability, phosphoamino acid analysis, mutation, deletion, and substrate analysis . These properties have implications in elucidation of the complexity of the ethylene signal transduction pathway and understanding of ethylene functions in plants. Genes Cells, 2004 Oct, 9(10), 891 - 903 Local exposure of phosphatidylethanolamine on the yeast plasma membrane is implicated in cell polarity; Iwamoto K et al.; Cell surface phosphatidylethanolamine (PE) of the yeast cell was probed by biotinylated Ro09-0198 (Bio-Ro), which specifically binds to PE and was visualized with fluorescein-labelled streptavidin . In Saccharomyces cerevisiae, the signals were observed at the presumptive bud site, the emerging small bud cortex, the bud neck of the late mitotic large-budded cells and the tip of the mating projection . In Schizosaccharomyces pombe, the signals were observed at one end or both ends of mono-nucleated cells and the division plane of the late mitotic cells . These sites were polarized ends in the yeast cells, implying that PE is exposed on the cell surface at cellular polarized ends . Treatment of S . cerevisiae cells with Ro09-0198 resulted in aberrant F-actin accumulation at the above sites, implying that limited surface exposure of PE is involved in the polarized organization of the actin cytoskeleton . Furthermore, S . cerevisiae ros3, dnf1 and dnf2 null mutants, which were known to be defective in the internalization of fluorescence-labelled PE, as well as the combinatorial mutants, were stained with Bio-Ro at the enlarging bud cortex, in addition to the Bio-Ro-staining sites of wild-type cells, suggesting that Ros3p, Dnf1p and Dnf2p are involved in the retrieval of exposed PE at the bud cortex. J Microbiol, 2004 Sep, 42(3), 233 - 8 Transcriptional regulation of the gene encoding gamma-glutamylcysteine synthetase from the fission yeast Schizosaccharomyces pombe; Kim SJ et al.; Transcriptional regulation of the Schizosaccharomyces pombe gamma-glutamylcysteine synthetase (GCS) gene was examined using the two GCS-lacZ fusion plasmids pUGCS101 and pUGCS102, which harbor 607 bp and 447 bp upstream regions, respectively . The negatively-acting sequence was located in the -607 approximately -447 bp upstream region of the GCS gene . The upstream sequence responsible for induction by menadione (MD) and L-buthionine-(S, R)-sulfoximine (BSO) resides in the -607 approximately -447 bp region, whereas the sequence which codes for nitric oxide induction is located within the -447 bp region, measured from the translational initiation point . Carbon source-dependent regulation of the GCS gene appeared to be dependent on the nucleotide sequence within -447 bp region . The transcription factor Pap1 is involved in the induction of the GCS gene by MD and BSO, but not by nitric oxide . Induction of the GCS gene occurring due to low glucose concentration does not depend on the presence of Pap1 . These data imply that induction by MD and BSO may be mediated by the Pap1 binding site, probably located in the -607 approximately -447 region, and also that the nitric oxide-mediated regulation of the S . pombe GCS gene may share a similar mechanism with its carbon-dependent induction . J Cell Sci, 2004 Oct 15, 117(Pt 22), 5293 - 302 Epub 2004 Sep 28. The GIN4 family kinase, Cdr2p, acts independently of septins in fission yeast; Morrell JL et al.; Two relatives of the GIN4 protein kinase family, Cdr1p and Cdr2p, exist in the yeast Schizosaccharomyces pombe . Although in Saccharomyces cerevisiae GIN4-related kinases influence septin ring organization and septin rings influence the localization and function of GIN4-related protein kinases, it is unknown whether this relationship is conserved in S . pombe . Here, we have probed the relationship between Cdr2p activity and septins and find that Cdr2p and septins are functionally independent . Cdr2p localizes in a cortical band overlying the nucleus during interphase, whose dimension is proportional to cell length, and to a medial ring structure in late mitosis . Both localizations are septin-independent and disrupted by treatment with filipin . Structure/function analysis indicates that the intracellular targeting domain of Cdr2p is largely contained within its non-catalytic C-terminus . Cdr2 protein kinase activity, while unimportant for its localization, is critical for its cell cycle function . Our data indicate that Cdr2p functions at two positions within the cell at discrete cell cycle stages to influence the timing of mitotic entry and cytokinesis, respectively. Nucleic Acids Res, 2004 Sep 27, 32(17), 5119 - 25 Print 2004. A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe; Alseth I et al.; One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability . It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe . A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage . Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair . The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway . The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites . Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro . Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway . We suggest that the main function of Apn2 in BER is to remove the resulting 3'-blocking termini following AP lyase cleavage by Nth1. J Biol Chem, 2004 Dec 10, 279(50), 52437 - 46 Epub 2004 Dec 10. Genome-wide analysis of pre-mRNA splicing: intron features govern the requirement for the second-step factor, Prp17 in Saccharomyces cerevisiae and Schizosaccharomyces pombe; Sapra AK et al.; Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation . The spliceosome, a ribonucleoprotein performs this critical function; however, precise roles for many of its proteins remain unknown . Genome-wide consequences triggered by the loss of a specific factor can elucidate its function in splicing and its impact on other cellular processes . We have employed splicing-sensitive DNA microarrays, with yeast open reading frames and intron sequences, to detect changes in splicing efficiency and global expression . Comparison of expression profiles, for intron-containing transcripts, among mutants of two second-step factors, Prp17 and Prp22, reveals their unique and shared effects on global splicing . This analysis enabled the identification of substrates dependent on Prp17 . We find a significant Prp17 role in splicing of introns which are longer than 200nts and note its dispensability when introns have a < or =13-nucleotide spacing between their branch point nucleotide and 3 ' splice site . In vitro splicing of substrates with varying branch nucleotide to 3 ' splice site distances supports the differential Prp17 dependencies inferred from the in vivo analysis . Furthermore, we tested the predicted dispensability of Prp17 for splicing short introns in the evolutionarily distant yeast, Schizosaccharomyces pombe, where the genome contains predominantly short introns . SpPrp17 was non-essential at all growth temperatures implying that functional evolution of splicing factors is integrated with genome evolution . Together our studies point to a role for budding yeast Prp17 in splicing of subsets of introns and have predictive value for deciphering the functions of splicing factors in gene expression and regulation in other eukaryotes. FEMS Yeast Res, 2004 Sep, 4(8), 841 - 7 Directional ligation of long-flanking homology regions to selection cassettes for efficient targeted gene-disruption in Candida albicans; Taneja V et al.; PCR-product directed gene disruption with a marker cassette having short homology regions is often used in Candida albicans . However, it is quite inefficient due to the high frequency of non-homologous recombination at non-targeted loci, which necessitates extensive screening to identify the correct disruptants . Thus, many PCR-based methods to introduce long flanking homology regions have been developed to increase the frequency of integration at the targeted loci . However, these methods are not that amenable for use with the widely employed C . albicans marker cassettes having direct repeats, as these repeats tend to recombine during PCR, resulting in shorter amplified products without the selection marker . To circumvent this limitation, we have developed a dinucleotide-sticky-end-ligation strategy to add one flanking homology region to one side of the selection cassette, and the other flanking homology region to the other side of the selection cassette . This method involves release of the selection cassette from the plasmid by digestion with two different restriction enzymes, followed by partial fill-in, to provide a unique two base overhang at each end of the cassette . The flanking homology regions, corresponding to the gene to be disrupted, are individually PCR-amplified, and treated with T4-DNA Polymerase in the presence of appropriate dNTPs to yield two base-5' overhangs . The primers used for the PCR have additional bases at the 5' ends such that after T4 DNA Polymerase treatment, the two flanks will have distinct overhangs compatible with the overhangs of the partially filled-in selection cassette . The selection cassette and the flanks are then ligated together and directly used to transform C . albicans . We have successfully used this method for disruption of several C . albicans genes . We have also used this method to recreate insertion mutations obtained with transposons to reconfirm the mutant phenotypes . This approach can be extended to other organisms like Schizosaccharomyces pombe which also require long flanking regions of homology for targeted gene disruption. Yeast, 2004 Sep, 21(12), 1005 - 19 Chr4, a Schizosaccharomyces pombe homologue of the Saccharomyces cerevisiae Chs4p/Skt5p protein, is related to septum formation and is required for the proper localization of Chs2; Matsuo Y et al.; In Saccharomyces cerevisiae, Chs4p directly interacts with chitin synthase III (Chs3p) to act as a post-translational regulator of the Chs3p complex . We identified four Chs4p homologous proteins in Schizosaccharomyces pombe which we named Chr1, Chr2, Chr3 and Chr4 (putative chitin synthase regulatory factor) . We assessed the functions of these proteins and found that while overproduction of Chr1, Chr2 or Chr3 did not affect the cellular morphology of wild-type Sz . pombe cells, overproduction of Chr4 caused the cells to form multi-septa and delayed their growth . All multiple disruptants of chr1, chr2, chr3 and chr4 grew normally under a variety of growth conditions . However, while chitin synthase II (Chs2) normally localizes exclusively at the septum, in many chr4-disrupted cells it was found in the cytoplasm and the septa . Chs2 did localize at the abnormal septa caused by the overproduction of chr4+ . Chr4-13Myc expression was unaffected by the different media or growth conditions in both wild-type and the chs2 disruptant . Chs2 expression was also unaltered by the absence of Chr4 . Moreover, Chr4-13Myc localized mostly at the tips and the septum during vegetative growth in chs2, chr1, chr2 and chr3 disruptants as well as in wild-type . Thus, chr4+ is involved in septum formation and is required for the proper localization of Chs2 at the septum in Sz . pombe . Copyright (c) 2004 John Wiley & Sons, Ltd. Yeast, 2004 Sep, 21(12), 1035 - 44 Schizosaccharomyces pombe ER oxidoreductin-like proteins SpEro1a p and SpEro1b p; Kettner K et al.; Endoplasmic reticulum oxidoreductins (Ero proteins) are essential for oxidation of protein disulphide isomerase (Pdi), which introduces disulphide bonds in target proteins . Contrary to the situation in Saccharomyces cerevisiae, with a single Ero protein (Ero1p), the genomes of Schizosaccharomyces pombe and of humans encode two Ero-like proteins . Here we show that both Sz . pombe proteins (SpEro1a p and SpEro1b p) are N-glycosylated and firmly associated with membranes of the secretory pathway . Surprisingly, only expression of SpEro1b p completely restores growth of the temperature-sensitive S . cerevisiae ero1-1 mutant, whereas SpEro1a p only partially complements this mutation . Upon expression in S . cerevisiae wild-type cells, SpEro1b p leads to a significantly increased resistance to reductive stress by dithiothreitol, whereas SpEro1a p has only a marginal effect . These data suggest that SpEro1b p is a functional homologue of the S . cerevisiae Ero1p . Copyright (c) 2004 John Wiley & Sons, Ltd. Curr Biol, 2004 Sep 21, 14(18), R806 - 18 Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells; Balasubramanian MK et al.; Cytokinesis is a temporally and spatially regulated process through which the cellular constituents of the mother cell are partitioned into two daughter cells, permitting an increase in cell number . When cytokinesis occurs in a polarized cell it can create daughters with distinct fates . In eukaryotes, cytokinesis is carried out by the coordinated action of a cortical actomyosin contractile ring and targeted membrane deposition . Recent use of model organisms with facile genetics and improved light-microscopy methods has led to the identification and functional characterization of many proteins involved in cytokinesis . To date, this analysis indicates that some of the basic components involved in cytokinesis are conserved from yeast to humans, although their organization into functional machinery that drives cytokinesis and the associated regulatory mechanisms bear species-specific features . Here, we briefly review the current status of knowledge of cytokinesis in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and animal cells, in an attempt to highlight both the common and the unique features . Although these organisms diverged from a common ancestor about a billion years ago, there are eukaryotes that are far more divergent . To evaluate the overall evolutionary conservation of cytokinesis, it will be necessary to include representatives of these divergent branches . Nevertheless, the three species discussed here provide substantial mechanistic diversity. Curr Biol, 2004 Sep 21, 14(18), R762 - 4 Phosphoinositides: older than we first thought? Cooke FT. Despite nearly 50 years of study, it is good to see that phosphoinositides are still capable of springing the odd surprise . Signaling by the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) was thought to be absent in yeast, but a recent paper now describes the presence of PtdIns(3,4,5)P(3) in Schizosaccharomyces pombe. Chin Med J (Engl), 2004 Sep, 117(9), 1321 - 5 Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase II beta subunit; Peng ZY et al.; BACKGROUND: Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine . We cloned a new gene, casein kinase II beta subunit, of Schistosoma japonicum (S . japonicum) and express it in Escherichia coli (E . coli) . METHODS: The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized . The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone . To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E . coli JM109 . The recombinant protein was analyzed by SDS-PAGE and Western-blot . RESULTS: A 908 bp cDNA was isolated from S . japonicum and identified to be casein kinase II beta subunit gene by sequence analysis . The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H . sapiens), Xenopus laevi (X . laevi), Drosophila melanogaster (D . melanogaster), Caenorhabditis elegan (C . elegan), and Schizosaccharomyces pombe (S . promber) respectively . The predicted molecular weight of the protein was 24.921 kDa . The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391 . This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E . coli JM109 . The recombinant protein could be recognized by the S . japonicum infected rabbit serum . CONCLUSION: The full-length cDNA sequences encoding S . japonicum casein kinase II beta subunit were firstly sequenced, cloned, and expressed in E . coli. Proc Natl Acad Sci U S A, 2004 Sep 28, 101(39), 14085 - 90 Epub 2004 Sep 15. swi1- and swi3-dependent and independent replication fork arrest at the ribosomal DNA of Schizosaccharomyces pombe; Krings G et al.; Replication forks are arrested at specific sequences to facilitate a variety of DNA transactions . Forks also stall at sites of DNA damage, and the regression of stalled forks without rescue can cause genetic instability . Therefore, unraveling the mechanisms of fork arrest and of rescue of stalled forks is of considerable general interest . In Schizosaccharomyces pombe, products of two mating-type switching genes, swi1 and swi3, participate in fork arrest at the mating-type switch locus . Here, we show that these proteins also act at three termini (Ter) also called replication fork barriers in the spacer regions of rDNA but not at a fourth site, RFP4, which is nonfunctional when present in a plasmid . Two of the Swi1p- and Swi3p-dependent sites were also dependent on the transcription terminator Reb1p . Furthermore, hydroxyurea-induced replication stress mimicked the effect of swi1 or swi3 mutations at these sites . A swi1 mutant that failed to arrest forks at the mating-type fork barrier RTS1 was functional at the rDNA Ter sites, suggesting some specificity of action . Both WT and mutant forms of Swi1p were physically localized at the Ter sites in vivo . The results support the notion that Swi1p and Swi3p act at several different protein-DNA complexes in the rDNA spacer regions to arrest replication but that not all fork barriers required their activity to arrest forks. Genes Dev, 2004 Oct 1, 18(19), 2359 - 67 Epub 2004 Sep 15. A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe; Sigova A et al.; The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1) . These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus . Here, we show that the introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S . pombe . That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription . RNAi in S . pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing . Thus, the RNAi machinery in S . pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein . Our findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways . Mol Cell Biol, 2004 Oct, 24(19), 8342 - 55 Swi1 and Swi3 are components of a replication fork protection complex in fission yeast; Noguchi E et al.; Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe . This fork pausing is required to initiate a recombination event that switches mating type . Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest . How Swi1 accomplishes these tasks is unknown . Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+) . The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1 . Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork . swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication . These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase . Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks . We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks. J Mol Biol, 2004 Oct 1, 342(5), 1353 - 8 SAM domains can utilize similar surfaces for the formation of polymers and closed oligomers; Ramachander R et al.; The mitogen-activated protein kinase (MAPK) Byr2 and its activator Ste4 are involved in the mating pheromone response pathway of Schizosaccharomyces pombe and interact via their SAM domains . SAM domains can self-associate to form higher-order structures, including dimers, polymers and closed oligomers . Ste4-SAM is adjacent to a trimeric leucine zipper domain and we have shown previously that the two domains together (Ste4-LZ-SAM) bind to a monomeric Byr2-SAM with high affinity (Kd approximately 20 nM), forming a 3:1 complex . Here, we map the surfaces of Byr2-SAM and Ste4-SAM that is involved the interaction . A set of 38 mutants of Byr2-SAM and 33 mutants of Ste4-SAM were prepared, covering most of the protein surfaces . These mutants were purified and screened for binding, yielding a map of residues that are required for binding and a complementary map of residues that are not required . We find that the interface maps to regions of the SAM domains that are known to be important for the formation of SAM polymers . These results indicate that SAM domains can create a variety of oligomeric architectures utilizing common binding surfaces. J Bioinform Comput Biol, 2004 Sep, 2(3), 589 - 93 Robustness of metabolic map reconstruction; Ahren DG et al.; With the ever increasing amount of genomic data available, the interest for generating biochemical pathways has grown tremendously . So far, mainly complete genomes have been used to reconstruct the biochemical pathways and their associated interactions . However, a large number of low coverage genomes, as well as other sources of partial genomic data, are currently available for many organisms . In order to be able to use incomplete data for metabolic reconstruction, the inherent properties of this procedure need to be investigated . In this short note, we describe the robustness and predictive power of metabolic reconstructions using partial information from Schizosaccharomyces pombe . We also discuss the implications of the results on reference genome projects as well as other large-scale sequencing data. Biochem Biophys Res Commun, 2004 Aug 20, 321(2), 424 - 31 The in vivo tyrosine phosphorylation level of yeast immunophilin Fpr3 is influenced by the LMW-PTP Ltp1; Magherini F et al.; Tyr-phosphorylation in Saccharomyces cerevisiae is essential in controlling the activity of MAP kinase regulating mating, pseudohyphal growth, and cell wall biosynthesis . Yeast serves as a model system for studying the biological function of many protein kinases and PTPs . Two LMW-PTP from yeast have been cloned, namely, Ltp1 from S . cerevisiae and Stp1 from Schizosaccharomyces pombe . The sequences of both enzymes are relatively similar to those of the mammalian LMW-PTP . Recently we showed that the yeast immunophilin Fpr3 interacts with Stp1 and its dephosphorylated state induces a growth defective phenotype . Here we show the phosphatase activity of Ltp1 on Fpr3 and we demonstrated that Tyr 184 is the residue phosphorylated on in vivo Fpr3 . We also described the marked activation of Ltp1 by adenine in S . cerevisiae proteome and determined in vivo the influence of tyrosine phosphorylation on Fpr3 localization. Biochem Biophys Res Commun, 2004 Sep 3, 321(4), 922 - 9 Differential expression and role of two dithiol glutaredoxins Grx1 and Grx2 in Schizosaccharomyces pombe; Chung WH et al.; Glutaredoxins are glutathione-specific thiol oxidoreductases . The regulation and the role of grx1(+) and grx2(+) genes encoding dithiol glutaredoxins were analyzed in Schizosaccharomyces pombe . When tested in the same genetic background including mating type, the grx1 null mutant became sensitive to hydrogen peroxide, whereas grx2 mutant became highly sensitive to paraquat, a superoxide generator . The grx1grx2 double mutant showed additive phenotype of each single mutant . The grx1(+) gene expression was induced by various stresses such as oxidants, salts, and heat, and increased in the stationary phase, whereas grx2(+) stayed constitutive . The induction was mediated via Spc1 MAP kinase path involving both Atf1 and Pap1 transcription factors . Sub-cellular fractionation as well as fluorescence microscopy revealed that Grx1 resides mainly in the cytosol, whereas Grx2 is in mitochondria . These results suggest distinct roles for Grx1 and Grx2 in S . pombe in mediating glutathione-dependent redox homeostasis. Biochem Biophys Res Commun, 2004 Sep 3, 321(4), 823 - 7 Phosphoinositide-dependent kinase-1 orthologues from five eukaryotes are activated by the hydrophobic motif in AGC kinases; Silber J et al.; Phosphoinositide-dependent kinase-1 (PDK1) mediates activation of many AGC kinases by docking onto a phosphorylated hydrophobic motif located C-terminal of the catalytic domain in the AGC kinase . The interaction shifts PDK1 into a conformation with increased catalytic activity and leads to autophosphorylation of PDK1 . We demonstrate here that addition of a hydrophobic motif peptide increases the catalytic activity of PDK1 orthologues from Homo sapiens, Aplysia californica, Arabidopsis thaliana, Schizosaccharomyces pombe (ksg1), and Saccharomyces cerevisiae (Pkh1 and Pkh2) 2- to 12-fold . Furthermore, the hydrophobic motif peptide increases autophosphorylation of PDK1 from Homo sapiens, S . pombe, and S . cerevisiae (Phk2) . Our results suggest that PDK1 interaction and activation by the hydrophobic motif of AGC kinases is a central mechanism in PDK1 function, which is conserved during eukaryotic evolution. J Microbiol, 2004 Mar, 42(1), 32 - 6 Schizosaccharomyces pombe rsm1 genetically interacts with spmex67, which is involved in mRNA export; Yoon JH; We have previously isolated three synthetic lethal mutants from Schizosaccharomyces pombe in order to identify mutations in the genes that are functionally linked to spmex67 with respect to mRNA export . A novel rsm1 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMexl . The rsm1 gene contains no introns and encodes a 296 amino-acid-long protein with the RING finger domain, a C3HC4 in the N-terminal half . The deltarsm1 null mutant is viable, but it showed a slight poly(A)+ RNA accumulation in the nucleus . Also, the combination of deltarsm1 and deltaspmex67 mutations confers synthetic lethality that is accompanied by the severe poly(A)+ RNA export defect . These results suggest that rsm1 is involved in mRNA export from the nucleus. Plant Mol Biol, 2004 Mar, 54(5), 653 - 70 Diversification of genes encoding mei2 -like RNA binding proteins in plants; Anderson GH et al.; A predominantly plant-based family of genes encoding RNA binding proteins is defined by the presence of a highly conserved RNA binding motif first described in the mei2 gene of the fission yeast Schizosaccharomyces pombe . In silico analyses reveal nine mei2 -like genes in Arabidopsis thaliana and six in Oryza sativa . These predicted genes group into four distinct clades, based on overall sequence similarity and subfamily-specific sequence elements . In situ analysis show that Arabidopsis genes from one of these clades, TEL1 and TEL2, are specifically expressed in central zone of the shoot apical meristem and the quiescent center of the root apical meristem, suggesting that they may somehow function to maintain indeterminacy in these tissues . By contrast, members of two sister clades, AML1 through AML5, are expressed more broadly, a trend that was confirmed by Q-PCR analysis . mei2 -like transcripts with similar sequences showed similar expression patterns, suggesting functional redundancy within the four clades . Phenotypic analyses of lines that contain T-DNA insertions to individual mei2 -like genes reveal no obvious phenotypes, further suggesting redundant activities for these gene products. Mol Biol Cell, 2004 Nov, 15(11), 5038 - 46 Epub 2004 Sep 08. The small subunit processome is required for cell cycle progression at G1; Bernstein KA et al.; Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops . We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells . We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis . Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated . Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth . However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle . This arrest was further supported by the lack of staining for proteins expressed in post-G1 . Similarly, synchronized cells depleted of SSU processome proteins did not enter G2 . This suggests that when ribosomes are no longer made, the cells stall in the G1 . Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur. J Biol Chem, 2004 Nov 19, 279(47), 49455 - 9 Epub 2004 Sep 08. The Soh1/MED31 protein is an ancient component of Schizosaccharomyces pombe and Saccharomyces cerevisiae Mediator; Linder T et al.; We here demonstrated that the Soh1/MED31 protein is a stable component of Mediator complex isolated from Schizosaccharomyces pombe and Saccharomyces cerevisiae . Bioinformatic analysis traces the Soh1/MED31 family of Mediator subunits to the point of major eukaryotic divergence, before the appearance of the canonical heptapeptide repeat structure of the RNA polymerase II C-terminal domain. DNA Seq, 2004 Feb, 15(1), 44 - 50 Isolation and molecular analysis of Umhda2 a gene encoding a histone deacetylase from Ustilago maydis; Gonzalez-Prieto JM et al.; By use of the polymerase chain reaction and synthetic oligonucleotides designed from conserved regions, we amplified a fragment of a gene from Ustilago maydis encoding a putative histone deacetylase . With this probe we isolated the full gene from a minigenomic library . The gene (designated as Umhda2) contains an open reading frame (ORF) of 1701bp encoding a protein of 566 amino acids . Multiple comparison analysis with other histone deacetylases suggests that the Umhda2 gene product belongs to the Rpd3-related family of proteins . The highest degree of homology with histone deacetylases from other organisms corresponded to Hdalp of Schizosaccharomyces pombe and Rpd3p of Saccharomyces cerevisiae with 64.2 and 62.2% of sequence similarity, respectively . It displayed a substantially lower similarity with another histone deacetylase from U . maydis (Hdalp, 52.4%) . Semi-quantitative RTPCR results indicate that the gene is transcriptionally up-regulated during the in vitro yeast-to-mycelium dimorphic transition. Curr Biol, 2004 Sep 7, 14(17), R722 - 30 SIN and the art of splitting the fission yeast cell; Krapp A et al.; The septation initiation network (SIN) triggers the onset of cytokinesis in the fission yeast Schizosaccharomyces pombe by promoting contraction of the medially placed F-actin ring . SIN signaling is regulated by the polo-like kinase plo1p and by cdc2p, the initiator of mitosis, and its activation is co-ordinated with other events in mitosis to ensure that cytokinesis does not begin until chromosomes have been separated . Though the SIN controls the contractile ring, the signal originates from the poles of the mitotic spindle . Recent studies suggest that the spindle pole body may act as a dynamic assembly site for active SIN signaling complexes . In the budding yeast Saccharomyces cerevisiae the counterpart of the SIN, called the MEN, mediates both mitotic exit and cytokinesis, in part through regulating activation of the phosphoprotein phosphatase Cdc14p . Flp1p, the S . pombe ortholog of Cdc14p, is not essential for mitotic exit, but may contribute to an orderly mitosis-G1 transition by regulating the destruction of the mitotic inducer cdc25p. J Cell Sci, 2004 Sep 15, 117(Pt 20), 4769 - 78 Epub 2004 Aug 31. Requirement for Schizosaccharomyces pombe Top3 in the maintenance of chromosome integrity; Win TZ et al.; In Schizosaccharomyces pombe, topoisomerase III is encoded by a single gene, top3(+), which is essential for cell viability and proper chromosome segregation . Deletion of rqh1(+), which encodes the sole RecQ family helicase in S . pombe, suppresses the lethality caused by loss of top3 . Here, we provide evidence suggesting that the lethality in top3 mutants is due to accumulation of aberrant DNA structures that arise during S phase, as judged by pulsed-field gel electrophoresis . Using a top3 shut-off strain, we show here that depletion of Top3 activates the DNA damage checkpoint associated with phosphorylation of the checkpoint kinase Chk1 . Despite activation of this checkpoint, top3 cells exit the arrest but fail to undergo faithful chromosome segregation . However, these mitotic defects are secondary to chromosomal abnormalities that lead to the lethality, because advance into mitosis did not adversely affect cell survival . Furthermore, top3 function is required for maintenance of nucleolar structure, possibly due to its ability to prevent recombination at the rDNA loci . Our data are consistent with the notion that Top3 has a key function in homologous recombinational repair during S phase that is essential for ensuring subsequent fidelity of chromosome segregation. Chromosoma, 2004 Sep, 113(3), 145 - 56 Epub 2004 Aug 03. Schizosaccharomyces pombe replication protein Cdc45/Sna41 requires Hsk1/Cdc7 and Rad4/Cut5 for chromatin binding; Dolan WP et al.; Cdc45 is a conserved protein required for firing of replication origins and processive DNA replication . We used an in situ chromatin-binding assay to determine factors required for fission yeast Cdc45p chromatin binding . Assembly of the pre-replicative complex is essential for Cdc45p chromatin binding, but pre-replicative complex assembly occurs independently of Cdc45p . Fission yeast Cdc45p associates with MCM proteins in asynchronously growing cells and cells arrested in S phase by hydroxyurea, but not in cells arrested at the G2/M transition . Both hsk1+ (the fission yeast CDC7 homologue) and rad4+/ cut5+ (the fission yeast DPB11 homologue) are required for Cdc45p chromatin binding . Cdc45p also remains chromatin-bound in mutants that fail to recover from replication arrest . In summary, Cdc45p chromatin binding requires an intact pre-replicative complex as well as signaling from both the Dbf4-dependent kinase and cyclin-dependent kinases. Carbohydr Res, 2004 Sep 13, 339(13), 2255 - 65 Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy; Sugawara T et al.; Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, a (1-->6)-beta-d-glucan, and a alpha-galactomannan . To refine the structures of these polysaccharides, cell-wall glucans of S . pombe were extracted, fractionated, and analyzed by NMR spectroscopy . S . pombe cells were treated with 3% NaOH, and alkali-soluble and insoluble fractions were prepared . The alkali-insoluble fraction was treated with 0.5M acetic acid or Zymolyase 100T to yield an alkali-insoluble, acetic acid-insoluble fraction, an alkali-insoluble, Zymolyase-insoluble fraction, and an alkali-insoluble, Zymolyase-soluble fraction . (13)C NMR and 2D-NMR spectra disclosed that the cell wall of S . pombe is composed of three types of glucans, specifically, a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, which may either be linear or slightly branched, and a highly branched (1-->6)-beta-d-glucan, in addition to alpha-galactomannan . The highly branched (1-->6)-beta-d-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1-->3)-beta-branched (1-->6)-beta-d-glucan with more branches than that of Saccharomyces cerevisiae . Flexibility of these polysaccharides in the cell wall was analyzed by (13)C NMR spectra in D(2)O . The data collectively indicate that (1-->3)-alpha- and (1-->3)-beta-d-glucans are rigid and contribute to the cell shape, while the highly branched (1-->6)-beta-d-glucan and alpha-galactomannan are flexible. DNA Repair (Amst), 2004 Oct 5, 3(10), 1363 - 74 Identification and characterization of the rlp1+, the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe; Khasanov FK et al.; A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified . Rlp1 shows homology to RecA-like proteins, and is the third S . pombe Rad51 paralog besides Rhp55 and Rhp57 . The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif . The rlp1Delta mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes . In contrast to other recombinational repair mutants, the rlp1Delta mutant does not exhibit sensitivity to UV light and mitomycin C (MMC) . Mitotic recombination is moderately reduced in rlp1 mutant . Epistatic analysis of MMS and IR-sensitivity of rlp1Delta mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+, rhp55+, and rad22+ genes . Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein . We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse. Antimicrob Agents Chemother, 2004 Sep, 48(9), 3268 - 71 The melaminophenyl arsenicals melarsoprol and melarsen oxide interfere with thiamine metabolism in the fission yeast Schizosaccharomyces pombe; Schweingruber ME; The melaminophenyl arsenical melarsoprol is the main drug used against late-stage sleeping sickness caused by Trypanosoma brucei subspecies . Its active metabolite in the human body is melarsen oxide . Here, it is shown that this metabolite inhibits growth of the fission yeast Schizosaccharomyces pombe and that its toxicity can be abolished efficiently by thiamine (vitamin B(1)), thiamine analogues, and the pyrimidine moiety of the thiamine molecule . Uptake of melarsen oxide is mediated by a membrane protein (car1p), which is involved in the uptake of thiamine and its pyrimidine moiety . Melarsoprol is taken up by cells in a thiamine- and car1p-dependent manner but is not toxic to cells. Nucleic Acids Res, 2004 Aug 18, 32(15), 4421 - 8 Print 2004. A novel type of silencing factor, Clr2, is necessary for transcriptional silencing at various chromosomal locations in the fission yeast Schizosaccharomyces pombe; Bjerling P et al.; The mating-type region of the fission yeast Schizosaccharomyces pombe comprises three loci: mat1, mat2-P and mat3-M . mat1 is expressed and determines the mating type of the cell . mat2-P and mat3-M are two storage cassettes located in a 17 kb heterochromatic region with features identical to those of mammalian heterochromatin . Mutations in the swi6+, clr1+, clr2+, clr3+, clr4+ and clr6+ genes were obtained in screens for factors necessary for silencing the mat2-P-mat3-M region . swi6+ encodes a chromodomain protein, clr3+ and clr6+ histone deacetylases, and clr4+ a histone methyltransferase . Here, we describe the cloning and characterization of clr2+ . The clr2+ gene encodes a 62 kDa protein with no obvious sequence homologs . Deletion of clr2+ not only affects transcriptional repression in the mating-type region, but also centromeric silencing and silencing of a PolII-transcribed gene inserted in the rDNA repeats . Using chromatin immunoprecipitation, we show that Clr2 is necessary for histone hypoacetylation in the mating-type region, suggesting that Clr2 acts upstream of histone deacetylases to promote transcriptional silencing. Mol Biol Cell, 2004 Nov, 15(11), 4960 - 70 Epub 2004 Aug 18. Schizosaccharomyces pombe RanGAP homolog, SpRna1, is required for centromeric silencing and chromosome segregation; Kusano A et al.; We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP . Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation . In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S . pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S . pombe Ran homolog), rescued a lethality of Sprna1ts . Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres . Consistently, Sprna1ts was defective in gene-silencing at the centromeres . But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation . snf2SR+ rescued a centromeric silencing defect and Deltaclr4+ was synthetic lethal with Sprna1ts . Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR . Loss of SpRna1 activity, therefore, caused chromosome missegregation. Nucleic Acids Res, 2004 Aug 17, 32(14), 4400 - 10 Print 2004. Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations; Davidson MK et al.; Identification of genes required for segregation of chromosomes in meiosis (scm) is difficult because in most organisms high-fidelity chromosome segregation is essential to produce viable meiotic products . The biology of fission yeast Schizosaccharomyces pombe facilitates identification of such genes . Insertional mutagenesis was achieved by electroporation of linear ura4+ DNA into cells harboring a ura4 deletion . Approximately 1000 stable transformants were screened individually for the production of elevated frequencies of aneuploid spore colonies . Twenty-two candidates were subjected to a secondary screen for cytological defects . Five mutants exhibited significant levels of aberrant meiotic chromosome segregation, but were proficient for mating and completion of meiosis . Each mutant's phenotype cosegregated with its respective ura4+ transgene . The mutations were recessive and defined five complementation groups, revealing five distinct genes (scm1, scm2, scm3, scm4 and scm5) . Southern blotting revealed single-site integration in each transformant, indicating that insertional mutagenesis is useful for generating single-locus scm mutations linked to a selectable marker . The transgene insertion points were refractory to analysis by inverse-PCR . Molecular and real-time PCR analyses revealed the presence of multiple, truncated copies of ura4+ at each integration site . Thus, electroporation-mediated insertional mutagenesis in S.pombe is preceded by exonucleolytic processing and concatomerization of the transforming DNA. J Biol Chem, 2004 Oct 22, 279(43), 44987 - 95 Epub 2004 Aug 16. Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2; Chi Y et al.; Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling . We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2 . We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin . In contrast, Ca2+ and Cu2+ are inhibitory . We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2) . Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction . Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated . The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5 . The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4 . Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways . The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation. Nucleic Acids Res, 2004 Aug 10, 32(14), 4297 - 305 Print 2004. Position-dependent function of a B block promoter element implies a specialized chromatin structure on the S.cerevisiae U6 RNA gene, SNR6; Kaiser MW et al.; The Saccharomyces cerevisiae U6 RNA gene, SNR6, is transcribed by RNA polymerase III (Pol III), but lacks the intragenic B block promoter element found in most other Pol III transcription units . Rather, the SNR6 B block element is located 120 bp downstream of the terminator . In contrast, the Schizosaccharomyces pombe U6 RNA gene has an intragenic B block sequence in a short intron . We show that the S.pombe U6 intron, when inserted into SNR6, can functionally replace the downstream B block in vitro but not in vivo . The in vivo expression defect is caused by at least three different effects of the insertion: (i) the S.pombe intron is inefficiently spliced in S.cerevisiae due to the short distance between the 5' splice site and branchpoint; (ii) the S.pombe B block sequence is suboptimal for S.cerevisiae; and (iii) a B block does not function well within the context of the SNR6 intron, especially when the gene is present at its normal chromosomal locus rather than on a plasmid . This last observation suggests that the chromatin structure of the SNR6 locus favors utilization of a downstream B block element . We also provide evidence that splicing of U6 RNA reduces its activity, presumably due to alterations in U6 RNA structure, localization and/or assembly into the spliceosome. Nucleic Acids Res, 2004 Aug 09, 32(14), 4205 - 16 Print 2004. Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast; Ryu GH et al.; The Schizosaccharomyces pombe pfh1+ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing . Mutations in the pfh1+ gene suppress a temperature-sensitive allele of cdc24+, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing . In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism . We show that a full-length Pfh1 fusion protein is active as a monomer . The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate . The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1+ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38 . Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities . These results indicate that the simultaneous loss-of-function mutations of pfh1+ and dna2+ (or cdc24+) are essential to restore the growth defect . Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol -mediated lagging strand displacement DNA synthesis. Eukaryot Cell, 2004 Aug, 3(4), 944 - 54 The forkhead transcription factor Fkh2 regulates the cell division cycle of Schizosaccharomyces pombe; Bulmer R et al.; In eukaryotes the regulation of gene expression plays a key role in controlling cell cycle progression . Here, we demonstrate that a forkhead transcription factor, Fkh2, regulates the periodic expression of cdc15(+) and spo12(+) in the M and G(1) phases of the cell division cycle in the fission yeast Schizosaccharomyces pombe . We also show that Fkh2 is important for several cell cycle processes, including cell morphology and cell separation, nuclear structure and migration, and mitotic spindle function . We find that the expression of fkh2(+) is itself regulated in a cell cycle-dependent manner in G(1) coincident with the expression of cdc18(+), a Cdc10-regulated gene . However, fkh2(+) expression is independent of Cdc10 function . Fkh2 was found to be phosphorylated during the cell division cycle, with a timing that suggests that this posttranslational modification is important for cdc15(+) and spo12(+) expression . Related forkhead proteins regulate G(2) and M phase-specific gene expression in the evolutionarily distant Saccharomyces cerevisiae, suggesting that these proteins play conserved roles in regulating cell cycle processes in eukaryotes. Gene, 2004 Aug 18, 338(1), 85 - 91 Preferential loss and gain of introns in 3' portions of genes suggests a reverse-transcription mechanism of intron insertion; Sverdlov AV et al.; In an attempt to gain insight into the dynamics of intron evolution in eukaryotic protein-coding genes, the distributions of old introns, that are conserved between distant phylogenetic lineages, and new, lineage-specific introns along the gene length, were examined . A significant excess of old introns in 5'-regions of genes was detected . New introns, when analyzed in bulk, showed a nearly flat distribution from the 5'- to the 3'-end . However, analysis of new intron distributions in individual genomes revealed notable lineage-specific features . While in intron-poor genomes, particularly yeast Schizosaccharomyces pombe (Sp), the 5'-portions of genes contain a significantly greater number of new introns than the 3'-portions, the intron-rich genomes of humans and Arabidopsis show the opposite trend . These observations seem to be compatible with the view that introns are both lost and inserted in 3'-terminal portions of genes more often than in 5'-portions . Overrepresentation of 3'-terminal sequences among cDNAs that mediate intron loss appears to be the most likely explanation for the apparent preferential loss of introns in the distal parts of genes . Preferential insertion of introns in the 3'-portions suggests that introns might be inserted via a reverse-transcription-mediated pathway similar to that implicated in intron loss . This mechanism could involve duplication of a portion of the coding region during reverse transcription followed by homologous recombination and subsequent rapid sequence divergence in the copy that becomes a new intron. Yeast, 2004 Jul 30, 21(10), 867 - 81 Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe; Iwaki T et al.; To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant . The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae . We characterized the fission yeast mutant end4 Delta as well as ypt7 Delta, which is deficient in vacuolar fusion and, hence, endocytosis . The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3-GFP were inhibited in the end4 mutant . Deletion of end4 resulted in pleiotropic phenotypes consistent with F-actin depolarization, including high temperature sensitivity, abnormal morphology and mating defects . Extensive missorting of carboxypeptidase Y was detected in the ypt7 mutant; however, little missorting was detected in the end4 mutant . These results indicate that End4p is essential for the internalization process and Ypt7p affects endocytosis at a post-internalization step after the intersection of the endocytic and the vacuolar protein-sorting pathways in fission yeast. Genes Cells, 2004 Aug, 9(8), 671 - 84 Meiosis induced by inactivation of Pat1 kinase proceeds with aberrant nuclear positioning of centromeres in the fission yeast Schizosaccharomyces pombe; Chikashige Y et al.; Nuclear organization of chromosomes proceeds with significant changes during meiosis . In the fission yeast Schizosaccharomyces pombe, centromeres are clustered at the spindle-pole body (SPB) during the mitotic cell cycle; however, during meiotic prophase telomeres become clustered to the SPB and centromeres dissociate from the SPB . We followed the movement of telomeres, centromeres and sister chromatids in living S . pombe cells that were induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis) . Time-course observation in living cells determined the temporal order of DNA synthesis, telomere clustering, centromere separation and meiotic chromosome segregation . When meiosis was induced by Pat1 inactivation at the G1 phase of mitosis, telomeres clustered to the SPB as per normal meiosis, but in most cells the centromeres remained partially associated with the SPB . When meiosis was initiated at the G2 phase by Pat1 inactivation, both telomeres and centromeres retained their mitotic nuclear positions in the majority of cells . These results indicate that the progression of meiosis induced by Pat1 inactivation is aberrant from normal meiosis in some events . As Pat1 inactivation is often useful to induce S . pombe cells synchronously into meiosis, the temporal order of chromosomal events determined here will provide landmarks for the progression of meiosis downstream the Pat1 inactivation. EMBO J, 2004 Sep 1, 23(17), 3548 - 58 Epub 2004 Aug 05. Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination; Budzowska M et al.; Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition . Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals . However, the role of many CCC genes in higher eukaryotes remains elusive . Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation . In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable . These cells displayed hypersensitivity to various DNA-damaging agents . Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage . However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency . In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents. J Biol Chem, 2004 Oct 15, 279(42), 43574 - 80 Epub 2004 Aug 05. On the slowing of S phase in response to DNA damage in fission yeast; Kumar S et al.; Eukaryotic cells slow their progression through S phase upon DNA damage . The mechanism that leads to this slowing is called the intra-S-phase checkpoint . Previous studies demonstrated that in the fission yeast Schizosaccharomyces pombe this checkpoint is mediated by a pathway that includes Rad3 (similar to human ATR and ATM) and Cds1 (similar to human Chk1 and Chk2) . Here we present evidence that a major downstream target of this pathway is the cyclin-dependent kinase, Cdc2 . We also present evidence suggesting that the intra-S-phase checkpoint makes a relatively minor contribution to the survival of cells with damaged DNA. Curr Biol, 2004 Aug 10, 14(15), 1330 - 40 Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B; Khodjakov A et al.; INTRODUCTION: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs) . To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe . RESULTS: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity . By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B . Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate . In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut . Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment . Spindle midzone fragments not connected to either of the two spindle poles also elongated . Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles . CONCLUSIONS: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation . By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression . Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast. J Cell Sci, 2004 Aug 15, 117(Pt 18), 4265 - 75 Epub 2004 Aug 03. AtSGP1, AtSGP2 and MAP4K alpha are nucleolar plant proteins that can complement fission yeast mutants lacking a functional SIN pathway; Champion A et al.; In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is signalled via the septation initiation network (SIN) involving several protein kinases and a GTPase . Arabidopsis thaliana and Brassica napus proteins homologous to fission yeast spg1p (AtSGP1, AtSGP2), cdc7p (AtMAP3K epsilon 1, AtMAP3K epsilon 2, BnMAP3K epsilon 1) and sid1p (AtMAP4K alpha 1, AtMAP4K alpha 2, BnMAP4K alpha 2) exhibit a significant similarity . The plant proteins AtSGP1/2 and BnMAP4K alpha 2 are able to complement the S . pombe mutant proteins spg1-B8 and sid1-239, respectively and to induce mutisepta when overexpressed in wild-type yeast . Yeast two-hybrid assays demonstrated interactions both between plant proteins and between plant and yeast proteins of the SIN pathway . However, the primary structure of the proteins as well as the partial complementation of yeast mutants indicates that plant homologous proteins and their yeast counterparts have diverged during evolution . Real-time RT-PCR studies demonstrated plant SIN-related gene expression in all organs tested and a co-expression pattern during the cell cycle, with a higher accumulation at G(2)-M . During interphase, the plant SIN-related proteins were found to co-localise predominantly in the nucleolus of the plant cells, as shown by fusions to green fluorescent protein . These data suggest the existence of a plant SIN-related pathway. Chromosoma . 2004 Aug 3; {Epub ahead of print} Schizosaccharomyces pombe replication protein Cdc45/Sna41 requires Hsk1/Cdc7 and Rad4/Cut5 for chromatin binding; Dolan WP et al.; Cdc45 is a conserved protein required for firing of replication origins and processive DNA replication . We used an in situ chromatin-binding assay to determine factors required for fission yeast Cdc45p chromatin binding . Assembly of the pre-replicative complex is essential for Cdc45p chromatin binding, but pre-replicative complex assembly occurs independently of Cdc45p . Fission yeast Cdc45p associates with MCM proteins in asynchronously growing cells and cells arrested in S phase by hydroxyurea, but not in cells arrested at the G2/M transition . Both hsk1(+) (the fission yeast CDC7 homologue) and rad4(+)/ cut5(+) (the fission yeast DPB11 homologue) are required for Cdc45p chromatin binding . Cdc45p also remains chromatin-bound in mutants that fail to recover from replication arrest . In summary, Cdc45p chromatin binding requires an intact pre-replicative complex as well as signaling from both the Dbf4-dependent kinase and cyclin-dependent kinases. Chromosome Res, 2004, 12(6), 535 - 42 The roles of histone modifications and small RNA in centromere function; Ekwall K; Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi) . Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function . The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion . The chromatin structure of both domains is regulated epigenetically . In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role . In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein . Swi6 then directly mediates cohesin binding to the centromeric heterochromatin . Recently, a surprising link was made between heterochromatin formation and RNAi. Mol Cell Biol, 2004 Aug, 24(16), 7235 - 48 Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality; Hopkins KM et al.; The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage . Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals . Mrad9(-/-) cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity . These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9(+/+) controls . Mrad9(-/-) cells could initiate but not maintain gamma-ray-induced G(2) delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed . Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9(-/-) cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved . Mrad9(+/-) mice were generated, but heterozygous intercrosses failed to yield Mrad9(-/-) pups, since embryos died at midgestation . Furthermore, Mrad9(-/-) mouse embryo fibroblasts were not viable . These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development. Biochim Biophys Acta, 2004 Jul 23, 1658(1-2), 133 - 40 Structural insight into the cooperativity between catalytic and noncatalytic sites of F1-ATPase; Falson P et al.; F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites . The molecular mechanism of such cooperativity is currently unknown . To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe . These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative cooperativity . The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide . Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative cooperativity . In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface . The properties of these mutants suggest that negative cooperativity occurs through interactions between neighbor alpha- and beta-subunits . Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site . We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated. Int J Food Microbiol, 2004 Sep 1, 95(2), 119 - 26 Yeast ecology of Kombucha fermentation; Teoh AL et al.; Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria . Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha . Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests . During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha . The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii . While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology . Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species. Proteins, 2004 Sep 1, 56(4), 808 - 20 Function-dependent clustering of orthologues and paralogues of cyclophilins; Galat A; The 18 kDa archetypal cyclosporin-A binding protein, cyclophilin-A, has multiple paralogues in the human genome . Only 18 of those paralogues have been detected as mRNAs or proteins whose masses vary from 18 to 354 kDa, whereas the functional significance of the open reading frames (ORFs) encoding other paralogues of cyclophilin-A remains unknown . The genomes of Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae encode different numbers of the cyclophilin paralogues, some of which are orthologous to the human cyclophilins . A library of novel algorithms was developed and used for computation of the conservation levels for hydrophobicity and bulkiness profiles, and amino acid compositions (AACs) of 303 aligned sequences of cyclophilins . The majority of the paralogues and orthologues encoded in these 6 genomes differ considerably from each other . Some of the orthologues and paralogues have high correlation coefficients (CCFs) for pairwise compared hydrophobicity and bulkiness profiles, and whose AACs differ to a low degree . Convergence of these three properties of the polypeptide chain and apparent conservation of the typical sequence hallmarks and parameters allowed for the clustering of the functionally related orthologues and paralogues of the cyclophilins . The clustering method allowed for sorting out the cyclophilins into several distinct classes . Analyses of the overlapping clusters of sequences permitted delineation of some hypothetical pathways that might have led to the creation of certain paralogues of cyclophilins in the eukaryotic genomes . Antonie Van Leeuwenhoek, 2004 Aug, 86(2), 135 - 47 Bioinformatic analysis of the link between gene composition and expressivity in Saccharomyces cerevisiae and Schizosaccharomyces pombe; Fuglsang A; The compositional non-randomness was studied in genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe . In both species, codon usage is well correlated with expressivity (measured as the codon adaptation index) . Both species generally display higher nucleotide non-randomness in the group of highly expressed genes than in the lowly expressed genes . The highly expressed genes in both species are furthermore characterized by marked peaks in non-randomness at N=3 upstream of start codons, N=2 downstream of start codons and at N=1 and N=7 downstream of stop codons, indicating that these nucleotides may be key elements in translational regulation . Intragenic variation in codon usage was also observed to be linked to expressivity . It is suggested that the firm link between expressivity and codon usage calls for codon optimization . Based on bioinformatic calculations, examples of proteins are given for which codon optimizations might be relevant. Genetics, 2004 Jul, 167(3), 1143 - 53 Alleles of the hotspot cog are codominant in effect on recombination in the his-3 region of Neurospora; Yeadon PJ et al.; There are two naturally occurring functional alleles of the recombination hotspot cog, which is located 3.5 kb from the his-3 locus of Neurospora crassa . The presence of the cog+ allele in a cross significantly increases recombination in the his-3 region compared to a cross homozygous for the cog allele . Data obtained shortly after discovery of cog+ suggested that it was fully dominant to cog . However, a dominant cog+ conflicts with observations of hotspots in Saccharomyces cerevisiae and Schizosaccharomyces pombe, in which recombination is initiated independently of homolog interactions, and suggests recombination mechanisms may differ in Neurospora and yeast . We present evidence that cog alleles are codominant in effect on both allelic recombination in his-3 and crossing over between loci flanking his-3 . In addition, we show that genetic background variation has at least a twofold effect on allelic recombination . We speculate that variation in genetic background, together with the complexities of recombination in crosses bearing close mutant alleles, accounts for the previous conclusion that cog+ is dominant to cog. Genetics, 2004 Jul, 167(3), 1095 - 107 Functional dissection of the gamma-tubulin complex by suppressor analysis of gtb1 and alp4 mutations in Schizosaccharomyces pombe; Tange Y et al.; In fission yeast, gamma-tubulin (encoded by the gtb1+ gene), Alp4 (Spc97/GCP2), and Alp6 (Spc98/GCP3) are essential components of the gamma-tubulin complex . We isolated gtb1 mutants as allele-specific suppressors of temperature-sensitive alp4 mutations . Mutation sites in gtb1 mutants and in several alp4 alleles were determined . The majority of substituted amino acids were mapped to a small area on the predicted surface of the gamma-tubulin molecule that might directly interact with the Alp4 protein . The cold sensitivity of gamma-tubulin mutants was almost completely suppressed by an alpha-tubulin mutation and partially suppressed by a low concentration of thiabendazole, a microtubule assembly inhibitor . Other gtb1 mutants had increased resistance to this drug . Gel-filtration and immunoprecipitation analyses suggested that the mutant gamma-tubulin formed an altered gamma-tubulin complex with increased stability compared to wild-type gamma-tubulin . In most gtb1 mutants, sexual development was impaired, and aberrant asci that contained an irregular spore shape and number were produced . In contrast, spore formation was not appreciably damaged in some alp4 and alp6 mutants, even at temperatures where vegetative proliferation was substantially defective . These results suggested that the function of the gamma-tubulin complex or the requirement of each component of the complex is differentially regulated between the vegetative and sexual phases of the life cycle in fission yeast . In addition, genetic data indicated intimate functional connections of gamma-tubulin with several kinesin-like proteins. Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1621 - 6 Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe; Jeong HT et al.; The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized . We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1) . Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation . The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p . We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S . pombe cells . An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p . A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm . The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S . pombe. Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1489 - 99 Genetic analysis of chs1+ and chs2+ encoding chitin synthases from Schizosaccharomyces pombe; Matsuo Y et al.; To explore the function of chitin in Schizosaccharomyces pombe, we have cloned chs1+ and chs2+, encoding putative chitin synthases, based on sequences in the Sanger Centre database . The synthetic lethal phenotype of the S . cerevisiae chs1 chs2 chs3 mutant was complemented by expression of S . pombe chs1+ or chs1+, indicating that both chs1+ and chs2+ in fact encode chitin synthase . The homothallic Deltachs1 strain formed abnormal asci that contained 1, 2, or 3 spores, while the Deltachs2 strain had no noticeable phenotype . The chs1 chs2 double disruptant looked similar phenotypically to the Deltachs1 strain . The Chs2-GFP fusion protein predominantly localized at the septum after the septum was formed during vegetative growth . The level of chs2+ mRNA increased just before the septum was formed . Levels of Chs2-13Myc synthesis also changed during the cell cycle . Thus, chs1+ is required for proper spore formation, and chs2+ is perhaps involved in septum formation. Structure (Camb), 2004 Jun, 12(6), 999 - 1013 Structure of the actin crosslinking core of fimbrin; Klein MG et al.; Filamentous actin is organized into bundles and orthogonal networks by the fimbrin/alpha-actinin superfamily of F-actin crosslinking proteins . The crystal structure of the Arabidopsis thaliana and Schizosaccharomyces pombe fimbrin cores provides the first description of a functional F-actin crosslinking protein and highlights the compact and distinctly asymmetric organization of the fimbrin molecule, in which the two actin binding domains present distinct surfaces to solvent . The mapping of functionally important residues onto the structure affords new insights into the binding process and provides additional constraints which must be accommodated by models for F-actin binding and crosslinking . Most strikingly, this work provides unique insight into the mechanistic features of conditional-lethal mutants and their extragenic suppressors, which highlight conformational and dynamic properties required for fimbrin function . These results underscore the power of jointly considering structural and genetic suppressor data for obtaining unexpected and biologically relevant mechanistic information. Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1396 - 403 Epub 2004 Jul 21. Structure, crystal packing and molecular dynamics of the calponin-homology domain of Schizosaccharomyces pombe Rng2; Wang CH et al.; Schizosaccharomyces pombe Rng2 is an IQGAP protein that is essential for the assembly of an actomyosin ring during cytokinesis . Rng2 contains an amino-terminal calponin-homology (CH) domain, 11 IQ repeats and a RasGAP-homology domain . CH domains are known mainly for their ability to bind F-actin, although they have other ligands in vivo and there are only few examples of actin-binding single CH domains . The structures of several CH domains have already been reported, but this is only the third report of an actin-binding protein that contains a single CH domain (the structures of calponin and EB1 have been reported previously) . The 2.21 A resolution crystal structure of the amino-terminal 190 residues of Rng2 from Br- and Hg-derivatives includes 40 residues (150-190) carboxyl-terminal to the CH domain that resemble neither the extended conformation seen in utrophin, nor the compact conformation seen in fimbrin, although residues 154-160 form an unstructured coil which adopts a substructure similar to dystrophin residues 240-246 in the carboxyl-terminal portion of the CH2 domain . This region wraps around the stretch of residues that would be equivalent to the proposed actin-binding site ABS1 and ABS2 from dystrophin . This distinctive feature is absent from previously published CH-domain structures . Another feature revealed by comparing the two derivatives is the presence of two loop conformations between Tyr92 and Arg99. J Biol Chem, 2004 Sep 17, 279(38), 39636 - 44 Epub 2004 Jul 22. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain; Pike BL et al.; Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks . To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain . Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks . However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks . The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta) . Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint . The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase . Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways. Biotechnol Lett, 2004 Jun, 26(11), 933 - 7 Flow process for electroextraction of intracellular enzymes from the fission yeast, Schizosaccharomyces pombe; Ganeva V et al.; Flow treatment of the yeast, Schizosaccharomyces pombe, with high intensity electric field pulses released intracellular enzymes such as glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase . Over 70% of the total activity was liberated within 4 h after pulse application . The optimal field intensities were considerably higher than that needed for irreversible plasma membrane permeabilization. J Cell Sci, 2004 Aug 1, 117(Pt 17), 3875 - 86 Epub 2004 Jul 20. Activation of the pheromone-responsive MAP kinase drives haploid cells to undergo ectopic meiosis with normal telomere clustering and sister chromatid segregation in fission yeast; Yamamoto TG et al.; Meiosis is a process of importance for sexually reproducing eukaryotic organisms . In the fission yeast Schizosaccharomyces pombe, meiosis normally proceeds in a diploid zygote which is produced by conjugation of haploid cells of opposite mating types . We demonstrate that activation of the pheromone-responsive MAPK, Spk1, by the ectopic expression of a constitutively active form of Byr1 (MAPKK for Spk1) induced the cells to undergo meiosis while in the haploid state . Moreover, the induction of meiosis required Mei2 (a key positive regulator of meiosis), but did not require Mei3; Mei3 is normally required to inactivate the Pat1 kinase (a negative regulator of Mei2) thereby allowing Mei2 to drive meiosis . Therefore, expression of a constitutively active form of Byr1 activates Mei2 without the need of Mei3 . In cells induced to undergo meiosis by activating the Spk1 MAPK signaling pathway, telomeres clustered at the spindle pole body (SPB) and centromeres detached normally from the SPB during meiotic prophase, and the cells showed the correct segregation of sister chromatids during meiotic divisions . In contrast, in meiosis induced by inactivation of Pat1, sister chromatids segregate precociously during the first meiotic division . Thus, these results suggest that activation of Spk1 drives meiosis in S . pombe. J Cell Sci, 2004 Aug 1, 117(Pt 17), 3897 - 910 Epub 2004 Jul 20. The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe; Mishra M et al.; Fission yeast mutants defective in actomyosin ring formation and function exhibit a prolonged G2 delay following cytokinesis failure . This G2 delay depends on the SIN, a signaling network essential for cytokinesis, and the non-essential Cdc14p family phosphatase, Clp1p/Flp1p and has been proposed to signify a cytokinesis checkpoint mechanism . However, the physiological relevance of this proposed Clp1p/Flp1p-dependent checkpoint is unclear because all previous studies were carried out using mutations in essential actomyosin ring components under fully restrictive conditions and thus these cells would have died regardless of the presence of the checkpoint . Here we show that delays in cytokinesis caused by minor perturbations to different components of the cytokinetic machinery, which normally cause only mild defects, become lethal when Clp1p/Flp1p is inactivated . In addition, we show that Clp1p/Flp1p does not function simply to inhibit further rounds of nuclear division, but also allows damaged actomyosin rings to be maintained to facilitate completion of cell division . Ectopic activation of the SIN significantly bypasses the requirement of Clp1p/Flp1p for G2 delay as well as for completion of cytokinesis . We conclude that the Clp1p/Flp1p-dependent cytokinesis checkpoint provides a previously unrecognized cell survival advantage when the cell division apparatus is mildly perturbed. Eur J Biochem, 2004 Aug, 271(15), 3208 - 14 Structural basis of charge transfer complex formation by riboflavin bound to 6,7-dimethyl-8-ribityllumazine synthase; Koch M et al.; The amino acid residue tryptophan 27 of 6,7-dimethyl-8-ribityllumazine synthase of the yeast Schizosaccharomyces pombe was replaced by tyrosine . The structures of the W27Y mutant protein in complex with riboflavin, the substrate analogue 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, and the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine, were determined by X-ray crystallography at resolutions of 2.7-2.8 A . Whereas the indole system of W27 forms a coplanar pi-complex with riboflavin, the corresponding phenyl ring in the W27Y mutant establishes only peripheral contact with the heterocyclic ring system of the bound riboflavin . These findings provide an explanation for the absence of the long wavelength shift in optical absorption spectra of riboflavin bound to the mutant enzyme . The structures of the mutants are important tools for the interpretation of the unusual physical properties of riboflavin in complex with lumazine synthase. J Cell Biol, 2004 Jul 19, 166(2), 205 - 11 Epub 2004 Jul 12. A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast; Mitra P et al.; The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI{3,4,5}P3) . We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1) . This was an unexpected finding because yeast (S . pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes . Indeed, PI(3,4,5)P3 has not been detected in yeast . Surprisingly, upon deletion of ptn1 in S . pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S . pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels . By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S . pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p . These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases . Copyright The Rockerfeller University Press Acta Biochim Biophys Sin (Shanghai), 2004 Jul, 36(7), 443 - 9 Inositol and phosphatidylinositol mediated mediated glucose derepression, gene expression and invertase secretion in yeasts; Chi ZM et al.; Glucose repression occurs in many yeast species and some filamentous fungi, and it represses the expression and secretion of many intracellular and extracellular proteins . In recent years, it has been found that many biochemical reactions in yeast cells are mediated by phosphatidylinositol (PI)-type signaling pathway . However, little is known about the relationships between PI-type signaling and glucose repression, gene expression and invertase secretion in yeasts . Many evidences in our previous studies showed that glucose repression, invertase secretion, gene expression and cell growth were mediated by inositol and PI in Saccharomyces and Schizosaccharomyces . The elucidation of the new regulatory mechanisms of protein secretion, gene expression and glucose repression would be an entirely new aspect of inositol and PI-type signaling regulation in yeasts. RNA, 2004 Aug, 10(8), 1200 - 14 Epub 2004 Jul 09. mRNA deadenylation by PARN is essential for embryogenesis in higher plants; Reverdatto SV et al.; Deadenylation of mRNA is often the first and rate-limiting step in mRNA decay . PARN, a poly(A)-specific 3' --> 5' ribonuclease which is conserved in many eukaryotes, has been proposed to be primarily responsible for such a reaction, yet the importance of the PARN function at the whole-organism level has not been demonstrated in any species . Here, we show that mRNA deadenylation by PARN is essential for viability in higher plants (Arabidopsis thaliana) . Yet, this essential requirement for the PARN function is not universal across the phylogenetic spectrum, because PARN is dispensable in Fungi (Schizosaccharomyces pombe), and can be at least severely downregulated without any obvious consequences in Metazoa (Caenorhabditis elegans) . Development of the Arabidopsis embryos lacking PARN (AtPARN), as well as of those expressing an enzymatically inactive protein, was markedly retarded, and ultimately culminated in an arrest at the bent-cotyledon stage . Importantly, only some, rather than all, embryo-specific transcripts were hyperadenylated in the mutant embryos, suggesting that preferential deadenylation of a specific select subset of mRNAs, rather than a general deadenylation of the whole mRNA population, by AtPARN is indispensable for embryogenesis in Arabidopsis . These findings indicate a unique, nonredundant role of AtPARN among the multiple plant deadenylases. J Biol Chem, 2004 Oct 1, 279(40), 41594 - 602 Epub 2004 Jul 06. A cooperative role for Atf1 and Pap1 in the detoxification of the oxidative stress induced by glucose deprivation in Schizosaccharomyces pombe; Madrid M et al.; In Schizosaccharomyces pombe, glucose concentrations below a certain threshold trigger the stress-activated protein kinase (SAPK) signal transduction pathway and promote increased transcription of Atf1-dependent genes coding for the general stress response . Removal of glucose specifically induces the nuclear accumulation of green fluorescent protein-labeled Pap1 (GFP-Pap1) and the expression of genes dependent on this transcription factor . In contrast, depletion of the nitrogen source triggers the SAPK pathway but does not activate Pap1-dependent gene transcription, indicating that carbon stress rather than growth arrest leads to an endogenous oxidative condition that favors nuclear accumulation of Pap1 . The reductant agents glutathione or N-acetylcysteine suppress the nuclear accumulation of GFP-Pap1 induced by glucose deprivation without inhibiting the activation of the MAPK Sty1 . In addition, cells expressing a mutant GFP-Pap1 unable to accumulate into the nucleus upon hydrogen peroxide-mediated oxidative stress failed to show this protein into the nucleus in the absence of glucose . These results support the concept of a concerted action between the SAPK pathway and the Pap1 transcription factor during glucose exhaustion by which glucose limitation induces activation of the SAPK pathway prior to the oxidative stress caused by glucose deprivation . The ensuing induction of Atf1-dependent genes (catalase) decreases the level of hydroperoxides allowing Pap1 nuclear accumulation and function . Congruent with this interpretation, glucose-depleted cells show higher adaptive response to exogenous oxidative stress than those maintained in the presence of glucose. Bioorg Med Chem, 2004 Aug 1, 12(15), 4285 - 99 2-pyrones possessing antimicrobial and cytotoxic activities; Fairlamb IJ et al.; The 2-pyrone sub-unit is found in a number of natural products possessing broad spectrum biological activity . Such compounds are validated as being capable of binding to specific protein domains and able to exert a remarkable range of biological effects . In an effort to identify synthetic 2-pyrones with interesting biological effects, herein we report the synthesis and biological evaluation of 4-substituted-6-methyl-2-pyrones . Synthetic routes to 4-alkyl/alkenyl/aryl/alkynyl-6-methyl-2-pyrones have been developed utilising Sonogashira, Suzuki and Negishi cross-coupling starting from readily available 4-bromo-6-methyl-2-pyrone . Specific conditions for each organometallic protocol were required for successful cross-coupling . In particular, a triethylamine/acetonitrile--base/solvent mixture was crucial to Sonogashira alkynylation of 4-bromo-6-methyl-2-pyrone, whereas thallium carbonate was a mandatory base for the Suzuki cross-coupling of trialkylboranes . The 2-pyrones demonstrate potent inhibitory activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Schizosaccharomyces pombe and Botrytis cinerea . The growth inhibitory activities of selected 2-pyrones were determined in A2780 human ovarian carcinoma and K562 human chronic myelogenous leukaemia cell lines using an in vitro cell culture system (MTT assay) . These studies demonstrate that 4-phenylethynyl-, 4-tetrahydropyranylpropargyl ether- and 4-ethynyl-6-methyl-2-pyrones have excellent potential as a new class of anticancer agents. Curr Biol, 2004 Jul 13, 14(13), 1181 - 6 Positioning and elongation of the fission yeast spindle by microtubule-based pushing; Tolic-Norrelykke IM et al.; In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development . We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe . We hypothesized that astral microtubules exert mechanical force on the S . pombe spindle and thus help align the spindle with the major axis of the cell . Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy . Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules . We found that astral microtubules push on the spindle poles in S . pombe, in contrast to the pulling forces observed in a number of other cell types . Further, laser dissection of the spindle midzone induced spindle collapse inward . This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone . Broken spindles recovered and mitosis completed as usual . We propose a model of spindle centering and elongation by microtubule-based pushing forces. Genetics, 2004 Jun, 167(2), 593 - 605 Conserved and nonconserved proteins for meiotic DNA breakage and repair in yeasts; Young JA et al.; During meiosis DNA double-strand breaks initiate recombination in the distantly related budding and fission yeasts and perhaps in most eukaryotes . Repair of broken meiotic DNA is essential for formation of viable gametes . We report here distinct but overlapping sets of proteins in these yeasts required for formation and repair of double-strand breaks . Meiotic DNA breakage in Schizosaccharomyces pombe did not require Rad50 or Rad32, although the homologs Rad50 and Mre11 are required in Saccharomyces cerevisiae; these proteins are required for meiotic DNA break repair in both yeasts . DNA breakage required the S . pombe midmeiosis transcription factor Mei4, but the structurally unrelated midmeiosis transcription factor Ndt80 is not required for breakage in S . cerevisiae . Rhp51, Swi5, and Rad22 + Rti1 were required for full levels of DNA repair in S . pombe, as are the related S . cerevisiae proteins Rad51, Sae3, and Rad52 . Dmc1 was not required for repair in S . pombe, but its homolog Dmc1 is required in the well-studied strain SK1 of S . cerevisiae . Additional proteins required in one yeast have no obvious homologs in the other yeast . The occurrence of conserved and nonconserved proteins indicates potential diversity in the mechanism of meiotic recombination and divergence of the machinery during the evolution of eukaryotes. J Agric Food Chem, 2004 Jul 14, 52(14), 4529 - 34 Effect of Schizosaccharomyces pombe on aromatic compounds in dry sherry wines containing high levels of gluconic acid; Peinado RA et al.; Volatile compounds have been determined in control dry sherry wines and those supplemented with gluconic acid, which were inoculated with the Schizosaccharomyces pombe 1379 (ATCC 26760) yeast strain . These compounds were grouped, according to volatiles exhibiting the identical odor quality, into nine groups of the same odor character (aromatic series) as a way of establishing the aroma profile for the studied wines . Control and supplemented wines showed changes in the balsamic, spicy, roasty, and fruity aromatic series, and tasters judged the aroma as typical of wines subjected to biological aging . This fission yeast may be used as a treatment to reduce gluconic acid contents in wines obtained from rotten grapes, making feasible the incorporation of these wines into the biological aging process . In addition, this procedure may also help to accelerate the traditional biological aging in sherry winemaking due to the contribution of some specific compounds by S . pombe to the wine. J Mol Biol, 2004 Jul 23, 340(5), 981 - 9 The Schizosaccharomyces pombe open promoter bubble: mammalian-like arrangement and properties; Choi WS et al.; The fission yeast Schizosaccharomyces pombe is often used as a genetic system to model processes that apply to higher cells . Here S.pombe was used to study promoter DNA opening and transcription initiation by RNA polymerase II . The melted region within the adh promoter is about 20 bp in size and has the start site near its center . This arrangement is similar to that at the AdML promoter but different from that in Saccharomyces cerevisiae . Although expression of human TFIIB shifts the start site to the nearby human position, it does not change the location of the bubble . The start site shift is directed by the C terminus of human TFIIB, in contrast to expectations from S.cerevisiae . The creation of the bubble requires the ATPase motifs of XPB . Overall, the data show that promoter melting and initiation in fission yeast is much more similar to humans than to budding yeast. J Biol Chem, 2004 Sep 10, 279(37), 38409 - 14 Epub 2004 Jun 30. Homo-oligomerization is the essential function of the tandem BRCT domains in the checkpoint protein Crb2; Du LL et al.; BRCT (BRCA1 C terminus) domains are frequently found as a tandem repeat in proteins involved in DNA damage responses, such as Saccharomyces cerevisiae Rad9, human 53BP1 and BRCA1 . Tandem BRCT domains mediate protein-protein and protein-DNA interactions . However, the functional significance of these interactions is largely unknown . Here we report the oligomerization of Schizosaccharomyces pombe checkpoint protein Crb2 through its tandem BRCT domains . Truncated Crb2 without BRCT domains is defective in DNA damage checkpoint signaling . However, addition of either of two heterologous dimerization motifs largely restores the functions of truncated Crb2 without BRCT domains . Replacement of Crb2 BRCT domains with a dimerization motif also renders cells resistant to the dominant negative effect of overexpressing Crb2 BRCT domains . These results demonstrate that the crucial function of the tandem BRCT domains is to oligomerize Crb2. Mol Cell Biol, 2004 Jul, 24(14), 6379 - 92 The fission yeast Nup107-120 complex functionally interacts with the small GTPase Ran/Spi1 and is required for mRNA export, nuclear pore distribution, and proper cell division; Bai SW et al.; We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1 . In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S . pombe proteins are localized at the nuclear envelope . Biochemical data confirmed the organization of these nucleoporins within conserved complexes . Although examination of the S . cerevisiae and S . pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts . Unexpectedly, microscopic analyses of some of the S . pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S . pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants . Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S . pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway. Mol Cell Biol, 2004 Jul, 24(14), 6215 - 30 Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast; Nakamura TM et al.; Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX . Histone H2A is similarly regulated in Saccharomyces cerevisiae . The phosphorylated forms of H2AX and H2A, known as gamma-H2AX and gamma-H2A, are thought to be important for DNA repair, although their evolutionarily conserved roles are unknown . Here, we investigate gamma-H2A in the fission yeast Schizosaccharomyces pombe . We show that formation of gamma-H2A redundantly requires the ATR/ATM-related kinases Rad3 and Tel1 . Mutation of the SQE motif to AQE (H2A-AQE) in the two histone H2A genes caused sensitivity to a wide range of genotoxic agents, increased spontaneous DNA damage, and impaired checkpoint maintenance . The H2A-AQE mutations displayed a striking synergistic interaction with rad22Delta (Rad52 homolog) in ionizing radiation (IR) survival . These phenotypes correlated with defective phosphorylation of the checkpoint proteins Crb2 and Chk1 and a failure to recruit large amounts of Crb2 to damaged DNA . Surprisingly, the H2A-AQE mutations substantially suppressed the IR hypersensitivity of crb2Delta cells by a mechanism that required the RecQ-like DNA helicase Rqh1 . We propose that gamma-H2A modulates checkpoint and DNA repair through large-scale recruitment of Crb2 to damaged DNA . This function correlates with evidence that gamma-H2AX regulates recruitment of several BRCA1 carboxyl terminus domain-containing proteins (NBS1, 53BP1, MDC1/NFBD1, and BRCA1) in mammals. J Cell Sci, 2004 Jul 1, 117(Pt 15), 3343 - 51 S . pombe meiotic linear elements contain proteins related to synaptonemal complex components; Lorenz A et al.; The fission yeast Schizosaccharomyces pombe does not form synaptonemal complexes (SCs) in meiotic prophase nuclei . Instead, thin threads, the so-called linear elements (LEs), are observed at the corresponding stages by electron microscopy . Here, we demonstrate that S . pombe Rec10 is a protein related to the Saccharomyces cerevisiae SC protein Red1 and that it localizes to LEs . Moreover, a homologue to S . cerevisiae Hop1 does exist in S . pombe and we show by in situ immunostaining that it, and the kinase Mek1 (a homologue of which is also known to be associated with SCs), localizes to LEs . These observations indicate the evolutionary relationship of LEs with the lateral elements of SCs and suggest that these structures might exert similar functions in S . cerevisiae and S . pombe. Mol Cell, 2004 Jul 2, 15(1), 129 - 39 A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase; Veal EA et al.; Oxidative stress-induced cell damage is an important component of many diseases and ageing . In eukaryotes, activation of JNK/p38 stress-activated protein kinase (SAPK) signaling pathways is critical for the cellular response to stress . 2-Cys peroxiredoxins (2-Cys Prx) are highly conserved, extremely abundant antioxidant enzymes that catalyze the breakdown of peroxides to protect cells from oxidative stress . Here we reveal that Tpx1, the single 2-Cys Prx in Schizosaccharomyces pombe, is required for the peroxide-induced activation of the p38/JNK homolog, Sty1 . Tpx1 activates Sty1, downstream of previously identified redox sensors, by a mechanism that involves formation of a peroxide-induced disulphide complex between Tpx1 and Sty1 . We have identified conserved cysteines in Tpx1 and Sty1 that are essential for normal peroxide-induced Tpx1-Sty1 disulphide formation and Tpx1-dependent regulation of peroxide-induced Sty1 activation . Thus we provide new insight into the response of SAPKs to diverse stimuli by revealing a mechanism for SAPK activation specifically by oxidative stress. J Inorg Biochem, 2004 Jul, 98(7), 1229 - 37 The adrenodoxin-like ferredoxin of Schizosaccharomyces pombe mitochondria; Schiffler B et al.; The single mitochondrial type I {2Fe-2S} ferredoxin of the fission yeast Schizosaccharomyces pombe is produced as the carboxy terminal part of the electron-transfer-protein 1 (etp1) and cleaved off during mitochondrial import {Biochemistry 41 (2002) 2311-2321} . The UV/Vis (UV-visible) spectrum of the purified recombinant ferredoxin domain (etp1(fd)) expressed in Escherichia coli is similar to those of bovine Adx in the oxidized as well as in the reduced state . EPR (electronic paramagnetic resonance) studies revealed a correctly incorporated iron-sulfur cluster of the axial type . The redox potential of this protein was determined to be -353 mV, which is considerably lower than that of adrenodoxin (Adx, -273 mV) . Several lines of evidence indicate that the protein forms dimers under physiological and denaturating conditions . Interestingly, the fission yeast ferredoxin could be shown to be active as an electron carrier in heterologous redox systems . It is able to transfer electrons to horse heart cytochrome c and to bovine cytochromes P450(scc) (CYP11A1) and P450(11 beta) (CYP11B1), thereby receiving electrons from bovine NADPH-dependent Adx reductase . The kinetics of substrate conversion in the etp1(fd)-supported CYP11A1 and CYP11B1-dependent systems mediated was studied. J Cell Sci, 2004 Jul 15, 117(Pt 16), 3489 - 98 Epub 2004 Jun 22. DNA damage checkpoint maintenance through sustained Chk1 activity; Latif C et al.; The G2 DNA damage checkpoint prevents mitotic entry in the presence of DNA damage . This requires the activation of the phosphoinositide-3-kinase-related protein kinases ATR and ATM in human cells and the ATR homologue Rad3 in the fission yeast Schizosaccharomyces pombe . Rad3 activates the effector protein kinase Chk1 by phosphorylation . However, in fission yeast, inactivation of Rad3 following checkpoint activation has no impact on checkpoint duration . This demonstrates that Rad3 is not required for checkpoint maintenance and that the processes of checkpoint initiation and maintenance are distinct . Chk1 is required for checkpoint initiation but its role in checkpoint maintenance has not been investigated . We show here that Chk1 kinase activity is rapidly induced following irradiation and is maintained for the duration of a checkpoint arrest . On entry to mitosis, there is a transient decrease in Chk1 activity and phosphorylation, but Chk1 activity remains higher than that observed in unirradiated cells . We have generated temperature-sensitive alleles of chk1, which phenocopy chk1 deletion at the non-permissive temperature . Using these alleles, we have shown that inactivation of Chk1 during a checkpoint arrest leads to premature checkpoint termination, resulting in catastrophic mitoses that are a hallmark of checkpoint failure . Therefore, unlike Rad3, Chk1 is an important determinant of both checkpoint initiation and maintenance. Nucleic Acids Res, 2004 Jun 21, 32(11), 3325 - 39 Print 2004. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination; Saito TT et al.; We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I . Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo . This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae . Mcp7 proteins are detected on meiotic chromatin . The phenotypes of mcp7Delta cells are similar to those of meu13Delta cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology . However, a delay in initiation of meiosis I chromosome segregation of mcp7Delta cells is not so conspicuous as meu13Delta cells, and no meiotic delay is observed in mcp7Deltameu13Delta cells . Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Delta cells, whereas Meu13 becomes less stable in mcp7Delta cells . Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation . Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination. Mech Dev, 2004 Jul, 121(7-8), 629 - 37 Medaka and zebrafish, an evolutionary twin study; Furutani-Seiki M et al.; Comparison of two related species is one of the most successful approaches to decipher general genetic principles in eukaryotes . This is best illustrated in yeast, where the model systems Saccharomyyces . cervisiae and Schizosaccharomyces . pombe have been examined . Powerful forward genetics in both species, species-specific differences in biological features and the phylogenetic distance between the two species, make them well suited for a comparative approach . Recent whole genome sequencing has also facilitated comparative genomics of these simple eukaryotes . It is now possible to go a step further using higher eukaryotes . A duplication of the genome at the base of the teleost radiation, facilitated evolution of almost 25,000 fish species, more than half of all vertebrate species together . Two teleost genetic model systems have emerged in the past few decades: zebrafish, in which large-scale mutagenesis has been successfully performed, and Medaka, a Japanese killifish with a century of history in genetics and now, as reported in this issue, many induced mutations . In this review we will illustrate how comparison of these two model species, Medaka and zebrafish, can reveal conserved and species-specific genetic and molecular mechanisms underlying vertebrate development. Yeast, 2004 Jun, 21(8), 613 - 7 A simple and efficient procedure for transformation of Schizosaccharomyces pombe; Morita T et al.; We describe a simple and efficient procedure for transformation of Schizosaccharomyces pombe . Sz . pombe colonies grown on minimal (SD) plates were directly removed and suspended in a 100 microl reaction mixture containing 70 microl PLATE solution (50% polyethylene glycol-4000, 100 mM lithium acetate, 10 mM Tris-HCl, pH 4.9, and 1 mM EDTA), 10 microl plasmid DNA (1 microg), 10 microl carrier DNA (100 microg) and 10 microl sterile distilled water . After incubation at 30 degrees C for 1 h followed by heat shock treatment at 42 degrees C for 15 min, the reaction mixture was spread on a selection plate . The transformation efficiency obtained using the procedure was approximately 8000 transformants/microg DNA . The method is simple and time-saving, making it especially useful for a large number of samples and when a high transformation efficiency is not required . FEBS Lett, 2004 Jun 18, 568(1-3), 129 - 34 Two different dihydroorotate dehydrogenases from yeast Saccharomyces kluyveri; Zameitat E et al.; Genes for two structurally and functionally different dihydroorotate dehydrogenases (DHODHs, EC 1.3.99.11), catalyzing the fourth step of pyrimidine biosynthesis, have been previously found in yeast Saccharomyces kluyveri . One is closely related to the Schizosaccharomyces pombe mitochondrial family 2 enzymes, which use quinones as direct and oxygen as the final electron acceptor . The other one resembles the Saccharomyces cerevisiae cytosolic family 1A fumarate-utilizing DHODH . The DHODHs from S . kluyveri, Sch . pombe and S . cerevisiae, were expressed in Escherichia coli and compared for their biochemical properties and interaction with inhibitors . Benzoates as pyrimidine ring analogs were shown to be selective inhibitors of cytosolic DHODs . This unique property of Saccharomyces DHODHs could appoint DHODH as a species-specific target for novel anti-fungal therapeutics. Nat Genet, 2004 Aug, 36(8), 809 - 17 Epub 2004 Jun 13. Periodic gene expression program of the fission yeast cell cycle; Rustici G et al.; Cell-cycle control of transcription seems to be universal, but little is known about its global conservation and biological significance . We report on the genome-wide transcriptional program of the Schizosaccharomyces pombe cell cycle, identifying 407 periodically expressed genes of which 136 show high-amplitude changes . These genes cluster in four major waves of expression . The forkhead protein Sep1p regulates mitotic genes in the first cluster, including Ace2p, which activates transcription in the second cluster during the M-G1 transition and cytokinesis . Other genes in the second cluster, which are required for G1-S progression, are regulated by the MBF complex independently of Sep1p and Ace2p . The third cluster coincides with S phase and a fourth cluster contains genes weakly regulated during G2 phase . Despite conserved cell-cycle transcription factors, differences in regulatory circuits between fission and budding yeasts are evident, revealing evolutionary plasticity of transcriptional control . Periodic transcription of most genes is not conserved between the two yeasts, except for a core set of approximately 40 genes that seem to be universally regulated during the eukaryotic cell cycle and may have key roles in cell-cycle progression. Mol Biol Cell, 2004 Aug, 15(8), 3903 - 14 Epub 2004 Jun 11. Role of the alpha-glucanase Agn1p in fission-yeast cell separation; Dekker N et al.; Cell division in the fission yeast Schizosaccharomyces pombe yields two equal-sized daughter cells . Medial fission is achieved by deposition of a primary septum flanked by two secondary septa within the dividing cell . During the final step of cell division, cell separation, the primary septum is hydrolyzed by an endo-(1,3)-beta-glucanase, Eng1p . We reasoned that the cell wall material surrounding the septum, referred to here as the septum edging, also must be hydrolyzed before full separation of the daughter cells can occur . Because the septum edging contains (1,3)-alpha-glucan, we investigated the cellular functions of the putative (1,3)-alpha-glucanases Agn1p and Agn2p . Whereas agn2 deletion results in a defect in endolysis of the ascus wall, deletion of agn1 leads to clumped cells that remained attached to each other by septum-edging material . Purified Agn1p hydrolyzes (1,3)-alpha-glucan predominantly into pentasaccharides, indicating an endo-catalytic mode of hydrolysis . Furthermore, we show that the transcription factors Sep1p and Ace2p regulate both eng1 and agn1 expression in a cell cycle-dependent manner . We propose that Agn1p acts in concert with Eng1p to achieve efficient cell separation, thereby exposing the secondary septa as the new ends of the daughter cells. Eukaryot Cell, 2004 Jun, 3(3), 632 - 45 The Kip3-like kinesin KipB moves along microtubules and determines spindle position during synchronized mitoses in Aspergillus nidulans hyphae; Rischitor PE et al.; Kinesins are motor proteins which are classified into 11 different families . We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans . Relatedness analyses based on the motor domains grouped them into nine families . In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases . The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low . A disruption of kipB demonstrated that it is not essential for vegetative growth . kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules . kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis . Meiosis and ascospore formation were not affected . Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4, suggesting an important but redundant role of KipB in mitosis . KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends . Eukaryot Cell, 2004 Jun, 3(3), 610 - 9 Suppressors of an adenylate cyclase deletion in the fission yeast Schizosaccharomyces pombe; Stiefel J et al.; Schizosaccharomyces pombe utilizes two opposing signaling pathways to sense and respond to its nutritional environment . Glucose detection triggers a cyclic AMP signal to activate protein kinase A (PKA), while glucose or nitrogen starvation activates the Spc1/Sty1 stress-activated protein kinase (SAPK) . One process controlled by these pathways is fbp1+ transcription, which is glucose repressed . In this study, we isolated strains carrying mutations that reduce high-level fbp1+ transcription conferred by the loss of adenylate cyclase (git2delta), including both wis1- (SAPK kinase) and spc1- (SAPK) mutants . While characterizing the git2delta suppressor strains, we found that the git2delta parental strains are KCl sensitive, though not osmotically sensitive . Of 102 git2delta suppressor strains, 17 strains display KCl-resistant growth and comprise a single linkage group, carrying mutations in the cgs1+ PKA regulatory subunit gene . Surprisingly, some of these mutants are mostly wild type for mating and stationary-phase viability, unlike the previously characterized cgs1-1 mutant, while showing a significant defect in fbp1-lacZ expression . Thus, certain cgs1- mutant alleles dramatically affect some PKA-regulated processes while having little effect on others . We demonstrate that the PKA and SAPK pathways regulate both cgs1+ and pka1+ transcription, providing a mechanism for cross talk between these two antagonistically acting pathways and feedback regulation of the PKA pathway . Finally, strains defective in both the PKA and SAPK pathways display transcriptional regulation of cgs1+ and pka1+, suggesting the presence of a third glucose-responsive signaling pathway . Genes Cells, 2004 Jun, 9(6), 561 - 74 Sorting nexin homologues are targets of phosphatidylinositol 3-phosphate in sporulation of Schizosaccharomyces pombe; Koga T et al.; Schizosaccharomyces pombe defective in phosphatidylinositol (PtdIns) 3-kinase shows various defects in forespore membrane formation, including onset, growth orientation, and closure . Downstream factors of PtdIns 3-kinase in this system were explored . Among various phox homology (PX) domain-containing proteins, Vps5p and Vps17p, homologues of sorting nexins, were found to be required for efficient sporulation . Cells defective in these proteins showed a disordered growth orientation of the forespore membrane, as is the case with Deltapik3 cells . Vps5p and Vps17p with mutations in the PX domains failed to suppress the defects of their relevant disruptants . Vps5p and Vps17p migrated toward the the forespore membrane in a pik3+-dependent manner, suggesting that these proteins may interact with PtdIns(3)P . Electron-microscopic analysis revealed that the forespore membrane fails to engulf the nucleus in some of these cells, accumulating vesicle-like bodies similar to those seen in Deltaspo3 cells . These results suggest that Vps5p and Vps17p are the targets of PtdIns(3)P in vesicle transport required for onset of the forespore membrane formation . Copyright Blackwell Publishing Limited J Cell Biol, 2004 Jun 7, 165(5), 697 - 707 Regulation of a formin complex by the microtubule plus end protein tea1p; Feierbach B et al.; The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis . In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth . Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends . Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables . Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end . Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly . Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization . Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth . Copyright the Rockefeller University Press Eur J Biochem, 2004 Jun, 271(12), 2561 - 72 Mediator is required for activated transcription in a Schizosaccharomyces pombe in vitro system; Tamayo E et al.; RNA polymerase II (RNAPII) requires a set of general transcription factors - TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH - to initiate transcription from a gene promoter in vitro . General transcription factors have been isolated from Saccharomyces cerevisiae, rat, human and Drosophila, and their corresponding cDNAs have been cloned . In this report, we describe a reconstituted in vitro transcription system that consists of the following preparations of factors from the yeast Schizosaccharomyces pombe: affinity-purified RNAPII, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF . We show that this system can support basal transcription from the adenovirus major late promoter when purified RNAPII is used and activated transcription when the RNAPII holoenzyme (RNAPII plus the Mediator proteins) is included in the reaction . In contrast, the TATA binding protein-associated factors had no effect on transcriptional activation in our Sc . pombe system . These results indicate that Sc . pombe uses the same set of general transcription factors as other eukaryotes and that the Mediator is involved in activated transcription. J Biol Chem, 2004 Aug 20, 279(34), 35644 - 55 Epub 2004 Jun 01. Five genes involved in biosynthesis of the pyruvylated Galbeta1,3-epitope in Schizosaccharomyces pombe N-linked glycans; Andreishcheva EN et al.; The N-linked galactomannans of Schizosaccharomyces pombe have pyruvylated Galbeta1,3-(PvGal) caps on a portion of the Galalpha1,2-residues in their outer chains (Gemmill, T . R., and Trimble, R . B . (1998) Glycobiology 8, 1087-1095) . PvGal biosynthesis was investigated by ethyl methanesulfonate mutagenesis of S . pombe, followed by the isolation of cells devoid of negatively charged N-glycans by Q-Sepharose exclusion and failure to bind human serum amyloid P component, which acts as a lectin for terminal PvGal residues . Mutant glycans were characterized by lectin binding, saccharide composition, exoglycosidase sensitivity, and NMR spectroscopy . Restoration of the cell surface negative charge by complementation with an S . pombe genomic library led to the identification of five genes involved in PvGal biosynthesis, which we designated pvg1-pvg5 . Pvg1p may be a pyruvyltransferase, since NMR of pvg1(-) mutant N-glycans revealed the absence of only the pyruvyl moiety . Pvg2p-Pvg5p are crucial for attachment of the Galbeta1,3-residue that becomes pyruvylated . Pvg3p is predicted to be a member of the beta1,3-galactosyltransferase family, and Pvg3p-green fluorescent protein labeling was consistent with Golgi localization . Predicted Pvg1p and Pvg3p functions imply that Galbeta1,3-is added to the galactomannans and is then pyruvylated in situ, rather than by an en bloc addition of PvGalbeta1,3-caps to the outer chain . Pvg4p-green fluorescent protein targeted to the nucleus, and its sequence contains a MADS-box DNA-binding and dimerization domain; however, it does not appear to solely control transcription of the other identified genes . Pvg2p and/or Pvg5p may contribute to an enzyme complex . Whereas a functional role for the PvGal epitope in S . pombe remains unclear, it is nonessential for either cell growth or mating under laboratory conditions. J Biol Chem, 2004 Jul 30, 279(31), 32079 - 86 Epub 2004 Jun 01. Cds1 phosphorylation by Rad3-Rad26 kinase is mediated by forkhead-associated domain interaction with Mrc1; Tanaka K et al.; The protein kinase Cds1 is an effector of the replication checkpoint in the fission yeast Schizosaccharomyces pombe . Cds1 is required to stabilize stalled replication forks, and it helps to prevent the onset of mitosis until the genome is fully replicated . Mrc1 (mediator of the replication checkpoint-1) and Rad3-Rad26 kinase are required for Cds1 activation, but exactly how Mrc1 mediates Cds1 activation is unknown . Here we show that Mrc1 is required for the initial threonine 11 phosphorylation of Cds1 by Rad3-Rad26 . Mrc1 specifically interacts with the forkhead-associated (FHA) domain of Cds1 in yeast two-hybrid assays . Mutations in the FHA domain that abolish this interaction also eliminate Thr-11 phosphorylation of Cds1 . Weak Thr-11 phosphorylation of a "kinase-dead" mutant of Cds1 is rescued by co-expression of wild type Cds1 . The requirement for Mrc1 in the replication checkpoint can be partially eliminated by expression of a Rad26-Cds1 fusion protein . These findings suggest that recognition of Mrc1 by the FHA domain of Cds1 serves to recruit Cds1 to Rad3-Rad26 . This interaction mediates the initial Thr-11 phosphorylation of Cds1 by Rad3-Rad26 with subsequent intermolecular phosphorylation events leading to full activation of Cds1. Genetics, 2004 May, 167(1), 77 - 91 A novel gene, msa1, inhibits sexual differentiation in Schizosaccharomyces pombe; Jeong HT et al.; Sexual differentiation in the fission yeast Schizosaccharomyces pombe is triggered by nutrient starvation or by the presence of mating pheromones . We identified a novel gene, msa1, which encodes a 533-aa putative RNA-binding protein that inhibits sexual differentiation . Disruption of the msa1 gene caused cells to hypersporulate . Intracellular levels of msa1 RNA and Msa1 protein diminished after several hours of nitrogen starvation . Genetic analysis suggested that the function of msa1 is independent of the cAMP pathway and stress-responsive pathway . Deletion of the ras1 gene in diploid cells inhibited sporulation and in haploid cells decreased expression of mating-pheromone-induced genes such as mei2, mam2, ste11, and rep1; simultaneous deletion of msa1 reversed both phenotypes . Overexpression of msa1 decreased activated Ras1(Val17)-induced expression of mam2 . Phenotypic hypersporulation was similar between cells with deletion of only rad24 and both msa1 and rad24, but simultaneous deletion of msa1 and msa2/nrd1 additively increased hypersporulation . Therefore, we suggest that the primary function of Msa1 is to negatively regulate sexual differentiation by controlling the expression of Ste11-regulated genes, possibly through the pheromone-signaling pathway. Mol Microbiol, 2004 Jun, 52(5), 1427 - 35 Activation of the redox sensor Pap1 by hydrogen peroxide requires modulation of the intracellular oxidant concentration; Vivancos AP et al.; The transcription factor Pap1 and the MAP kinase Sty1 are key regulators of hydrogen peroxide-induced responses in Schizosaccharomyces pombe . Pap1 can be activated quickly at low, but not high, hydrogen peroxide concentrations . The MAP kinase Sty1 has been reported to participate in Pap1 activation by the oxidant . Here, we provide biochemical and genetic evidence for the in vivo formation of a hydrogen peroxide-induced disulphide bond in Pap1, which precedes the rapid and reversible nuclear accumulation of the transcription factor . We show that activation of the Sty1 cascade before the oxidative insult, or overexpression of the Sty1-regulated genes ctt1 (encoding catalase) or gpx1 (encoding glutathione peroxidase), can accelerate Pap1 entry even at high doses of hydrogen peroxide . In fact, the lack of Sty1 impedes Pap1 nuclear localization, but only at high doses of the oxidant . We propose that, whereas low doses of hydrogen peroxide lead directly to Pap1 oxidation-activation, high concentrations of the oxidant initially activate the Sty1 pathway, with the consequent increase in scavenging enzymes, which in turn helps to decompose the excess of hydrogen peroxide and achieve an appropriate concentration for the subsequent activation of Pap1 . Our results also suggest that activation of Sty1 at high doses of hydrogen peroxide may also be required to trigger other antioxidant activities such as those reverting the overoxidation of cysteine residues at the Pap1 pathway. Yeast, 2004 May, 21(7), 593 - 603 Transcriptional and post-translational regulation of neutral trehalase in Schizosaccharomyces pombe during thermal stress; Paredes V et al.; In the fission yeast Schizosaccharomyces pombe, a heat shock enhances transcription of the ntp1(+) gene, encoding the hydrolytic enzyme neutral trehalase . As compared to wild-type cells, cells devoid of the MAP kinase Sty1p showed a strong decrease in ntp1(+) expression induced by the temperature upshift, indicating that the stress-activated protein kinase (SAPK) pathway regulates the expression of this gene during heat shock . The transcription factor Atf1p, which is the main downstream target for Sty1p in the SAPK pathway, appears to be involved in such control, since ntp1(+) expression under heat shock proved to be significantly blocked in atf1(+)-disrupted cells . Serial deletion and point mutation analyses of the ntp1(+) promoter, as well as electrophoretic mobility shift assays, revealed the existence of a CRE-like element as the target for Atf1p-mediated expression under thermal stress . The relevance of two putative HSE elements located in the ntp1(+) promoter was also investigated for their potential role in regulating ntp1(+) transcription during heat shock . The results support a model in which heat-induced Atf1p binding to the CRE-like element favours the subsequent interaction of the heat shock factor (HSF) with HSE elements in the ntp1(+) promoter . Unlike what happens under osmostress or oxidative treatments, Sty1p has no role in the post-translational activation of neutral trehalase induced by heat shock in the fission yeast . J Cell Sci, 2004 Jun 15, 117(Pt 14), 2887 - 95 Epub 2004 May 25. The nucleolus is involved in mRNA export from the nucleus in fission yeast; Ideue T et al.; To elucidate the mechanism of mRNA export from the nucleus, we isolated five novel temperature-sensitive mutants (ptr7 to ptr11) that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature in Schizosaccharomyces pombe . Of those, the ptr11 mutation was found in the top2(+) gene encoding DNA topoisomerase II . In addition to the nuclear accumulation of poly(A)(+) RNA, ptr11 exhibited the cut (cell untimely torn) phenotype at the nonpermissive temperature, like the previously isolated mutant, ptr4 . In these two mutants, cytokinesis occurred without prior nuclear division, resulting in cleavage of the undivided nuclei by the septum . To investigate the relationship between mRNA export defects and the cut phenotype observed in ptr4 and ptr11, we analyzed 11 other mutants displaying the cut phenotype and found that all these tested mutants accumulate poly(A)(+) mRNA in the aberrantly cleaved nuclei . Interestingly, nuclear accumulation of poly(A)(+) mRNA was observed only in the anucleolate nuclei produced by aberrant cytokinesis . In addition, nuc1, the S . pombe mutant exhibiting a collapsed nucleolus, trapped poly(A)(+) mRNA in the nucleolar region at the nonpermissive temperature . In ptr11 and nuc1, mRNA transcribed from the intron-containing TBP gene showed nuclear accumulation, but not transcripts from the intron-less TBP cDNA, suggesting that the export pathway differs between the spliced and unspliced TBP mRNAs . These findings support the notion that a subset of mRNAs in yeast is exported from the nucleus through transient association with the nucleolus. Methods, 2004 Jul, 33(3), 260 - 3 Assaying the DNA damage checkpoint in fission yeast; Dunaway S et al.; Cell cycle checkpoints exist to ensure the proper maintenance and stable inheritance of genomic information . The pathways that insure the faithful execution of these checkpoints are well conserved throughout evolution . In the fission yeast, Schizosaccharomyces pombe, a major cell cycle checkpoint exists that responds to the presence of damaged DNA and prevents this damage from being propagated to future generations . Fission yeast is an ideal system to investigate these pathways because there exist specific techniques that allow one to assay the fidelity of this DNA damage checkpoint pathway. Methods, 2004 Jul, 33(3), 245 - 51 Cell wall analysis; Perez P et al.; The cell wall is a rigid structure essential for survival of the fungal cell . Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents . Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis . The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins . Here, we describe a fast and reliable method to analyze changes in S . pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls . This approach provides a powerful means to analyze changes in (1,3)beta-glucan and (1,3)alpha-glucan, two main polysaccharides present in fungal cell walls . Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure. Methods, 2004 Jul, 33(3), 239 - 44 Tandem affinity purification and identification of protein complex components; Gould KL et al.; As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell . These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping . We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S . pombe . This approach can theoretically be applied to the entire proteome as has been done in S . cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins. Methods, 2004 Jul, 33(3), 226 - 38 Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe; Yoshida M et al.; The nuclear envelope is essential for compartmentalizing the nucleus from the cytoplasm in all eukaryotic cells . There is a tremendous flux of both RNA and proteins across the nuclear envelope, which is intact throughout the entire cell cycle of yeasts but breaks down during mitosis of animal cells . Transport across the nuclear envelope requires the recognition of cargo molecules by receptors, docking at the nuclear pore, transit through the nuclear pore, and then dissociation of the cargo from the receptor . This process depends on the RanGTPase system, transport receptors, and the nuclear pore complex . We provide an overview of the nuclear transport process, with particular emphasis on the fission yeast Schizosaccharomyces pombe, including strategies for predicting and experimentally verifying the signals that determine the sub-cellular localization of a protein of interest . We also describe a variety of reagents and experimental strategies, including the use of mutants and chemical inhibitors, to study nuclear protein import, nuclear protein export, nucleocytoplasmic protein shuttling, and mRNA export in fission yeast . The RanGTPase and its regulators also play an essential transport independent role in nuclear envelope re-assembly after mitosis in animal cells and in the maintenance of nuclear envelope integrity at mitosis in S . pombe . Several experimental strategies and reagents for studying nuclear size, nuclear shape, the localization of nuclear pores, and the integrity of the nuclear envelope in living fission yeast cells are described. Methods, 2004 Jul, 33(3), 220 - 5 Imaging green fluorescent protein fusions in living fission yeast cells; Tran PT et al.; The use of green fluorescent protein (GFP) fusions as biosensors for examining protein localization and dynamics has revolutionized cell biology . Here, we describe the methods developed for imaging of GFP-fusions in the fission yeast Schizosaccharomyces pombe using fluorescence microscopy, with a focus on the use of time-lapse imaging to analyze the dynamics of microtubules . We discuss the considerations in fluorescence microscopy, cell preparation, data acquisition, and image analysis appropriate for analysis of living cells. Methods, 2004 Jul, 33(3), 206 - 12 The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe; Rajagopalan S et al.; Conditional mutants are a vital tool for analysis of gene function . The use of temperature-sensitive mutants in Schizosaccharomyces pombe has significantly promoted understanding of many cellular processes . A portable heat-inducible amino-terminal degron (N-degron) for conditional degradation of a gene product has been previously described in Saccharomyces cerevisiae . This paper describes the adaptation of the N-degron method to create temperature-sensitive (ts) mutants in S . pombe . A ts derivative of the mouse dihydrofolate reductase with an amino-terminal arginine (Arg-DHFR(ts)) previously described in S . cerevisiae was fused to the N-terminus of Bir1p, a nuclear protein involved in mitotic chromosome segregation in S . pombe . This fusion allele, referred to as bir1-td, conferred a chromosome segregation defect at 36 degrees C, as with previously described alleles of bir1 . Deletion of the S . pombe E3 ubiquitin ligase (N-recognin), Ubr11p, reversed the temperature-dependent lethality of bir1-td, providing evidence for N-end rule mediated destruction of Bir1p . The methods we describe should therefore facilitate analysis of essential genes in fission yeast for which conditionally lethal mutants are unavailable. Methods, 2004 Jul, 33(3), 199 - 205 Strategies for gene disruptions and plasmid constructions in fission yeast; Wang L et al.; Molecular genetic analyses in Schizosaccharomyces pombe are greatly enhanced by our ability to delete chromosomal genes via homologous recombination and to introduce genes expressed from autonomous plasmids . In this paper, we describe a novel approach to generating marked deletion cassettes that bypasses the need for the long, PAGE-purified oligonucleotides required in the currently used PCR-based deletion approach . We also describe additional uses of this two-step PCR method for constructing chromosomal insertion cassettes . Finally, we describe how gap repair in S . pombe can facilitate plasmid constructions in a manner that circumvents the reliance on compatible restriction sites in the DNA molecules that are being joined . Several applications of this gap repair plasmid construction strategy are discussed. Methods, 2004 Jul, 33(3), 189 - 98 Choosing and using Schizosaccharomyces pombe plasmids; Siam R et al.; A wide range of plasmids has been developed for molecular studies in the fission yeast Schizosaccharomyces pombe . This includes general purpose episomes, expression vectors, epitope tagging plasmids, and integration vectors . This review describes the typical features of S . pombe vectors, including replication origins, positive and negative selection markers, and constitutive and inducible promoter systems . We will also discuss vectors with epitope tags and how these can be used to modify episomal or endogenous gene sequences . Considerations for choosing and using a plasmid are presented and specialized methods are described. Genes Dev, 2004 May 15, 18(10), 1154 - 64 Chk1 activation requires Rad9 S/TQ-site phosphorylation to promote association with C-terminal BRCT domains of Rad4TOPBP1; Furuya K et al.; To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult we investigated the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9 . C-terminal T412/S423 phosphorylation of Rad9 by Rad3(ATR) occurs in S phase without replication stress . Rad3(ATR) and Tel1(ATM) phosphorylate these same residues, plus additional ones, in response to DNA damage . In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4(TOPBP1) protein . Rad9-Rad4(TOPBP1) interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint . When the Rad9-T412/S423 are phosphorylated, Rad4(TOPBP1) coprecipitates with Rad3(ATR), suggesting that phosphorylation coordinates formation of an active checkpoint complex. Mol Biol Cell, 2004 Aug, 15(8), 3796 - 810 Epub 2004 May 14. Functional characterization of Dma1 and Dma2, the budding yeast homologues of Schizosaccharomyces pombe Dma1 and human Chfr; Fraschini R et al.; Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis . In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected . We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints . We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck . DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis . The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation . In addition, dma1Delta dma2Delta cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs . Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase. EMBO Rep, 2004 Jun, 5(6), 602 - 6 Epub 2004 May 14. Chemoattractant-induced Ras activation during Dictyostelium aggregation; Kae H et al.; Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli . Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development . Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras . We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG . We have found that the Ras-binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG . Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation-competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants . These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium. FEMS Microbiol Lett, 2004 May 15, 234(2), 379 - 85 Stress-dependent regulation of the gene encoding thioredoxin reductase from the fission yeast; Hong SM et al.; The unique putative gene for thioredoxin reductase (TrxR) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe . The determined DNA sequence carries 3125 bp, and encodes the plausible 322 amino acid sequence of TrxR with a molecular mass of 34,618 Da . The S . pombe cells harboring the cloned TrxR gene contain increased TrxR activity, and shows higher survivals on solid media with mercuric chloride or aluminum chloride . The 1526 bp upstream region was fused into promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid . The synthesis of beta-galactosidase from the fusion plasmid pYUTR10 was enhanced by menadione, mercuric chloride, hydrogen peroxide, aluminium chloride and sodium selenite . Menadione significantly enhanced the TrxR mRNA level in the S . pombe cells, which was detected by RT-PCR . Induction of the S . pombe TrxR gene by menadione and mercuric chloride occurs through the mediation of the transcription factor Pap1 . These results suggest that the S . pombe TrxR gene is one of the stress response-related genes . FEBS Lett, 2004 May 7, 565(1-3), 176 - 80 Analysis of the S . pombe signalling scaffold protein Cdc11p reveals an essential role for the N-terminal domain in SIN signalling; Krapp A et al.; The initiation of cytokinesis in the fission yeast Schizosaccharomyces pombe is signalled by the septation initiation network (SIN) . Signalling originates from the spindle pole body (SPB), where SIN proteins are anchored by a scaffold composed of cdc11p and sid4p . Cdc11p links the other SIN proteins to sid4p and the SPB . Homologues of cdc11p have been identified in Saccharomyes cerevisiae (Nud1p) and human cells (Centriolin) . We have defined functional domains of cdc11p by analysis of deletion mutants . We demonstrate that the C-terminal end of cdc11p is necessary for SPB localisation . We also show that the N-terminal domain is necessary and sufficient for signal transduction, since tethering of this domain to the SPB will substitute for cdc11p in SIN function. Mol Cell Endocrinol, 2004 Mar 31, 217(1-2), 249 - 54 Development of test systems for the discovery of selective human aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) inhibitors . Discovery of a new lead compound for the therapy of congestive heart failure, myocardial fibrosis and hypertension; Bureik M et al.; Two key players in adrenal steroid hormone biosynthesis are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2 that catalyze the final steps in the biosynthesis of cortisol and aldosterone, respectively . Overproduction of both hormones contributes to a number of severe diseases, as illustrated by the association of elevated aldosterone levels with hypertension and higher mortality in congestive heart failure, and by Cushing's syndrome as the clinical correlate of chronic hypercortisolism . Thus, CYP11B1 and CYP11B2 comprise new targets for drug treatment and selective inhibitors of both enzymes are of high pharmacological interest . To facilitate the search for such compounds, we have established novel test procedures using recombinant fission yeast strains that stably express these enzymes . The aim of this study was to compare the inhibition profiles displayed by these enzymes in established mammalian cell culture test systems to those obtained with the new fission yeast assays, and to evaluate the usefulness of the Schizosaccharomyces pombe strains as screening systems for the identification of novel lead compounds . Using these test systems, we were able to identify a new and very selective CYP11B2 inhibitor (SIAS-1) that displayed no detectable interference with CYP11B1 activity. Genes Dev, 2004 May 1, 18(9), 1007 - 21 Recruitment of NIMA kinase shows that maturation of the S . pombe spindle-pole body occurs over consecutive cell cycles and reveals a role for NIMA in modulating SIN activity; Grallert A et al.; Mitotic exit in Saccharomyces cerevisiae and septation in Schizosaccharomyces pombe are regulated by a conserved signaling network called the mitotic exit and septum initiation networks (SIN), respectively . The network is active on one of the two anaphase B spindle-pole bodies (SPBs) . Whereas the inherent asymmetry of growth by budding accounts for elements of the asymmetry in S . cerevisiae, it has been unclear how, or why, the pathway is asymmetric in S . pombe . We show that elements of SPB duplication in S . pombe are conservative, and that the SIN is active on the new SPB . SIN association with the new SPB persists after transient depolymerization of microtubules . The localization of the NIMA-related kinase, Fin1, reveals further complexity in SPB inheritance . Fin1 associates with the SPB bearing the older components in all cells and with the "new" SPB in half of the population . Fin1 only binds the new SPB when this new SPB has arisen from the duplication of an SPB that is two or more cycles old . Thus, each of the four SPBs generated over two consecutive cell cycles are different, because they have distinct fates in the next cell cycle . Fin1 binds the SPB once the SIN is active and the association requires the SIN inhibitors Byr4 and Cdc16 . Fin1 physically associates with Byr4 . Compromising Fin1 function leads to SIN activation on both anaphase B SPBs and promotes septation, indicating that Fin1 restrains SIN activity on the old SPB. Appl Bioinformatics, 2003, 2(1), 13 - 34 Innovation from reduction: gene loss, domain loss and sequence divergence in genome evolution; Braun EL; Analyses of genome sequences have revealed a surprisingly variable distribution of genes, reflecting the generation of novel genes, lateral gene transfer and gene loss . The impact of gene loss on organisms has been difficult to examine, but the loss of protein coding genes, the loss of domains within proteins and the divergence of genes have made surprising contributions to the differences among organisms . This paper reviews surveys of gene loss and divergence in fungal and archaeal genomes that indicate suites of functionally related genes tend to undergo loss and divergence . Instances of fungal gene loss highlighted here suggest that specific cellular systems have changed, such as Ca 2+ biology in Saccharomyces cerevisiae and peroxisome function in Schizosaccharomyces pombe . Analyses of loss and divergence can provide specific predictions regarding protein-protein interactions, and the relationship between networks of protein interactions and loss may form a part of a parametric model of genome evolution. J Cell Sci, 2004 May 1, 117(Pt 11), 2283 - 93 Suppression of a mitotic mutant by tRNA-Ala anticodon mutations that produce a dominant defect in late mitosis; Kimata Y et al.; Cold-sensitive dominant mutants scn1 and scn2 of Schizosaccharomyces pombe were isolated by their ability to suppress temperature-sensitive cut9-665 defective in an essential subunit (human Apc6/budding yeast Cdc16 ortholog) of anaphase promoting complex/cyclosome (APC/C) . APC/C mutants were defective in metaphase/anaphase transition, whereas single scn mutants showed the delay in anaphase spindle elongation at 20 degrees C . The scn mutants lost viability because of chromosome missegregation, and were sensitive to a tubulin poison . To understand the scn phenotypes, mutant genes were identified . Surprisingly, scn1 and scn2 have the same substitution in the anticodon of two different tRNA-Ala (UGC) genes . UGC was altered to UGU so that the binding of the tRNA-Ala to the ACA Thr codon in mRNA became possible . As cut9-665 contained an Ala535Thr substitution, wild-type Cut9 protein was probably produced in scn mutants . Indeed, plasmid carrying tRNA-Ala (UGU) conferred cold-sensitivity to wild-type and suppressed cut9-665 in a dominant fashion . The previously identified scn1(+) (renamed as scn3(+)) turned out to be a high copy suppressor for scn1 and scn2 . These are the first tRNA mutants that cause a mitotic defect. Mol Cell Biol, 2004 May, 24(10), 4309 - 20 The Schizosaccharomyces pombe HIRA-like protein Hip1 is required for the periodic expression of histone genes and contributes to the function of complex centromeres; Blackwell C et al.; HIRA-like (Hir) proteins are evolutionarily conserved and are implicated in the assembly of repressive chromatin . In Saccharomyces cerevisiae, Hir proteins contribute to the function of centromeres . However, S . cerevisiae has point centromeres that are structurally different from the complex centromeres of metazoans . In contrast, Schizosaccharomyces pombe has complex centromeres whose domain structure is conserved with that of human centromeres . Therefore, we examined the functions of the fission yeast Hir proteins Slm9 and the previously uncharacterised protein Hip1 . Deletion of hip1(+) resulted in phenotypes that were similar to those described previously for slm9 Delta cells: a cell cycle delay, synthetic lethality with cdc25-22, and poor recovery from nitrogen starvation . However, while it has previously been shown that Slm9 is not required for the periodic expression of histone H2A, we found that loss of Hip1 led to derepression of core histone genes expression outside of S phase . Importantly, we found that deletion of either hip1(+) or slm9(+) resulted in increased rates of chromosome loss, increased sensitivity to spindle damage, and reduced transcriptional silencing in the outer centromeric repeats . Thus, S . pombe Hir proteins contribute to pericentromeric heterochromatin, and our data thus suggest that Hir proteins may be required for the function of metazoan centromeres. Mol Cell Biol, 2004 May, 24(10), 4229 - 40 The splicing factor U2AF small subunit is functionally conserved between fission yeast and humans; Webb CJ et al.; The small subunit of U2AF, which functions in 3' splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated . Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AF(SM)) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function . Precursor mRNAs accumulate in S . pombe following U2AF(SM) depletion in a time frame consistent with a role in splicing . A comprehensive mutational analysis reveals that all three conserved domains are required for viability . Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical . Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant . Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer . In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues. Bioinformatics, 2004 Nov 1, 20(16), 2553 - 61 Epub 2004 Apr 29. Identification of DNA regulatory motifs using Bayesian variable selection; Tadesse MG et al.; MOTIVATION: Understanding the mechanisms that determine gene expression regulation is an important and challenging problem . A common approach consists of identifying DNA-binding sites from a collection of co-regulated genes and their nearby non-coding DNA sequences . Here, we consider a regression model that linearly relates gene expression levels to a sequence matching score of nucleotide patterns . We use Bayesian models and stochastic search techniques to select transcription factor binding site candidates, as an alternative to stepwise regression procedures used by other investigators . RESULTS: We demonstrate through simulated data the improved performance of the Bayesian variable selection method compared to the stepwise procedure . We then analyze and discuss the results from experiments involving well-studied pathways of Saccharomyces cerevisiae and Schizosaccharomyces pombe . We identify regulatory motifs known to be related to the experimental conditions considered . Some of our selected motifs are also in agreement with recent findings by other researchers . In addition, our results include novel motifs that constitute promising sets for further assessment . AVAILABILITY: The Matlab code for implementing the Bayesian variable selection method may be obtained from the corresponding author. Yeast, 2004 Apr 30, 21(6), 495 - 509 Identification of genes encoding putative nucleoporins and transport factors in the fission yeast Schizosaccharomyces pombe: a deletion analysis; Chen XQ et al.; In a systematic approach to study genes that are related to nucleocytoplasmic trafficking in the fission yeast Schizosaccharomyces pombe, the open reading frames (ORFs) of 26 putative nucleoporins and transport factors were deleted . Here we report the initial characterization of these deletion mutants . Of the 26 putative genes deleted, 14 were found to be essential for viability . Null mutations of essential genes resulted in failure to either complete one round or to sustain cell division . Four of the 14 essential genes, SPBC582.11c, SPBC17G9.04c, SPBC3B9.16c and SPCC162.08c, encode putative nucleoporins and a myosin-like protein with homologues NUP84, NUP85, NUP120 and MLP1, respectively, that are not required for viability in Saccharomyces cerevisiae, suggesting that their gene products perform critical functions in Sz . pombe . On the basis of combined drug sensitivity assays and genetic analysis we have identified five non-essential null mutants that were hypersensitive to the microtubule depolymerizing drug thiabendazole (TBZ) and exhibited a cut phenotype upon TBZ treatment, suggesting possible involvement in microtubule function . Three of the corresponding ORFs, SPCC18B5.07c, nup40 and SPAC1805.04, encode putative nucleoporins with low similarity to the S . cerevisiae nucleoporins NUP2p, NUP53p and NUP133p, respectively . Further genetic analysis revealed that one of the nucleoporin genes, nup40, and another gene, SPCC1322.06, encoding a putative importin-beta/Cse1p superfamily protein may have a spindle checkpoint function . Folia Microbiol (Praha), 2004, 49(1), 31 - 6 Characterization of chromate-sensitive and -tolerant mutants of Schizosaccharomyces pombe; Czako-Ver K et al.; Stable chromium(VI)-sensitive and -tolerant mutants were obtained by induced mutagenesis of Schizosaccharomyces pombe lysine and leucine auxotrophic heterothallic strains 6chr+ and 9chr+ . Eleven of them were selected for further studies . Fast transport of 51CrO4(2-) was detected in a representative sensitive mutant, chr-51S, while the tolerant mutant chr1-66T and the parental strain 6chr+ exhibited significantly lower 51CrO4(2-) uptake . The segregation of tetrads of three selected CrVI-tolerant mutants, chr1-66T, chr1-14T and chr2-04T, strongly indicated that tolerance was determined by single mutations . Random spore analysis proved that the mutations of chr1-66T and chr1-14T were allelic and the mutation of mutant chr2-04T was not allelic with the mutation of chr1-66T . Recombinants carrying the ura4D18 selective marker were created for transformation experiments . Two of them (chr1-661T and chr2-046T) can be used to clone and identify the genes responsible for their CrVI tolerance phenotype. FEMS Microbiol Rev, 2004 May, 28(2), 201 - 23 Genetic aspects of targeted insertion mutagenesis in yeasts; Klinner U et al.; Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae . The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research . Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S . cerevisiae . Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared . We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps . The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered . Specific features of some yeast species are discussed . In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S . cerevisiae are described. J Biol Chem, 2004 Jul 2, 279(27), 28744 - 55 Epub 2004 Apr 23. The Schizosaccharomyces pombe Pccs protein functions in both copper trafficking and metal detoxification pathways; Laliberte J et al.; Because copper is both an essential cofactor and a toxic metal, different strategies have evolved to appropriately regulate its homeostasis as a function of changing environmental copper levels . In this report, we describe a metallochaperone-like protein from Schizosaccharomyces pombe that maintains the delicate balance between essentiality and toxicity . This protein, designated Pccs, has four distinct domains . SOD activity assays reveal that the first three domains of Pccs are necessary and sufficient to deliver copper to its target, copper-zinc superoxide dismutase (SOD1) . Pccs domain IV, which is absent in Saccharomyces cerevisiae CCS1, contains seventeen cysteine residues, eight pairs of which are in a potential metal coordination arrangement, Cys-Cys . We show that S . cerevisiae ace1Delta mutant cells expressing the full-length Pccs molecule are resistant to copper toxicity . Furthermore, we demonstrate that the Pccs domain IV enhances copper resistance of the ace1Delta cells by an order of magnitude compared with that observed in the same strain expressing a pccs+ I-II-III allele encoding Pccs domains I-III . We consistently found that S . pombe cells disrupted in the pccs+ gene exhibit an increased sensitivity to copper and cadmium . Furthermore, we demonstrate that overexpression of pccs+ is associated with increased copper resistance in fission yeast cells . Taken together, our findings suggest that Pccs activates apo-SOD1 under copper-limiting conditions through the use of its first three domains and protects cells against metal ion toxicity via its fourth domain. Acta Biochim Pol, 2004, 51(1), 173 - 87 Functional and physical interactions of Krr1p, a Saccharomyces cerevisiae nucleolar protein; Gromadka R et al.; The Krr1 protein of Saccharomyces cerevisiae is involved in processing of pre-rRNA and assembly of pre-ribosomal 40S subunits . To further investigate the function of Krr1p we constructed a conditional cold sensitive mutant krr1-21, and isolated seven genes from Schizosaccharomyces pombe whose products suppressed the cold sensitive phenotype of krr1-21 cells . Among the multicopy suppressors we found genes coding for translation elongation factor EF-1alpha, a putative ribose methyltransferase and five genes encoding ribosomal proteins . Using the tandem affinity purification (TAP) method we identified thirteen S . cerevisiae ribosomal proteins interacting with Krr1p . Taken together, these results indicate that Krr1p interacts functionally as well as physically with ribosomal proteins . Northern blot analysis revealed that changes in the level of krr1-21 mRNA were accompanied by similar changes in the level of mRNAs of genes encoding ribosomal proteins . Thus, Krr1p and the genes encoding ribosomal proteins it interacts with seem to be coordinately regulated at the level of transcription. Eukaryot Cell, 2004 Apr, 3(2), 406 - 12 Cell division defects of Schizosaccharomyces pombe liz1- mutants are caused by defects in pantothenate uptake; Stolz J et al.; The liz1+ gene of the fission yeast Schizosaccharomyces pombe was previously identified by complementation of a mutation that causes abnormal mitosis when ribonucleotide reductase is inhibited . Liz1 has similarity to transport proteins from Saccharomyces cerevisiae, but the potential substrate and its connection to the cell division cycle remain elusive . We report here that liz1+ encodes a plasma membrane-localized active transport protein for the vitamin pantothenate, the precursor of coenzyme A (CoA) . Liz1 is required for pantothenate uptake at low extracellular concentrations . A lack of pantothenate uptake results in three phenotypes: (i) slow growth, (ii) delayed septation, and (iii) aberrant mitosis in the presence of hydroxyurea (HU) . All three phenotypes are suppressed by high extracellular concentrations of pantothenate, where pantothenate uptake occurs by passive diffusion . liz1Delta mutants are viable because they can synthesize pantothenate from uracil as an endogenous source . The use of uracil for both pantothenate biosynthesis and deoxyribonucleotide generation provides an explanation for the aberrant mitosis in the presence of HU . HU blocks ribonucleotide reductase, and we propose that the accumulation of ribonucleotides reduces uracil biosynthesis by feedback inhibition of aspartate transcarbamoylase . Thus, the addition of HU to liz1Delta mutants results in a shortage of pantothenate . Because liz1Delta mutants show striking similarities to mutants with defects in fatty acid biosynthesis, we propose that the shortage of pantothenate compromises fatty acid synthesis, resulting in slow growth and mitotic defects. Eukaryot Cell, 2004 Apr, 3(2), 302 - 10 pdf1, a palmitoyl protein thioesterase 1 Ortholog in Schizosaccharomyces pombe: a yeast model of infantile Batten disease; Cho SK et al.; Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human PPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins . PPT1 has orthologs in many species, including lower organisms and plants, but not in Saccharomyces cerevisiae . The fission yeast Schizosaccharomyces pombe contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the S . pombe Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p) . In the present study, we characterized this interesting gene (designated here as pdf1, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the pdf1 sequence . Strains bearing a deletion of the entire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid . Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH . The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (vps) mutants and vacuolar membrane H(+)-ATPase (vma) mutants in S . cerevisiae . Importantly, the Ppt1p-deficient phenotype is complemented by the human PPT1 gene . These results indicate that the function of PPT1 has been widely conserved throughout evolution and that S . pombe may serve as a genetically tractable model for the study of human infantile Batten disease. J Biol Chem, 2004 Jun 18, 279(25), 26126 - 33 Epub 2004 Apr 08. Heregulin regulates the ability of the ErbB3-binding protein Ebp1 to bind E2F promoter elements and repress E2F-mediated transcription; Zhang Y et al.; The ErbB3/4 ligand heregulin (HRG) profoundly affects cell growth and differentiation, but its mechanism of action is poorly understood . Ebp1, a protein isolated by its binding to ErbB3, inhibits cell growth and represses transcription of E2F-regulated cell cycle genes . Since Ebp1 shares 38% identity with a Schizosaccharomyces pombe DNA-binding protein, we postulated that Ebp1 could bind E2F consensus elements in an HRG-inducible manner, leading to transcriptional repression . We show here that GST-Ebp1 bound to the DNA sequence bound by the S . pombe protein . Whereas GST-Ebp1 alone failed to bind E2F1 promoter elements, Ebp1 contained in nuclear lysates associated with E2F1 consensus sequences in the E2F1 promoter . Endogenous Ebp1 was recruited to the E2F1 promoter in vivo as demonstrated by chromatin immunoprecipitation assays . Ebp1 bound E2F consensus oligonucleotides in association with E2F1, retinoblastoma protein, and HDAC2 . HRG regulated the association of Ebp1 with E2F promoter sequences and enhanced the ability of Ebp1 to repress transcription . Our findings suggest that Ebp1, by linking HRG activation of membrane receptors to E2F gene activity, may be a downstream modulator of the effects of HRG on cell cycle progression. Dev Cell, 2004 Apr, 6(4), 497 - 509 Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle; Zimmerman S et al.; Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton . In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus . Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs . We identify rsp1p, an MTOC protein required for eMTOC disassembly . In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced . Conversely, rsp1p overexpression inhibits eMTOC formation . Rsp1p is a J domain protein that interacts with an hsp70 . Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules. Evolution Int J Org Evolution, 2004 Feb, 58(2), 441 - 6 Essential eukaryotic core; Strobel GL et al.; The AIDs-related fungal pathogen Pneumocystis carinii is unusual in having a remarkably compact genome of 7.7 megabase pairs (mbp) whose small size presents the opportunity to identify the essential eukaryotic core of genes . The essential eukaryotic core is defined to be a collection of essential genes shared by all eukaryotes . Sequencing the 3' ends of more than 5500 cDNAs from P . carinii allowed us to identify about 200 genes shared with its nearest known but distant relative, Schizosaccharomyces pombe and also Saccharomyces cerevisiae, and with homologs known to be essential in S . pombe or S . cerevisae . As the cDNA library contains about one half of the P . carinii genes, the size of the essential eukaryotic core (approximately 400) is slightly larger than the prokaryotic core (265-350) being identified by studies of the bacterial pathogen Mycoplasma genitalium . The collection of genes in the essential eukaryotic core may prove useful in identifying new broad spectrum antifungal drug targets. J Mol Biol, 2004 Apr 23, 338(2), 241 - 55 Structural basis for telomeric single-stranded DNA recognition by yeast Cdc13; Mitton-Fry RM et al.; The essential budding yeast telomere-binding protein Cdc13 is required for telomere replication and end protection . Cdc13 specifically binds telomeric, single-stranded DNA (ssDNA) 3' overhangs with high affinity using an OB-fold domain . We have determined the high-resolution solution structure of the Cdc13 DNA-binding domain (DBD) complexed with a cognate telomeric ssDNA . The ssDNA wraps around one entire face of the Cdc13-DBD OB-fold in an extended, irregular conformation . Recognition of the ssDNA bases occurs primarily through aromatic, basic, and hydrophobic amino acid residues, the majority of which are evolutionarily conserved among budding yeast species and contribute significantly to the energetics of binding . Contacting five of 11 ssDNA nucleotides, the large, ordered beta2-beta3 loop is crucial for complex formation and is a unique elaboration on the binding mode commonly observed in OB-fold proteins . The sequence-specific Cdc13-DBD/ssDNA complex presents a complementary counterpoint to the interactions observed in the Oxytricha nova telomere end-binding and Schizosaccharomyces pombe Pot1 complexes . Analysis of the Cdc13-DBD/ssDNA complex indicates that molecular recognition of extended single-stranded nucleic acids may proceed via a folding-type mechanism rather than resulting from specific patterns of hydrogen bonds . The structure reported here provides a foundation for understanding the mechanism by which Cdc13 recognizes GT-rich heterogeneous sequences with both unusually strong affinity and high specificity. Curr Biol, 2004 Apr 6, 14(7), 579 - 84 Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S . pombe SPB; Morrell JL et al.; The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division . It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p . Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB . We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p . While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p . Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB . FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures . Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division. Plant Physiol Biochem, 2004 Jan, 42(1), 49 - 55 Expression of the fission yeast cell cycle regulator cdc25 induces de novo shoot formation in tobacco: evidence of a cytokinin-like effect by this mitotic activator; Suchomelova P et al.; During the last decade, the cell cycle and its control by cyclin-dependent kinases (CDKs) has been extensively studied in eukaryotes . The regulation of CDK activity includes, among others, its activation by Cdc25 phosphatase at G2/M . However, within the plant kingdom studies of this regulation have lagged behind and a plant cdc25 homologue has not been identified yet . Here, we report on the effects of transformation of tobacco (Nicotiana tabacum L., cv . Samsun) with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on de novo plant organ formation, a process dependent on rate and orientation of cell division . On shoot-inducing medium (low 1-naphthylacetic acid (NAA), high 6-benzylaminopurine (BAP)) the number of shoots formed on internode segments cultured from transgenic plants was substantially higher than in the non-transformed controls . Anatomical observations indicated that the shoot formation process was accelerated but with no changes in the quality and sequence of shoot development . Surprisingly, and in contrast to the controls, when on root-inducing medium (high NAA, low BAP) cultured segments from transgenic plants failed to initiate hardly any roots . Instead, they continued to form shoots at low frequencies . Moreover, in marked contrast to the controls, stem segments from transgenic plants were able to form shoots even without the addition of exogenous growth regulators to the medium . The results indicate that Spcdc25 expression in culture tobacco stem segments mimicked the developmental effects caused by an exogenous hormone balance shifted towards cytokinins . The observed cytokinin-like effects of Spcdc25 transformation are consistent with the concept of an interaction between cell cycle regulators and phytohormones during plant development. Mol Cell Biol, 2004 Apr, 24(8), 3262 - 76 Initiation of cytokinesis is controlled through multiple modes of regulation of the Sid2p-Mob1p kinase complex; Hou MC et al.; The Sid2p-Mob1p kinase complex is an important component of the septation initiation network (SIN) in the fission yeast Schizosaccharomyces pombe . However, regulation of this complex is still elusive . Here we show that Mob1p is required not only for the subcellular localization of Sid2p but also for its kinase activity . We identified a region at the amino terminus of Sid2p that is required for Mob1p binding and spindle pole body (SPB) localization . Deletion of this region abolishes Mob1p binding and diminishes SPB localization, whereas this region alone is sufficient to associate with Mob1p and SPBs . We further show that a similar region of the N terminus of the Sid2p-related protein kinase Orb6p binds to the Mob1p-related protein Mob2p, suggesting that this may be a conserved mode of interaction for this family of kinases . Phosphorylation of Ser402 and especially Thr578 is important for Sid2p function . Sid2p with a mutation of Thr578 to Ala (T578A) can no longer rescue sid2-250 mutant cells, and this results in reduction of Mob1p binding . Sid2p mutants mimicking phosphorylation at this site (T578D and T578E) can rescue sid2-250 cells, enhance Sid2p kinase activity, and partially rescue growth defects of upstream sin mutants . Interestingly, Sid2p, but not Mob1p, is self-associated . Our experiments suggest that self-associated Sid2p is inactive . This self-association is mediated by a region that overlaps with Mob1p and SPB binding sites . Overexpression of Mob1p is able to disrupt the self-association of Sid2p . Taken together, our results suggest that Sid2p kinase may utilize multiple modes of regulation including self-association, Mob1p binding, and phosphorylation to achieve its full activity at an appropriate time and place in the cell. Mol Cell Biol, 2004 Apr, 24(8), 3157 - 67 In vivo dynamics of Swi6 in yeast: evidence for a stochastic model of heterochromatin; Cheutin T et al.; The mechanism for transcriptional silencing of pericentric heterochromatin is conserved from fission yeast to mammals . Silenced genome regions are marked by epigenetic methylation of histone H3, which serves as a binding site for structural heterochromatin proteins . In the fission yeast Schizosaccharomyces pombe, the major structural heterochromatin protein is Swi6 . To gain insight into Swi6 function in vivo, we have studied its dynamics in the nucleus of living yeast . We demonstrate that, in contrast to mammalian cells, yeast heterochromatin domains undergo rapid, large-scale motions within the nucleus . Similar to the situation in mammalian cells, Swi6 does not permanently associate with these chromatin domains but binds only transiently to euchromatin and heterochromatin . Swi6 binding dynamics are dependent on growth status and on the silencing factors Clr4 and Rik1, but not Clr1, Clr2, or Clr3 . By comparing the kinetics of mutant Swi6 proteins in swi6(-) and swi6(+) strains, we demonstrate that homotypic protein-protein interactions via the chromoshadow domain stabilize Swi6 binding to chromatin in vivo . Kinetic modeling allowed quantitative estimation of residence times and indicated the existence of at least two kinetically distinct populations of Swi6 in heterochromatin . The observed dynamics of Swi6 binding are consistent with a stochastic model of heterochromatin and indicate evolutionary conservation of heterochromatin protein binding properties from mammals to yeast. Mol Cell Biol, 2004 Apr, 24(8), 3100 - 11 Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene; Rollins RA et al.; The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers . Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis . The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes . Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut . To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication. Biosci Biotechnol Biochem, 2004 Mar, 68(3), 545 - 50 A set of loxP marker cassettes for Cre-mediated multiple gene disruption in Schizosaccharomyces pombe; Iwaki T et al.; For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe . Here we describe a loxP-flanked ura4(+) cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S . pombe . This loxP-ura4-loxP cassette can be used for disruption of hmt1(+) as a model target gene . We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter . Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter . In addition, ura4(+) could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4 . The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S . pombe. Folia Biol (Praha), 2004, 50(1), 1 - 6 Eukaryotic operon genes can define highly conserved syntenies; Trachtulec Z; The synteny conservation of the members of eukaryotic operons was investigated by mapping their orthologues in Drosophila, human, and other eukaryotes . While the homologues of the operon members are generally not linked, some examples of highly conserved syntenies were found . The most significant synteny involves two members of one C . elegans operon, encoding fibrillarin and ribosomal protein S16 . Their homologues are linked in human, mouse, Drosophila, Anopheles gambiae, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Plasmodium falciparum, and Guillardia theta, but not in five other genomes . The distances between the genes are larger than in the nematode, suggesting the prevalence of intrachromosomal rearrangements. Can J Microbiol, 2004 Jan, 50(1), 61 - 6 Characterization and regulation of the gamma-glutamyl transpeptidase gene from the fission yeast Schizosaccharomyces pombe; Park HJ et al.; The structural gene for the putative gamma-glutamyl transpeptidase (GGT) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe . The determined sequence contained 3324 bp and encoded the predicted 630 amino acid sequence of GGT, which resembles counterparts in Homo sapiens, Rattus norvegicus, Saccharomyces cerevisiae, and Escherichia coli . The S . pombe cells harboring the cloned GGT gene showed about twofold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGT gene was functional . To monitor the expression of the S . pombe GGT gene, we fused the fragment 1085 bp upstream of the cloned GGT gene into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pGT98 . The synthesis of beta-galactosidase from the fusion plasmid in S . pombe cells was enhanced by treatments with NO-generating sodium nitroprusside (SN), L-buthionine-(S,R)-sulfoximine (BSO), and glycerol . The GGT mRNA level in the S . pombe cells was increased by SN and BSO . Involvement of Pap1 in the induction of the GGT gene by SN and BSO was observed. Biol Cell, 2004 Mar, 96(2), 169 - 79 The protein kinase kin1 is required for cellular symmetry in fission yeast; La Carbona S et al.; The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused . The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation . Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p . Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation . Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling . Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus . Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus . The resulting asymmetric cell division produces daughter cells with distinct shapes . Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules . Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell. J Biochem (Tokyo), 2004 Feb, 135(2), 225 - 30 Disruption of the plr1+ gene encoding pyridoxal reductase of Schizosaccharomyces pombe; Morita T et al.; Pyridoxal (PL) reductase encoded by the plr1(+) gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN) . The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B(6), or the excretion of PL as PN from yeast cells . In this study, a PL reductase-disrupted (plr1 Delta) strain was constructed and its phenotype was examined . The plr1 Delta cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media . In EMM, the plr1 Delta strain became flocculent at the late stationary phase for an unknown reason . The plr1 Delta cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1(+) gene, which maintained the flow of "PL --> PN --> PNP --> PLP" in the salvage synthesis of PLP . The total vitamin B(6) and pyridoxamine 5'-phosphate contents in the plr1 Delta cells were significantly lower than those in the wild-type ones . The percentages of the PLP amount as to the other vitamin B(6) compounds were similar in the two cell types . The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type . In contrast, PN was much higher in the latter than the former . The plr1 Delta cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL . The results showed that PL reductase encoded by the plr1(+ )gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis. Arch Microbiol, 2004 May, 181(5), 371 - 7 Epub 2004 Mar 24. Identification of thermostable glyoxalase I in the fission yeast Schizosaccharomyces pombe; Takatsume Y et al.; Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals . Here, the structural gene for glyoxalase I ( glo1(+)) from the fission yeast Schizosaccharomyces pombe was identified . Disruption of glo1(+) enhanced susceptibility to methylglyoxal, while expression of glo1(+) in a Delta glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde . The glo1(+) gene product was purified . The glyoxalase I of S . pombe was a monomeric enzyme with a molecular weight of 34000 and the k(cat)/ K(m) value for methylglyoxal was 4.3 x 10(7) M(-1) x min(-1) . Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn(2+), Co(2+), Ni(2+) and Mn(2+) . The glyoxalase I of S . pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60 degrees C for 4 h. FEMS Yeast Res, 2004 Mar, 4(6), 649 - 54 A distinct type of alcohol dehydrogenase, adh4+, complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe; Sakurai M et al.; In the fission yeast Schizosaccharomyces pombe, only one alcohol dehydrogenase gene, adh1(+), has been identified . To elucidate the influence of adh1(+) on ethanol fermentation, we constructed the adh1 null strain (delta adh1) . The delta adh1 cells still produced ethanol and grew fermentatively as the wild-type cells . Both DNA microarray and RT-PCR analysis demonstrated that this ethanol production is caused by the enhanced expression of a Saccharomyces cerevisiae ADH4-like gene product (SPAC5H10.06C named adh4(+)) . Since the strain lacking both adh1 and adh4 genes (delta adh1 delta adh4) showed non-fermentative retarded growth, only these two ADHs produce ethanol for fermentative growth . This is the first observation that a S . cerevisiae ADH4-like alcohol dehydrogenase functions in yeast ethanol fermentation. FEMS Yeast Res, 2004 Mar, 4(6), 619 - 24 On the role of Trk1 and Trk2 in Schizosaccharomyces pombe under different ion stress conditions; Calero F et al.; Trk1 and Trk2 are the major K(+) transport systems in Schizosaccharomyces pombe . Both transporters individually seem to be able to cope with K(+) requirements of the cells under normal conditions, since only the double mutant shows defective K(+) transport and defective growth at limiting K(+) concentrations . We have studied in detail the role of SpTrk1 and SpTrk2 under different ion stress conditions . Results show that the strain with only Trk1 (trk1(+)) is less sensitive to Li(+) and to hygromycin B, it grows better at low K(+) and it survives longer in a medium without K(+) than the strain expressing only Trk2 (trk2(+)) . We conclude that Trk1 contributes more efficiently than Trk2 to the performance of the fission yeast under ion stress conditions . In the wild type both trk1(+) and trk2(+) genes are expressed and probably collaborate for the performance of the cells. Mol Biol Rep, 2004 Mar, 31(1), 23 - 30 Stress-dependent regulation of the gene encoding gamma-glutamylcysteine synthetase from the fission yeast; Kim SJ et al.; Glutathione (GSH), an important antioxidant involved in stress response, is synthesized in two sequential reactions . Gamma-glutamylcysteine synthetase (GCS) catalyzes the first step in GSH biosynthesis, which is usually known to be rate-limiting . In this work, regulatory patterns of the GCS gene from the fission yeast Schizosaccharomyces pombe have been investigated . The 607 bp upstream region from the translational initiation point was amplified by the two synthetic primers . The amplified DNA was ligated into the BamHI/HindIII site of the shuttle vector YEp367R to generate the fusion plasmid pUGCS101 . The GCS-lacZ fusion gene construct was confirmed by restriction mapping and nucleotide sequencing . The GCS-lacZ fusion gene was used to study effects of various agents on the transcription of the GCS gene . The synthesis of beta-galactosidase from the fusion plasmid pUGCS101 was enhanced by metals, oxidative and nitrosative stresses, and glutathione-depleting agents . The GCS mRNA level in the wildtype S . pombe cells was significantly elevated by the treatment with sodium nitroprusside or menadione, which was detected by RT-PCR . It was also induced by low concentrations of glucose and sucrose . These results suggest that the expression of S . pombe GCS gene is regulated by various stresses and carbon sources. Antonie Van Leeuwenhoek, 2004 Feb, 85(2), 85 - 92 Accumulation and release of the osmolyte glycerol is independent of the putative MIP channel Spac977.17p in Schizosaccharomyces pombe; Kayingo G et al.; Schizosaccharomyces pombe accumulates glycerol as an osmotic regulatory solute in response to hyper-osmotic conditions . Upon a decrease in the external osmolarity, the intracellular glycerol levels should be adjusted in order to attain osmotic homeostasis . In this study, the patterns and kinetics of glycerol export from S . pombe were investigated . Upon a decrease in external osmolarity, glycerol was rapidly exported from cells to the external medium . The amount of glycerol released from the cells was proportional to the degree of change in the external osmolarity . The export process was well controlled and was not affected by reduced temperature . This points to S . pombe controlling glycerol export using specialized facilitating proteins as has been found in Saccharomyces cerevisiae where a MIP family channel protein Fps1p is involved . Analysis of the S . pombe databases revealed a putative transport protein (Spac977.17p) with homology to glycerol channel proteins of the MIP family . However, expression of the gene into the S . cerevisiae strain lacking a glycerol channel protein (fps1Delta mutant), did not complement the defect in glycerol export during hypo-osmotic stress . Deletion of spac977.17, did not affect glycerol accumulation or release in S . pombe . The patterns and kinetics of glycerol release in the mutant were similar to those of the wild type strains suggesting that the export process is independent of Spac977.17p, the only putative MIP family glycerol channel homologue in S . pombe . While the process of glycerol export in response to hypo-osmotic stress is similar to budding yeast, the underlying molecular mechanism in S . pombe appears distinct from that described in S . cerevisiae . Further studies are needed to elucidate the physiological role of the Spac977.17p channel. Genetika, 2004 Jan, 40(1), 26 - 36 {Effect of superexpression of DNA-binding protein heterochromatin Abp1p on frequency of loss of minichromosomes and growth of Schizosaccharomyces pombe with mutations of the gene coding cofactor D}; Fedianina OS et al.; Mitotic chromosome segregation is partly determined by interaction between microtubules (MTs) and the kinetochores of sister chromatids . The precise mechanism of the interaction between kinetochores and MTs remains unclear . This process has been studied in fission yeast Schizosaccharomyces pombe by analyzing interaction between genes encoding kinetochore components, such as DNA-binding protein Abp1p, and genes whose protein products affect the dynamics of MTs, such as cofactor D of tubulin dimer assembly . Analysis of cell growth and minichromosome loss frequency has demonstrated that mutations in the gene of cofactor D, especially mutation tsm1-512, increase the rate of minichromosome loss and the sensitivity to changes in Abp1p concentration in cells compared to wild-type cells Probably, mutations alp1-1315 and tsm1-512 of the cofactor D gene cause defects in the kinetochore-MT interaction. Cell Cycle, 2004 May, 3(5), 529 - 33 Epub 2004 May 01. Resisting arrest: recovery from checkpoint arrest through dephosphorylation of Chk1 by PP1; den Elzen N et al.; The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance . Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry . Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1 . This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release . Here we discuss the implications of this finding on DNA damage checkpoint signaling, and speculate on models for checkpoint maintenance and release. DNA Repair (Amst), 2004 Apr 1, 3(4), 429 - 39 SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae; Onoda F et al.; SMC6 (RHC18) in Saccharomyces cerevisiae, which is a homologue of the Schizosaccharomyces pombe rad18+ gene and essential for cell viability, encodes a structural maintenance of chromosomes (SMC) family protein . In contrast to the rest of the SMC family of proteins, Smc1-Smc4, which are the components of cohesin or condensin, little is known about Smc6 . In this study, we generated temperature sensitive (ts) smc6 mutants of budding yeast and characterized their properties . One ts-mutant, smc6-56, ceased growth soon after up-shift to a non-permissive temperature, arrested in the late S and G2/M phase, and gradually lost viability . smc6-56 cells at a permissive temperature showed a higher sensitivity than wild-type cells to various DNA damaging agents including methyl methanesulfonate (MMS) . The rad52 smc6-56 double mutant showed a sensitivity to MMS similar to that of the rad52 single mutant, indicating that Smc6 is involved in a pathway that requires Rad52 to function . Moreover, no induction of interchromosomal recombination and sister chromatid recombination was observed in smc6-56 cells, which occurred in wild-type cells upon exposure to MMS. Microbiol Mol Biol Rev, 2004 Mar, 68(1), 1 - 108, table of contents Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism; Borkovich KA et al.; We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa . Seven major areas of Neurospora genomics and biology are covered . First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized . The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors . The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination . In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis . Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses . The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle . The seventh section covers topics relevant to animal and plant pathogenesis and human disease . The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease. Mol Biol Cell, 2004 May, 15(5), 2287 - 301 Epub 2004 Mar 05. Identification and characterization of two novel proteins affecting fission yeast gamma-tubulin complex function; Venkatram S et al.; The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe . To better understand its roles, we have purified the S . pombe gamma-tubulin complex . Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms . We show that both Mbo1p and Gfh1p localize to microtubule organizing centers . Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification . In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function . This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S . pombe. Mol Cell Proteomics, 2004 Jun, 3(6), 596 - 607 Epub 2004 Mar 05. Proteomic study for the cellular responses to Cd2+ in Schizosaccharomyces pombe through amino acid-coded mass tagging and liquid chromatography tandem mass spectrometry; Bae W et al.; Cadmium (Cd(2+)) is one of well-known toxic heavy metal ions . To gain a global understanding how Cd(2+) affects cells at the molecular level, we systematically studied the cellular response of the fission yeast Schizosaccharomyces pombe to Cd(2+) using our integrated proteomic strategy of amino acid-coded mass tagging (AACT) and liquid chromatography-tandem mass spectrometry . Our proteome-wide investigation unequivocally identified 1133 S . pombe proteins . Of which, the AACT-based quantitative analysis revealed 106 up-regulated and 55 down-regulated proteins on the Cd(2+) exposure . The most prevalent functional class in the up-regulated proteins, approximately 28% of our profile, was the proteins involved in protein biosynthesis, showing a time-dependent biphasic expression pattern characteristic with rapid initial induction and later repression . Most significantly, 27 proteins functionally classified as cell rescue and defense were up-regulated for oxygen and radical detoxification, heat shock response, and other stress response . Furthermore, the large precursor sequence coverage of our AACT approach allowed us to unequivocally identify and quantitate different isozymes for glutathione S-transferase, which have close similarity in their amino acid sequence . Our quantitative dataset also showed that 80% of the up-regulated proteins found in the S . pombe response were different from those in the Saccharomyces cerevisiae response . The function of some of the key identifications was validated through biochemical assays . It is very interesting that the induction of cysteine synthase expression was not observed in our study, although it has been proven as a critical enzyme to supply free cysteines for the enhancing synthesis of Cd(2+)-sequestering molecules such as glutathione and phytochelatins in plants and some yeasts . Our quantitative proteomic result instead suggested that, as an alternative mechanism for the detoxification of Cd(2+), S . pombe produced significantly higher level of inorganic sulfide to immobilize cellular Cd(2+) as a form of CdS nanocrystallites capped with glutathione and/or phytochelatins. J Mol Biol, 2004 Mar 19, 337(2), 243 - 53 A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe; Eisenhaber B et al.; The fungal transamidase complex that executes glycosylphosphatidylinositol (GPI) lipid anchoring of precursor proteins has overlapping but distinct sequence specificity compared with the animal system . Therefore, a taxon-specific prediction tool for the recognition of the C-terminal signal in fungal sequences is necessary . We have collected a learning set of fungal precursor protein sequences from the literature and fungal proteomes . Although the general four segment scheme of the recognition signal is maintained also in fungal precursors, there are taxon specificities in details . A fungal big-Pi predictor has been developed for the assessment of query sequence concordance with fungi-specific recognition signal requirements . The sensitivity of this predictor is close to 90% . The rate of false positive prediction is in the range of 0.1% . The fungal big-Pi tool successfully predicts the Gas1 mutation series described by C . Nuoffer and co-workers, and recognizes that the human PLAP C terminus is not a target for the fungal transamidase complex . Lists of potentially GPI lipid anchored proteins for five fungal proteomes have been generated and the hits have been functionally classified . The fungal big-Pi prediction WWW server as well as precursor lists are available at Nucleic Acids Res, 2004 Mar 01, 32(4), 1480 - 91 Print 2004. The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast; Doe CL et al.; Homologous recombination is important for the repair of double-strand breaks and daughter strand gaps, and also helps restart stalled and collapsed replication forks . However, sometimes recombination is inappropriate and can have deleterious consequences . To temper recombination, cells have employed DNA helicases that unwind joint DNA molecules and/or dissociate recombinases from DNA . Budding yeast Srs2 is one such helicase . It can act by dissociating Rad51 nucleoprotein filaments, and is required for channelling DNA lesions to the post-replication repair (PRR) pathway . Here we have investigated the role of Srs2 in controlling recombination in fission yeast . Similar to budding yeast, deletion of fission yeast srs2 results in hypersensitivity to a range of DNA damaging agents, rhp51-dependent hyper-recombination and synthetic sickness when combined with rqh1- that is suppressed by deleting rhp51, rhp55 or rhp57 . Epistasis analysis indicates that Srs2 and the structure-specific endonuclease Mus81-Eme1 function in a sub-pathway of PRR for the tolerance/repair of UV-induced damage . However, unlike in Saccharomyces cerevisiae, Srs2 is not required for channelling lesions to the PRR pathway in Schizosaccharomyces pombe . In addition to acting as an antirecombinase, we also show that Srs2 can aid the recombinational repair of camptothecin-induced collapsed replication forks, independently of PRR. Mol Cell Biol, 2004 Mar, 24(6), 2324 - 31 Lub1 participates in ubiquitin homeostasis and stress response via maintenance of cellular ubiquitin contents in fission yeast; Ogiso Y et al.; Ubiquitin-dependent proteolysis plays a pivotal role in stress responses . To investigate the mechanisms of these cellular processes, we have been studying Schizosaccharomyces pombe mutants that have altered sensitivities to various stress conditions . Here, we showed that Lub1, a homologue of Ufd3p/Zzz4p/Doa1p in budding yeast, is involved in the regulation of ubiquitin contents . Disruption of the lub1+ gene resulted in monoubiquitin as well as multiubiquitin depletion without change in mRNA level and in hypersensitivity to various stress conditions . Consistently, overexpression of genes encoding ubiquitin suppressed the defects associated with lub1 mutation, indicating that the phenotypes of the lub1 mutants under stress conditions were due to cellular ubiquitin shortage at the posttranscriptional level . In addition, the lub1-deleted cells showed aberrant functions in ubiquitin/proteasome-dependent proteolysis, with accelerated degradation of ubiquitin . Also Cdc48, a stress-induced chaperon-like essential ATPase, was found to interact with Lub1, and this association might contribute to the stabilization of Lub1 . Our results indicated that Lub1 is responsible for ubiquitin homeostasis at the protein level through a negative regulation of ubiquitin degradation. Genome Res, 2004 Mar, 14(3), 451 - 8 Eukaryotic regulatory element conservation analysis and identification using comparative genomics; Liu Y et al.; Comparative genomics is a promising approach to the challenging problem of eukaryotic regulatory element identification, because functional noncoding sequences may be conserved across species from evolutionary constraints . We systematically analyzed known human and Saccharomyces cerevisiae regulatory elements and discovered that human regulatory elements are more conserved between human and mouse than are background sequences . Although S . cerevisiae regulatory elements do not appear to be more conserved by comparison of S . cerevisiae to Schizosaccharomyces pombe, they are more conserved when compared with multiple other yeast genomes (Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces bayanus) . Based on these analyses, we developed a sequence-motif-finding algorithm called CompareProspector, which extends Gibbs sampling by biasing the search in regions conserved across species . Using human-mouse comparison, CompareProspector identified known motifs for transcription factors Mef2, Myf, Srf, and Sp1 from a set of human-muscle-specific genes . It also discovered the NFAT motif from genes up-regulated by CD28 stimulation in T-cells, which implies the direct involvement of NFAT in mediating the CD28 stimulatory signal . Using Caenorhabditis elegans-Caenorhabditis briggsae comparison, CompareProspector found the PHA-4 motif and the UNC-86 motif . CompareProspector outperformed many other computational motif-finding programs, demonstrating the power of comparative genomics-based biased sampling in eukaryotic regulatory element identification. Bioinformatics, 2004 Mar 1, 20(4), 569 - 75 Epub 2004 Jan 22. Enrichment of transcriptional regulatory sites in non-coding genomic region; Xue W et al.; MOTIVATION: Over-represented k-mers in non-coding genomic regions often lead to identification of potential transcriptional regulatory sites (TRS) . This phenomenon has been employed by many algorithms to predict TRS in silico . Yet, the improvement of these algorithms should be based on deeper understanding of the enrichment feature . To obtain a general distributional profile of TRS in different regions of genomes as well as in different genomes, we here performed a systematic analysis on the over-representation of TRS in intergenic regions and gene upstream regions of yeasts and viral genomes, and the distributional pattern of TRS in intergenic and intron regions of the Drosophila genome . We also explored the way to evaluate the accuracy of TRS consensus sequences by measuring their enrichment . RESULTS: To measure enrichment, a statistical background model was introduced by comparing TRS frequency in certain regions of genome to either the frequency in the whole genome or the frequency in exon region . This model was applied to different classes of non-coding genomic regions in four genomes . Most of the TRS were observed to be over-represented in the intergenic regions of the Saccharomyces cerevisiae, Schizosaccharomyces pombe and Epstein-Barr virus (EBV) genomes . The enrichment of S.cerevisiae TRS in the 600 bp upstream region of genes was also significant . In Drosophila genome, TRS did not show enrichment in intergenic and intron regions when TRS frequency in the whole genome was taken as background, as we did in other genomes . However, when we took TRS frequency in exon region as background, over 70% TRS are over-represented in those two classes of non-coding regions . This fact indicates the existence of transcriptional regulatory signals in introns . The analysis of some S.cerevisiae TRS, which have inconsistent consensus sequences with different levels of enrichment in intergenic region, suggests the possibility of evaluating the accuracy of experimentally determined TRS by measuring their enrichment in non-coding genomic regions. EMBO J, 2004 Apr 21, 23(8), 1792 - 1803 Epub 2004 Feb 26. Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot; Yamada T et al.; Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration . Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1.Pcr1 . Chromatin remodeling occurs meiotically around M26 . We examined the roles of HATs and ADCRs in chromatin remodeling around M26 . Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis . SpGcn5, the S . pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo . Deletion of gcn5(+) caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency . The snf22(+) (a Swi2/Snf2-ADCR homologue) deletion and snf22(+)gcn5(+) double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26 . These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner. Dev Genes Evol, 2004 Mar, 214(3), 149 - 58 Epub 2004 Feb 17. A description of the Mei2-like protein family; structure, phylogenetic distribution and biological context; Jeffares DC et al.; The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus . We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences . Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs . The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs . RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains . Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes . While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation. Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 984 - 90 Cdk inhibitor ste9p/srw1p is involved in response to protein synthesis inhibition in fission yeast; Sakai T; It remains unknown whether the cell cycle system responds properly to protein synthesis inhibition . In this paper I report finding in Schizosaccharomyces pombe that partially deleted elongation factor 3 genes rescue various mitotic catastrophe mutants depending on deltaste9 in a dominant-negative manner . In response to protein synthesis inhibitors, deltaste9 and some other mutants delay halting the cell cycle at G2-M and the combined cdc2-M26 deltaste9 mutant greatly loses viability . It is suggested that cell cycle be positively controlled in an ste9-dependent manner before essential factors for viability and other important functions are exhausted when protein synthesis is inhibited. Biochem J, 2004 Jun 1, 380(Pt 2), 441 - 8 Cell viability and secretion of active proteins in Schizosaccharomyces pombe do not require the chaperone function of calnexin; Marechal A et al.; Folding of newly synthesized proteins within the ER (endoplasmic reticulum) is a rate-limiting step in protein secretion . Thus ER molecular chaperones and foldases have a major impact in determining the rate and yield of these crucial cellular processes . Calnexin is a key ER chaperone implicated in the folding, retention and targeting for degradation of proteins that go through the secretory pathway . Calnexin molecules contain a highly conserved central domain (hcd) that has been proposed to be involved in the interaction with folding substrates and other chaperones . To gain a better understanding of the roles played by calnexin in the secretory pathway, we examined the efficiency of fission yeast (Schizosaccharomyces pombe) strains expressing calnexin mutants to secrete different model proteins . Remarkably, calnexin hcd-deletion mutants, although devoid of detectable chaperone activity in vitro, confer viability and cause a considerable increase in the secretion of heterologous cellulase . Surprisingly the quality-control efficiency, measured as the activity/amount ratio of secreted model protein, was not severely reduced in these calnexin hcd-deletion mutant strains . Our results indicate that the essential function of calnexin does not reside in its role in the folding or in the retention of misfolded proteins . These observations suggest the existence of a highly stringent quality control mechanism in the ER of S . pombe that might reduce the secretion efficiency of endogenous proteins. J Am Chem Soc, 2004 Mar 3, 126(8), 2368 - 71 Guanosine distribution and oxidation resistance in eight eukaryotic genomes; Friedman KA et al.; Reactive oxygen species that attack DNA are continuously generated in living cells . Both the guanosine (G) mole fraction and its distribution should affect the stability of genomes and their parts to oxidation . At a lesser G content, genomes should be more oxidation resistant or "ennobled" . Oxidant scavenging by G's in nonessential parts of introns and intergenic domains should decrease G oxidation in the essential exons . To determine whether genomes are indeed ennobled and whether oxidant-scavenging domains exist in genomes, the relative rates of guanosine oxidation in average exons, introns, and intergenic domains were estimated . Comparison among genomes indicated that average exons are ennobled in the genomes of Caenorhabditis (worm), Arabidopsis (plant), Saccharomyces (yeast), Schizosaccharomyces (yeast), and Plasmodium (malaria parasite), and that average introns and intergenic domains are ennobled in these genomes and in the genome of Drosophila (fly) . The exon oxidation rates estimated for these genomes were less than the rate for the hypothetical "standard" genome, with a 0.25 mole fraction of uniformly distributed G . For Plasmodium the rate was half of that estimated for the standard genome . Average exons were not ennobled in the human or fly genomes; their G distributions were comparable to that in the standard genome . Instead, their exons were situated between introns and intergenic domains that could protect them by oxidant scavenging, the G's of their introns and intergenic domains outnumbering those of their exons 50-fold in humans and 4-fold in flies . The G distribution in the Encephalitozoon (parasite) genome was not protective relative to that of the standard genome. Biosci Biotechnol Biochem, 2004 Feb, 68(2), 306 - 11 Characterization of two genes encoding putative cysteine synthase required for cysteine biosynthesis in Schizosaccharomyces pombe; Fujita Y et al.; Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe . An examination of the S . pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases . Disruption of one of these candidates, cys1a+ (SPBC36.04), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype . Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells . These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S . pombe cells . We constructed a bacterial-S . pombe shuttle vector containing cys1a+ as a selective marker gene . The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein. EMBO Rep, 2004 Mar, 5(3), 311 - 6 Epub 2004 Feb 06. eIF4E isoform 2 in Schizosaccharomyces pombe is a novel stress-response factor; Ptushkina M et al.; Cap-binding proteins of the elF4E family are generally involved in mediating ribosome recruitment to capped mRNA via an interaction with the initiation factor elF4G . However, Schizosaccharomyces pombe has two elF4E isoforms, one of which (elF4E2, encoded by tif452) has a relatively low affinity for elF4G . We show that tif452 is required for specific stress responses . An S . pombe, tif452delta mutant manifests slow growth under conditions of nutrient, temperature and salt stress . elF4E2 shows a distinct subcellular distribution to elF4E1, the cap-binding factor that is required for mainstream translation . In response to salt stress, the cellular level of elF4E2 increases, whereas the amount of intact elF4G decreases, leaving elF4E2 as the predominant elF4E isoform in a cell deficient in ElF4G . The presence of elF4E2 modifies the competence of S . pombe ribosomes to translate mRNAs with structured leaders in vivo . The tif452 promoter has putative stress-response (T-rich) motifs, whereas elF4E2 seems to be a new type of stress-response factor. J Biol Chem, 2004 Apr 23, 279(17), 17434 - 42 Epub 2004 Feb 12. Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions; Thakurta AG et al.; Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe . Here we show that S . pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential . We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67 . These residues map within the known NTF2-like fold of TAP (amino acids 371-551) . We show that the homologs of these two NESs are present and are functionally conserved in TAP . The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S . pombe Nup159 and Nup98 but not with human p62 . Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S . pombe and in HeLa cells . Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S . pombe strain, suggesting that the NESs can function in the absence of p15 . These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex. J Cell Sci, 2004 Feb 29, 117(Pt 6), 907 - 18 A non-chromosomal factor allows viability of Schizosaccharomyces pombe lacking the essential chaperone calnexin; Collin P et al.; Calnexin is a molecular chaperone playing key roles in protein folding and the quality control of this process in the endoplasmic reticulum . We, and others, have previously demonstrated that cnx1(+), the gene encoding the calnexin homologue in Schizosaccharomyces pombe, is essential for viability . We show that a particular cnx1 mutant induces a novel mechanism allowing the survival of S . pombe cells in the absence of calnexin/Cnx1p . Calnexin independence is dominant in diploid cells and is inherited in a non-Mendelian manner . Remarkably, this survival pathway, bypassing the necessity for calnexin, can be transmitted by transformation of cell extracts into a wild-type naive strain, thus implicating a non-chromosomal factor . Nuclease and UV treatments of cells extracts did not obliterate transmission of calnexin independence by transformation . However, protease digestion of extracts did reduce the appearance of calnexin-independent cells, indicating that a protein element is required for calnexin-less viability . We discuss a model in which this calnexin-less survival mechanism would be activated and perpetuated by a protein component acting as a genetic element. Lett Appl Microbiol, 2004, 38(3), 191 - 6 A simple and reliable method for rapid production and purification of Pseudomonas aeruginosa haemolytic phospholipase C; Dubouix A et al.; AIMS: To design a simple method to produce active recombinant Pseudomonas aeruginosa haemolytic phospholipase C (PLC) . METHOD AND RESULTS: Pseudomonas aeruginosa PLC is a virulence factor mainly involved in inflammatory and cytotoxic responses . While ammonium sulphate purification requires large amounts of bacterial suspensions and leads to low yields, production of recombinant protein in Escherichia coli is no more successful because of frequent inclusion bodies and accumulation of inactive PLC in the periplasmic space . Using an inducible system based on the glucose-repressed inv1 promoter in the yeast Schizosaccharomyces pombe, we were able to produce up to 10 IU ml(-1) of pure toxin within 24 h . CONCLUSIONS: This work describes the first method to easily get recombinant haemolytic PLC . SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a powerful tool to study the mechanisms leading to its cellular toxicity. Eukaryot Cell, 2004 Feb, 3(1), 82 - 90 DNA repair functions that control sensitivity to topoisomerase-targeting drugs; Malik M et al.; DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation . Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate . Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents . Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs . We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment . S . pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II . In contrast to results observed with Saccharomyces cerevisiae, S . pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents . The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors . Finally, repair genes (such as the S . pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs . Since the pattern of sensitivity is distinct from that seen with other systems (such as the S . cerevisiae system), our results highlight the usefulness of S . pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases. Eukaryot Cell, 2004 Feb, 3(1), 27 - 39 ADAM family protein Mde10 is essential for development of spore envelopes in the fission yeast Schizosaccharomyces pombe; Nakamura T et al.; We report the identification of Schizosaccharomyces pombe mde10+ as a gene possessing a FLEX element, which forms a binding site for the meiosis-specific transcription factor Mei4 . In fact, mde10+ is transcribed only in diploid cells that are induced to meiosis in a Mei4-dependent manner . Western blot analysis indicated that the epitope-tagged Mde10 protein accumulates transiently during meiosis and then rapidly decreases . Mde10 is a multidomain protein containing a metalloprotease catalytic domain, a disintegrin domain, a cysteine-rich domain, and membrane-spanning regions, all of which are shared by members of the mammalian ADAM family . A fusion protein of Mde10 and green fluorescent protein localized to the endoplasmic reticulum during meiosis and was located at the peripheral region of spores at the end of meiosis . An mde10Delta deletion mutant showed no apparent defects in meiosis, sporulation, or spore germination . However, the mutant spores exhibited an aberrant surface appearance, in which the ragged outer spore wall was lost to a large extent . Furthermore, mde10Delta spores were found to be less tolerant to ethanol and diethyl ether than were wild-type spores . The mutagenic replacement of the conserved glutamic acid in the putative protease active site with an alanine residue did not affect the surface morphology or the resistance of spores to environmental stress . Our observations indicate that Mde10 is important in the development of the spore envelope, although this function of Mde10 seems to be independent of its metalloprotease activity. J Basic Microbiol, 2004, 44(1), 36 - 41 Electrophoretic karyotype of two Micromucor species; Nagy A et al.; Electrophoretic karyotype analysis was applied to obtain information on the organisation and intrageneric variability of the nuclear genome in three Micromucor isolates of two different species (M . isabellina and M . ramanniana) . A protoplast formation protocol, conditions for the preparation of highly-intact chromosome-size DNA molecules and for the separation of DNA molecules were established . The chromosomal banding patterns revealed substantial variability among the isolates: 11 to 14 chromosomal mobility groups were resolved . The DNA in the Micromucor chromosomes were rather small; their estimated sizes were calculated to be between 2.60 and 0.4 Mb . Using Saccharomyces cerevisiae and Schizosaccharomyces pombe as size standard, the minimum total genome sizes were estimated to be between 24.19 and 24.9 Mb. Mol Biol Cell, 2004 Apr, 15(4), 1656 - 65 Epub 2004 Feb 06. Mmd1p, a novel, conserved protein essential for normal mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe; Weir BA et al.; The mmd1 mutation causes temperature-sensitive growth and defects in mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe . In mutant cells, mitochondria aggregate at the two cell ends, with increased aggregation at elevated temperatures . Microtubules, which mediate mitochondrial positioning in fission yeast, seem normal in mmd1 cells at permissive temperature and after several hours at the nonpermissive temperature but display aberrant organization after prolonged periods at 37 degrees C . Additionally, cells harboring both mmd1 and ban5-4, a temperature-sensitive allele of alpha2-tubulin, display synthetic defects in growth and mitochondrial distribution . The mmd1 mutation maps to an open reading frame encoding a novel 35.7-kDa protein . The Mmd1p sequence features repeating EZ-HEAT motifs and displays high conservation with uncharacterized homologues found in a variety of organisms . Saccharomyces cerevisiae cells depleted for their MMD1 homologue show increased sensitivity to the antimicrotubule drug benomyl, and the S . cerevisiae gene complemented the S . pombe mutation . Mmd1p was localized to the cytosol . Mmd1p is the first identified component required for the alignment of mitochondria along microtubules in fission yeast.
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